Metazoa are multicellular animals having cells differentiated into tissues and organs and usually a digestive cavity and nervous system
If we consider the mature egg, either fertilized or parthenogenetic, as the starting point of the germ-cell cycle in the METAZOA, we may recognize seven or eight distinct periods as follows :
1. The segregation of the primordial germ cells ;
i. e., the formation of one or more primordial germ cells during the segmentation of the egg ;
2. Early multiplication of the primordial germ cells ;
3. A long period of "rest" characterized by cessa tion of cell division, either active or passive change of position, separation of the germ cells into two groups which become the definitive germ glands, accompanied by the general growth of the embryo until the larval stage is almost attained ;
4. Multiplication by mitosis of the primitive oogonia or spermatogonia to form a definite number (Miastor and perhaps others) or indefinite number (so far as we know) of oogonia or spermatogonia ;
5. In some cases the differentiation of oogonia into nurse cells and ultimate oogonia, and the spermatogonia into Sertoli cells and ultimate sper matogonia ;
6. The growth of the ultimate oogonia and spermatogonia to form primary obcytes and primary spermatocytes ;
7. Maturation ;
8. Fertilization (if not parthenogenetic) .
1. THE SEGREGATION OF THE PRIMORDIAL GERM CELLS. This phase of the germ-cell cycle is especially emphasized here and need be referred to only casually here. The mature eggs of animals are organized both morphologically and physiologically ; that is, differentiations have already taken place in their protoplasmic contents before they are ready to begin development. This organization determines what sort of divisions the egg will undergo during the cleavage stages. During cleavage certain parts of the cell contents become separated from other parts and thus the differentiated substances of the egg are localized in definite parts of the embryo. The contents of the cleavage cells likewise become differentiated as development proceeds, until finally the cells produced form two or three more or less definite germ layers. In some cases the egg always divides in the same way, and the history or "cell lineage" of the cells can be followed accurately, and the parts of the larva to which they give rise can be established. This is known as determinate cleavage in contrast to the indeterminate type in which there appears to be no relation between the cleavage cells and the structure of the egg or larva.
The degree of organization of the egg no doubt ac counts for the differences in cleavage; those of the determinate type being more fully organized than those of the indeterminate type.
The period when the primordial germ cells are established is probably due in part to the state of organization of the egg when development begins, and it is not strange, therefore, that the primordial germ cell may be completely segregated in certain eggs as early as the four-cell stage ; whereas in others germ cells have not been discovered until a late larval condition has been reached. An ever increasing number of species of animals is being added to those in which an early segregation of the germ cells has already been recorded. Nevertheless, there are certain zoologists who still question the general occurrence of an early segregation of the germ cells, but more careful investigations will probably establish the fact of early segregation in species in which this has not yet been demonstrated.
2. EARLY MULTIPLICATION OF THE PRIMORDIAL GERM CELLS. The number of germ cells present at the time of their first appearance in the embryo varies in different species. There may be one, as in the majority of cases, for example the fly, Miastor (Fig. 17), the nematode, Ascaris (Fig. 51), the crustacean, Cyclops (Fig. 48), and the arrow worm, Sagitta (Fig. 54) ; or a number, as in chrysome lid beetles (Fig. 36), certain parasitic HYMENOPTERA (Fig. 44), and vertebrates (Fig. 6). As a rule the primordial germ cell or cells increase in number by mitosis soon after they are segregated, and then cease to divide for a considerable interval. For example, in Miastor the single primordial germ cell produces eight; in the beetle Calligrapha multi punctata the original sixteen undergo two divisions resulting in sixty-four ; and in the chick Swift (1914) has counted as many as eighty-two at this stage.
We shall see later that the primordial germ cells are often characterized by the presence of certain cytoplasmic inclusions (the keimbahn-determinants) which are absent from the other cells of the embryo. These inclusions appear to be equally divided be tween the daughter cells so that each of the eight or sixty-four, as the case may be, is provided with an equal amount of the keimbahn-determinants.
3. PERIOD OF "REST" AND MIGRATION. By rest here is really meant cessation of division. During this period the germ cells either actively migrate or are passively carried by surrounding tissues to the position the germ glands occupy in the larva. In species possessing two germ glands the germ cells separate to form two groups, with, at least in some cases, an equal number in each group. Thus in Miastor the number in each group is four (Fig. 22) and in Calligrapha, thirty-two (Fig. 37). There is evidence that an active migration of germ cells occurs both in vertebrates and invertebrates. Figure 6 shows the positions of the germ cells in four species of vertebrates during their change of position. That the germ cells at this time are actively migrating by ameboid movements is the general opinion of investigators, since frequently these cells are ameboid in shape and the distance between the place of origin and the germinal ridge is too great to be traversed in any other way.
