In the female sex the first stage of the development of gametes is similar to that found in spermatogenesis – the oogonia—the cells eventually giving rise to the eggs— undergo proliferation by mitotic divisions. They then become oocytes and enter a period of growth.
Owing to the fact that the egg contributes the greater part of the substances used in development, growth plays a much greater role in oogenesis than in spermatogenesis. Also, the differentiation of the egg occurs simultaneously with growth, rather than after maturation.
The period of growth in the female gametes is a very prolonged one, and the increase in size is considerable. In frogs a young oocyte may be about 50 µm in diameter, and the fully developed egg in many species is between 1000 µm and 2000 µm in diameter. If Rana pipiens, in which the diameter of the mature egg is about 1500 µm, is taken as an example, the increase in size of the oocyte is by a factor of 27,000.
This growth takes place over a period of three years. The young oocytes start growing after the tadpoles metamorphose into young froglets. One-year-old and two-year-old frogs do not yet have mature eggs, but by the third year the eggs are ready and the frogs may spawn for the first time.
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Every year a new batch of oocytes is produced as the result of oogonial divisions, but these do not mature until three years later, so that oocytes of three generations may be contained in the ovary at the same time. The growth of the oocytes is fairly slow during the first two seasons but becomes much more rapid in the summer of the frog’s third year of life; so that by the autumn the eggs reach their maximum dimensions.
In other animals the growth of oocytes may proceed at a much higher rate and may take a shorter time for completion. In the hen the last rapid growth of the oocyte occurs in the 6 to 14 days preceding ovulation, and during this time the volume of the oocyte increases 200-fold.
In mammals the proliferation of the oogonia is restricted to the intrauterine period of life, and all the eggs produced by a mammalian female throughout her reproductive life are derived from oocytes already present at birth. The oocytes reach their full size in 16-day-old mice.
The eggs of mammals are, of course, much smaller than those of amphibians. The oocyte of a mouse grows from a size of about 20 µm in diameter into the ripe ovum of about 70 µm in diameter, an increase by a factor of only 43 as compared with an increase of 27,000 in the frog.
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The fully grown egg is a relatively large cell, always larger than the average somatic cell in the animal. When large quantities of foodstuffs are stored in the egg cell, it may attain giant proportions. The yolk of a hen’s egg, which represents the egg cell in this case, has an average weight of 55 gm. and is, of course, by no means the largest of its kind.
Very large eggs are also found in reptiles and in some sharks. However, the quantity of active cytoplasm in such large eggs is comparatively rather small, and the nucleus, although larger than in the somatic cells, never increases in proportion to the bulk of the whole egg.
In many groups of animals, notably in the chordates, the oocytes are surrounded during their entire growth and maturation stages by special cells of the ovary, the follicle cells. In mammals the follicle cells are derived from the germinal epithelium of the ovaries, and initially the young oocyte is surrounded by one layer of follicle cells, which form a simple cuboidal epithelium around the oocyte.
Later, the number of follicle cells increases greatly, the cells becoming arranged in several rows. As the egg approaches maturity, an eccentric cavity appears in the mass of the follicle cells. This cavity is filled with fluid secreted presumably by the cells of the follicle. The follicle at this stage is known as a Graafian follicle.
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The oocyte is surrounded by follicle cells not only in mammals but in other vertebrates as well, though due to the larger size of the egg the follicle cells are not so conspicuous. It is believed that the follicle cells actively assist the growth of the oocyte by secreting substances which are taken up by the oocyte.
The structural relationships between the follicle cells and the oocytes, revealed by electron microscopic studies in recent years, are very peculiar. Originally there is a simple apposition of the follicle cells and the oocyte, similar to the one existing between ordinary epithelial cells – the cytoplasmic membranes of the adjoining cells are separated only by a narrow gap of about 80 Å.
At certain points the two cytoplasmic membranes show a close connection in the form of desmosomes; here the plasma membranes of the adjoining cells are thickened and the space in between appears to be filled by a denser substance which presumably holds the two cell membranes together.
At a later stage a wider space appears between the follicle cells and the oocyte. The follicle cells, however, retain contact with the oocytes at the points where the desmosomes could be noticed in the previous stage. At these points the cytoplasm of the follicle cell becomes drawn out into elongated processes or microvilli, reaching across the space separating the follicle cell and the oocyte.