Professor B. M. Allen, who has made extensive studies of the germ cells of many species of vertebrates, makes the following statement regarding this phase of the germ-cell cycle :
"The sex-cells are migratory to a high degree. The path and time of their migration may vary greatly within a given group of animals, as illustrated by the case of Amia and Lepidosteus. While in the forms that I have studied they are first to be observed in the entoderm, I am quite open to conviction that in other forms they may migrate from this layer into the potential mesoderm before the two layers are separated, as shown by Wheeler in Petromyzon"
Swift (1914) has recently obtained evidence which seems to prove that not only do the germ cells of the chick migrate by ameboid movements but they enter the blood vessels and are distributed by the blood stream to all parts of the embryo and vascular area.
The migration of the germ cells has been noted in many invertebrates and has been fully described in chrysomelid beetles (Hegner, 1909a). In these insects the primordial germ cells are segregated at the posterior end of the egg at the time the blasto derm is formed (Fig. 36, C). The blastoderm is never completed just beneath them, but a canal, called the pole-cell canal, remains. Through this at a later embryonic stage the germ cells migrate by means of ameboid movements.
"As soon as the germ cells of Calligrapha have passed through the pole-cell canal, they lose their pronounced pseudopodia-like processes and become nearly spherical (Fig. 37, E) ; nevertheless, they undergo a decided change in position. They move away from the inner end of the pole-cell canal, and creep along between the yolk and the germ-band. Thus two groups are formed near the developing coelomic sacs; each group probably contains an equal number of cells. The smallest number I have counted in one group at this time is thirty; the largest number, thirty-four. As there is some difficulty in obtaining an accurate count, it seems probable that the sixty-four germ cells are equally divided and that each germ gland receives thirty-two. Some of the germ cells migrate not only laterally along the germ gland but also back toward the posterior end of the egg, where we find them forming narrow strands in the last abdominal segments. From this stage on, the germ cells are not very active ; they move closer to one another to form the compact germ glands. I was unable to determine whether the later movements of the germ cells are due to an active migration or to the tensions created by the growth of the surrounding tissues ; the latter seems the more probable" (Hegner, 1909a, p. 280).
It is thus evident that during the blastoderm stage the germ cells of this beetle are actually outside of the egg. How well this illustrates the theory of primary cellular differentiation, that is, the differentiation of germ cells from somatic cells, since the two sorts are here completely separated, the former constitut ing a group in contact with but not connected with the somatic cells. Later, as the germinal con tinuity hypothesis demands, the germ cells migrate into the embryo, there to be nourished, transported, and protected by the body until they are ready to separate from the somatic cells, and thus to give rise to a new generation.
4. PERIOD OF MULTIPLICATION. Soon after the germ cells aggregate to form more or less rounded groups lying in the position of the definitive germ glands mitotic division is resumed. At about this time also, the sex of the individual can often be determined by the shape of the germ-gland. Then both the testes and the ovaries acquire envelopes of the follicular cells, and frequently testicular cysts and ovarian tubes or chambers develop. The question of the origin of the follicular cells is still unsettled, but the evidence in most cases seems to favor the view that they are mesodermal.
The multiplication of the germ cells by mitosis continues rapidly from this time on. In only one case, so far as I am aware, do we know the actual number of germ cells produced by the primordial germ cell ; this is in Miastor, where typically sixty four oogonia are formed (Fig. 26). As the germ cells multiply they become smaller in size and the substances present in the primordial germ cell become divided among a large number of progeny. Thus at the beginning of the growth period each germ gland contains many oogonia or spermatogonia, and each of these contains a small fraction of the material present in the primordial germ cell, plus whatever substances may have been assimilated during the period of multiplication.