At the same time, the surface of the oocyte also produces numerous finger-like microvilli projecting into the space between the oocyte and follicle cells. The microvilli of the oocyte inter-digitate with those of the follicle cells.
With the light microscope, individual microvilli cannot be seen, and the zone of microvilli appears as a radially striated layer, which has long been known, in mammals, as the zona radiata. The presence of the microvilli greatly increases the surface area of the oocyte. In the frog oocyte the increase in surface area has been estimated to be by a factor of about 35. The increase in area appears to facilitate metabolic turnover between the oocyte and its environment.
Small inpocketings of the oocyte cytoplasm may often be observed at the base of the microvilli. These are interpreted as an indication that the oocyte is taking in fluids and dissolved substances from the space between itself and the follicle cells by means of pinocytosis or “cell drinking”.
Toward the time when the oocyte reaches its full size a denser material, often of fibrillar structure, appears in between the inter-digitating process of the oocyte and the follicle cells. The material becomes consolidated and eventually fills most of the space between the follicle cells and the oocyte, becoming the “primary” egg envelope.
In the last stages of oocyte maturation the cytoplasmic processes of the oocyte and of the follicle cells may be withdrawn, and the space between the two is then taken up by the primary egg envelope. This is the case in all vertebrates studied so far and particularly in mammals.
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In bivalve molluscs, however, the microvilli of the oocyte are not withdrawn, and at the time when the egg is released from the ovary, its primary envelope is still perforated by long processes of the egg cytoplasm; these persist even at the beginning of the cleavage. In fishes the perforations in the egg envelope which are left after the cytoplasmic processes are withdrawn become canals (“micropiles”) through which the spermatozoa can reach the egg.
The development on the surface of the oocyte of a system of microvilli which inter-digitate with cytoplasmic processes of the follicle cells is a general rule, but there are some exceptions, the most important of which are in the eggs of sea urchins.
The follicle cells in these animals are closely apposed to the surface of the oocyte, and no inter-digitating microvilli are present. Nevertheless, when the mature egg leaves the ovary it is surrounded by a thin layer of jelly, which must have been formed during the growth of the oocyte in the space between the oocyte and the surrounding follicle cells.
In some insects, molluscs, and annelids the relationship between the oocyte and the rest of the ovary is further complicated by the presence of special nurse cells which, together with follicle cells, take part in providing the nutrition for growing oocytes. The nurse cells are closely related in their origin to the egg cell and may be considered to be abortive oocytes.
In Drosophilia an oogonial cell gives rise, by four successive mitotic divisions, to 16 cells of which one becomes an oocyte and the other 15 become nurse cells. The nurse cells are thus siblings of the oocyte. The whole complex is surrounded by follicle cells. The mode of transfer of materials from nurse cells to the oocyte is radically different from the way matter is given off to the oocyte by the follicle cells.
No microvilli or cytoplasmic processes are developed at the interface between the oocyte and nurse cells. Instead, gaps may appear through the cell membranes of the oocyte and the nurse cell, and through such gaps the cytoplasm of the nurse cell pours into the oocyte. In any case, the nurse cells become used up during the growth of the oocyte, or they may be completely engulfed in the cytoplasm of the oocyte (in the snail Helix).
The materials used in the growth of the oocytes are to a large extent produced outside, in other parts of the body of the female, and brought to the gonads by way of the blood stream. In vertebrates the site of synthesis of egg proteins and phospholipids appears to be the liver. This may be proved by using precursors marked by a radioactive isotope atom such as 32P in disodium hydrogen phosphate.
When such a precursor was injected into the blood of a laying hen and the amount of radioactivity was measured in different tissues, it was found that after six hours the radioactivity was highest in the liver (164.0 units/g phosphate); the radioactivity was weaker in the blood (38.3 units/g phosphate), and very small indeed in the growing oocytes (0.62 units/g phosphate).
These results show that the injected phosphate was mainly taken up by the liver. Twelve hours later the radioactivity in the liver was down to 101.0 units/g phosphate but was increased slightly in the blood (to 45.4 units/g phosphate). There was a very large increase in radioactivity in the oocytes to 4.1 units/g phosphate. This indicates that the substances synthesized in the liver with inclusion of the injected phosphate found their way via the blood into the growing oocytes.
By direct chemical analysis it can be shown that a chemical constituent of the egg yolk (phosvitin) is present in the blood plasma of a laying hen. By use of immunological methods it was found that, in the frog, substances (antigens) identical to the ones found in the growing oocytes are present in the liver and in the blood serum.
A similar investigation has been performed with females of the cecropia silk moth (Plafysamia cecropia). It was proved by using immunological methods that a specific protein contained in the blood plasma of female pupae passes into the developing eggs and becomes a major part of the yolk of the egg.
As only larger molecules can be detected i by serological methods, the foregoing investigations serve to indicate not only that building materials such as amino acids are brought to the oocytes from without but that macromolecules (proteins, phosphoproteins, and phospholipids) can be supplied to the oocytes in the same way.
That building materials are supplied to the oocytes through the mediation of follicle cells and nurse cells (where such exist) can also be demonstrated by using radioactively marked precursors. Females of the common fly, Musca domestica, were injected with tritiated (containing radioactive hydrogen, 3H) cytidine, a nucleic acid precursor, or tritiated (3H-containing) histidine, a protein precursor.
After varying intervals the animals were fixed, the gonad sectioned, and the sections covered with a photographic film. The emulsion of the film shows black dots where the film is hit by electrons shooting out from the radioactive hydrogen atoms in the tissues.
Thirty minutes after the injection, the radioactively labeled amino-acid molecules are lodged predominantly in the follicle cells, but after three hours, radioactivity in the follicle cells disappears, and intense radioactivity is present in oocyte cytoplasm, mainly in the surface layer, thus showing that the labeled material has been passed from the follicle cells to the oocyte.
The nucleic acid precursors, on the other hand, appear to be taken primarily into the nuclei of the nurse cells, which are intensely radioactive one hour after the injection.
The newly synthesized radioactively labeled ribonucleic acid soon passes out of the nuclei into the cytoplasm of the nurse cells, so that in five hours the nuclei are practically free of radioactivity. From the cytoplasm of the nurse cells the labeled RNA then passes into the cytoplasm of the oocyte through the gaps connecting the nurse cells to the oocyte.
A comparison of work done on the growth of avian, amphibian, and insect oocytes shows that the mechanism of food supply to the oocyte in these animals is rather similar. There is evidence, on the other hand, that in some animals the synthesis of yolk proteins occurs inside the oocytes, and consequently, the materials necessary for yolk formation enter the oocytes in a simpler form.
Nuclear Activity during Growth of Oocyte:
Simultaneously with the growth of the oocyte its nucleus enters into the prophase of the meiotic divisions; the homologous chromosomes pair together similarly to what occurs in the primary spermatocytes. The subsequent stages of meiosis, however, are postponed until the end of the period of growth.
Instead, the nucleus of the oocyte increases in size, though not nearly to the same extent as the cytoplasm. The increase in size is due mainly to the production of large amounts of nuclear sap, so that the nuclei of advanced oocytes appear to be bloated with fluid and are usually referred to as germinal vesicles.
The chromosomes at the same time may increase in length, but the amount of deoxyribonucleic acid in the chromosomes does not increase in proportion to the enlargement of the nucleus. As a result, the nuclei in this stage are difficult to stain with the usual agents, such as methyl green or Feulgen reagent.
In oocytes of animals having large eggs, the chromosomes acquire a very characteristic appearance; thin threads or loops are “thrown out” transverse to the main axis of the chromosomes, making the chromosomes look like lampbrushes, thus the name lampbrush chromosomes.
It is believed that the loops represent actual sections of the chromosome which have become completely despiralized, and that this is a favorable condition for the main activity of the genes, namely, to synthesize the messenger RNA, which is subsequently to control the synthesis of proteins in the cell.
By using a radioactively labeled RNA precursor (uridine), it has actually been proved that RNA synthesis occurs on the loops of the lampbrush chromosomes. With the aid of very fine micro-chemical methods, the chromosomal RNA in oocytes has been found to have a different base composition (that is, a different ratio of adenine and uracil to guanine and cytosine) than the base ratio of ribosomal RNA, but it fairly closely resembles the base ratio of the chromosomal DNA (with thymidine replacing uracil).