5. THE ORIGIN OF NURSE CELLS AND SERTOLI CELLS. Germ cells receive nourishment during the growth period in many ways, e.g., from nurse cells, follicle cells, or directly from the blood. The origin of the nurse cells and follicle cells is important since in a few cases the germ cells themselves are known to give rise to them. There is thus a second differentiation whereby somatic cells (follicle cells or nurse cells) become differentiated from germ cells (oogonia or spermatogonia) . In some species, such as Miastor, we can prove without question that both the nurse cells and follicle cells are of mesodermal origin, and that the germ cells give rise only to germ cells. On the other hand, there are instances in both vertebrates and invertebrates of a common origin of germ cells and somatic cells from oogonia and spermatogonia. Perhaps the most striking examples are the differentiation of the nurse cells and ultimate oogonia in the water beetle, Dytiscus, and the differentiation of the Sertoli cells and ulti mate spermatogonia in man. (See Chapter V.) Haecker (1912) distinguishes' between a somato-ger minative period and a true germinative period ; the former is that during which the primordial germ cells are established and the latter that of the differentiation of nurse cells and ova.
6. THE GROWTH PERIOD. The last divisions of the oogonia and spermatogonia are followed by the growth of these cells. The extent of this growth depends, in the case of the female, upon whether or not the mature egg is to be supplied with an abundance of nutritive material. Nurse cells, fol licle cells, and circulating fluids may all assist in the enlargement of the oogonia. If the eggs are small, sufficient nutriment is supplied by surrounding liquids and no special nurse cells are required ; but larger eggs either become surrounded by follicle cells which nourish them and with which they are often intimately connected by protoplasmic bridges, or special nurse cells are provided. In the primitive type of ovary, such as exists in most ccelenterates, any of the cells surrounding the oogonium may function as nurse cells and even neighboring oogonia are engulfed by the oogonium that is successful in the struggle for development. A more definite mechan ism exists in higher organisms, where one or more cells become differentiated for the special purpose of supplying nutriment consisting of either their own substance or of material elaborated by them and then transferred to the egg. The egg of the annelid, Ophryotrocha, for example, is accompanied by a single nurse cell ; that of Myzostoma is provided with two, one at either end; and the eggs of certain insects are more or less intimately connected with groups of cells in definite nurse chambers (Fig. 46).
The growth of an oogonium may be well illus trated by that of the potato beetle.
The general arrangement of the cells in the ovary of an adult beetle is shown in Fig. 7. The terminal chamber of the ovarian tubule contains three kinds of cells : (1) nurse cells (n.c), (2) young oocytes (y.o) and growing oocytes, and (-3) epithelial cells. The nurse cells and oocytes are both derived from the oogonia ; the epithelial cells are of mesodermal origin.
The positions of the stages to be described are indicated in the diagram (Fig. 7) and the nuclear
and cytoplasmic structures are shown in Fig. 8. Two oocytes and a neighboring epithelial cell from position A in Fig. 7 are shown in Fig. &,A.
The nuclei of the oocytes are large and contain a dis tinct spireme ; the cytoplasm is small in amount and ap parently homogeneous. After a short period of growth the oocytes form a linear series in the ovarian tubule and become connected with the spaces between the nurse cells by means of egg strings (Fig. 7, e.s) through which the nu tritive streams flow into the oocytes. One of the young est of these oocytes is repre sented in Fig. 8, B (position B in Fig. 7). The nucleus is no larger than in those of the earlier stage; its chromatin forms a reticulum, and a dis tinct nucleolus is present. The cytoplasm, on the other hand, has trebled in amount and within it are embedded a number of spherical bodies which stain with crystal violet after Benda's method, and appear to be mitochondrial in nature. At a slightly later stage (Fig. 8, C ; position C in Fig. 7) the nucleus is larger and contains several small spherical chromatic bodies besides the nucleolus. The cytoplasm has increased more rapidly in volume and a corresponding increase in the number of mitochondrial granules has also taken place. Further growth results in an increase in the volume of both nucleus and cytoplasm (Fig. 8, D; position D in Fig. 7), and a slight increase in the number of mitochondria. Whether these bodies developed de novo or by division of the preexisting granules could not be determined.