These observations are in keeping with the assumption that the RNA synthesized on the loops of the lampbrush chromosomes is, base for base, a copy of the chromosomal DNA, in other words, that it becomes a carrier of genetic information contained in the chromosomes. This genetic information is then presumably passed into the oocyte cytoplasm, where proteins are being synthesized in the course of the growth of the oocyte.
The nucleoli in the germinal vesicle seem to be actively involved in the metabolism of the growing oocyte, as they are concerned with the synthesis of ribosomal RNA. The nucleolus of the oocyte increases greatly in size and becomes very conspicuous against the background of the vesicular nucleus.
In many animals, particularly amphibians, instead of one large nucleolus, numerous smaller nucleoli are formed in the germinal vesicle. Most of these become localized on the periphery of the nucleus, immediately underneath the nuclear membrane.
The formation of numerous nucleoli is the result of a very peculiar phenomenon that occurs in the oocytes of at least some animals. It is the outward expression of an increase in the number of genes coding for ribosomal nucleic acids, an occurrence which is due to the need for producing large amounts of these acids during the growth of the oocyte.
The genes for ribosomal nucleic acids, even in somatic cells, are present in a number of identical copies. In the frog, Xenopus laevis, the genes coding for the two main molecules which go into the formation of a ribosome, the 18S and the 28S RNA’s, are represented in a haploid chromosome set about 450 times. The third gene, coding for the smaller 5S ribosomal RNA, is present in some 20,000 copies.
In normal mitosis the ribosomal genes are duplicated in the same way as other genes, preserving their numbers in the daughter cells. In oocytes, however, the genes for 18S and 28S RNA’s are multiplied, without mitosis taking place, by a factor of several hundred. This increase in the number of genes without mitosis is referred to as amplification of the genes concerned.
The genes which code for the 18S and 28S rRNA’s are located in a special sector of a chromosome responsible for the formation of the nucleolus, the “nucleolus organizer.” The amplification of the ribosomal genes produces numerous nucleolus organizers and is responsible for the large number of nucleoli in amphibian oocytes.
The genes coding for the 5S rRNA, which are scattered on different chromosomes, are not amplified, perhaps because their number is already so high in somatic chromosomes. Ribosomal gene amplification has also been recorded in a worm and a mollusc, although the degree of amplification is much lower (by a factor of 5) and does not lead to the formation of additional nucleoli.
There are many reports, in both the older and more recent literature, that nucleic acids, in particular ribonucleic acid, pass out of the nucleus into the cytoplasm during the growth of the oocyte. In some cases large masses of nucleolar material, visible with the light microscope, have been seen passing through gaps in the nuclear membrane. In practically every animal studied with the electron microscope, electron dense material, which could be recognized as RNA, has been seen passing through the nuclear pores into the cytoplasm.
In the oocyte cytoplasm the RNA can be detected in the form of abundant ribosomes through either cyto-chemical methods or electron microscopy. Increased quantities are first seen in the vicinity of the nucleus, and later, some RNA becomes concentrated toward the periphery of the cytoplasm. As the yolk platelets begin to accumulate at the periphery of the oocyte, the area of cytoplasm rich in ribonucleic acid becomes restricted to the deeper parts of the cytoplasm.
There can be no doubt that messenger and transfer RNA are also released from the nucleus during the growth of the oocyte, although this cannot be demonstrated by morphological methods. Evidence concerning the presence of these types of RNA in the cytoplasm of the mature egg will be presented in a different connection.
Of considerable importance is the presence of deoxyribonucleic acid—DNA—in the egg cytoplasm. Quite substantial amounts of DNA have been found in the cytoplasm of mature eggs of amphibians, insects, and sea urchins.
Recent studies have shown that in amphibian oocytes the cytoplasmic DNA is contained in the mitochondria. Though the amount of DNA per mitochondrion is very small, the total amount per mature oocyte may be considerable (200 times greater than the amount of DNA contained in a tetraploid set of somatic chromosomes in Xenopus laeuis).