In succeeding stages growth is very rapid. The cytoplasm (Fig. 8, E; position E in Fig. 7) still remains homogeneous except for the mitochondria, which increase slightly in size and become situated as a rule near the periphery. The nucleus at this time contains a large number of chromatin granules and a diffuse reticulum. Part of an older oocyte is shown in Fig. 8, F (position F in Fig. 7) ; the cytoplasm has assumed a reticular appearance; the mitochondrial granules are present in greater numbers, and the nucleus is larger, oval in shape, and contains a distinct reticulum with many chromatin bodies of various sizes. A still older oocyte (Fig. 8, G ; position G in Fig. 7) is interesting particularly because of the rapid increase in the mitochondria and the localization of these near the periphery. From this stage on the character of the contents changes until, as shown in Fig. 7, the central part of the oocyte consists of homogeneous cytoplasm (cy), and the outer region of the cytoplasm is crowded with granules and spherical bodies of various sizes. Apparently the mitochondria lying near the periphery (Fig. 8, H) increase in size, gradually losing their affinity for the crystal violet stain and swelling up until they constitute the large yolk globules so numerous in the mature egg. All stages in the evolution of these bodies are illustrated at this time as represented in Fig. 8, H. In the meantime material is brought into the egg through the egg string from the nurse cells, thus probably adding several sorts of granules to the contents of the oocyte.
The growth period in the male germ-cell cycle is not so striking as in the female, since many spermatozoa of small size are produced, whereas only comparatively few large eggs develop. An increase in the size of the ultimate spermatogonia may occur, however, but the multiplication and growth periods are not nearly so distinct as in the case of the oogonia. In testes which are composed of cysts of spermatogonia there is evidence in some cases that all of the germ cells in a single cyst are descendants of a single spermatogonium. The proof for this seems certain in the potato beetle, where I have been able to follow the formation of the cysts by means of an uninterrupted series of stages (Hegner, 1914a).
7. MATURATION. Maturation or the ripening of the eggs and spermatozoa comprises a series of events which results in a reduction in the number of chromosomes and the amount of chromatin in the germ cells. Typically, both male and female germ cells divide twice during the process of maturation, and as shown in Fig. 9 these divisions result in the production of four functional spermatozoa in the male, and one functional egg and three polar bodies (abortive eggs) in the female. This increase in the number of cells is not, however, the most I'm portant phase of the maturation process, since a large part of our knowledge of the physical basis of heredity has been derived from studies of the behavior of the chromatin at this time. This subject will be dealt with more fully in Chapter IX, and for the present only a brief account of events need be given.
The first thing to be noted is that the mitoses leading to the division of the germ cells during maturation differ from those of ordinary cell multiplication. The germ cells, when they are ready for the maturation divisions, are known as primary oocytes and primary spermatocytes. The nuclei of these cells possess the complete or diploid number of chromosomes, characteristic of somatic cells ; but after maturation the eggs and spermatozoa contain only one-half of the original diploid number, or the haploid number. These mitoses are consequently called reducing or meiotic. The details of these mitoses differ in male and female germ cells and in different species of animals.
During and at the close of the growth period in the male the chromatin granules form a spireme which condenses at one side of the nucleus, a condition known as synizesis. After a time the spireme again spreads throughout the nucleus, but is now divided into segments, the chromosomes, which are only haploid in number. The reduction from the diploid to the haploid number is brought about by the union of the chromosomes in pairs, a condition called synapsis. Each of the haploid chromosomes thus consists of two of the diploid chromosomes and is said to be bivalent. That one of the chromosomes of each pair is of maternal origin, i.e., is a descendant of a chromosome present in the egg at the time of fertilization, and the other of pater nal origin, i.e., a descendant of one brought into the egg by the spermatozoon, seems to be well established. The final act of fertilization, therefore, occurs at this point in the germ-cell cycle an act of much greater significance than that of the union of the egg and spermatozoon. Furthermore, there is considerable evidence that the chromosomes differ one from another and that in synapsis corresponding (homologous) chromosomes unite. The importance of such a union from a theoretical standpoint will be discussed later.
The nuclei now prepare for the two maturation mitoses. In many nematodes, annelids, and arthro pods these are characterized by the formation of tetrads. Divisions of this sort may be illustrated as in Fig. 10. The diploid number of chromosomes is for convenience supposed to be four, as in the sper matogonium A. During the spermatogonial divisions these divide as in B, so that each daughter cell receives the diploid number, four. After synapsis, however, each of the haploid chromosomes of the primary spermatocyte is seen to be divided into four parts, thus forming in this case two tetrads (C). During the division of the primary spermatocyte, as shown in D, E, and F, half of each tetrad, or two dyads, passes to each daughter cell. The division of the daughter cells, which are known as secondary spermatocytes (G H), results in the separation of the two parts of each dyad so that each of the four spermatids (H) receives one member of each original tetrad or two monads. Thus the chromosomes (monads) of the spermatids (H) are already formed in the primary spermatocytes (C) by two divisions ; whereas the nuclear and cell divisions do not occur until later. The spermatids (H), which proceed to metamorphose into spermatozoa, possess, there fore, only two chromosomes, i.e., one-half of the number present in the spermatogonia and somatic cells.
Tetrad formation does not occur in most animals ; but usually the members of the bivalent chromosomes become separated on the first maturation spindle, the pairs appearing U-, F-, or ring-shaped, as in Fig. 62. Each secondary spermatocyte receives one-half of each haploid, bivalent chromosome. The second maturation mitosis then ensues, during which each daughter cell is provided with one-half of each chromosome as in ordinary mitotic division. Be cause of the peculiar behavior of the chromosomes the first division is often called the heterotype, whereas the second is known as the homotype divi sion. The final results are the same whether tetrads are formed or not, each spermatid containing the haploid number of chromosomes.
The maturation of the egg differs in no very Important respects from the process as it has been described in the male cells. Tetrads may or may not be formed according to the species, and the mature egg and polar bodies each contain the haploid number of chromosomes. Two phases of the maturation of the egg may be referred to here : (1) when the nucleus of the primary oocyte prepares for division a considerable amount of chromatin separates from the chromosomes and is lost in the cytoplasm. The size of the chromosomes is thus diminished, but no entire chromosomes are lost. (2) The cellular divisions are very unequal, the polar bodies being very small as compared with the rest of the egg. The chromatin content of the polar bodies, however, is equal to that of the much larger egg. In the male all of the four spermatids are functional, but in the female only the egg survives, the polar bodies de-enerating. As a rule two polar bodies are produced, but in certain cases of parthenogenesis (rotifers, CLADOCERA, OSTRACODA, and aphids) only one is formed. Rarely the first polar body divides into two.
8. FERTILIZATION. Eggs that develop partheno genetically are ready to begin a new germcell cycle as soon as they become mature; but the eggs of the majority of species must be fertilized before they are able to develop. Fertilization may be de fined as the fusion of an egg with a spermatozoon and the resulting processes of rearrangement of the egg contents which result in the formation of a uninuclear cell, the zygote. As a rule one spermatozoon only enters the egg (monospermy) ; but in a few species (certain insects, selachians, tailed amphibians, reptiles, and birds) many spermatozoa may normally fuse with the egg (physiological polyspermy). The sper matozoon, which consists usually of three rather dis tinct parts, the head, the middle piece, and tail, may become entirely embedded within the egg sub stance, or the tail may be left outside, or, in exceptional cases, only the head succeeds in entering.
The union of the egg and spermatozoon may occur before, during, or after the polar body formation (Fig. 11). If the spermatozoon enters before the maturation of the egg is completed (A), its head transforms into a nucleus equal in size to that of the egg (CO ; the middle piece dissolves, giving rise to a centrosome which inaugurates the formation of a spindle with asters (B) ; and the tailpiece apparently takes no active part in the fertilization processes. The middle piece also does not seem to be necessary for the formation of the centrosomes and asters. The nucleus of the spermatozoon and that of the mature egg approach each other and come into contact between the asters (C). Then the nuclear walls dissolve ; a spireme which segments into the haploid number of chromosomes is produced by each nucleus, and the first cleavage spindle of the developing egg results. This spindle bears the haploid number of chromosomes from the spermatozoon and a like number from the egg nucleus and thus the diploid or somatic number of chromosomes is regained.
When the spermatozoon enters an egg which has completed polar-body formation, the head does not
have time to transform into a nucleus as large as the egg nucleus, but nevertheless fuses with the latter (Fig. 11, D, E, F). Although the two nuclei are very unequal in size, they possess an equal amount of chromatin and furnish an equal number of chromosomes to the first cleavage spindle.
As already indicated, perhaps the most essential phase in the fertilization process does not occur until the homologous maternal and paternal chromosomes unite during synapsis, when the germ cells of the new individual become mature. The immediate results of fertilization are : (1) the inauguration of the development of the egg, (2) the increase of the chromosomes from the haploid to the diploid (somatic) number, and (3) the union of hereditary substances from, as a rule, two individuals.
This completes the last stage in the germ-cell cycle of animals. Many extremely important and interesting phases of the subject have had to be omitted from the account. Certain of these will be more fully discussed in succeeding chapters, es pecially those concerned with the early history of the germ cells during embryological development, but for the details of the nutrition, growth, matura tion, and fertilization of the germ cells, the reader must be referred to other sources (Wilson, 1900; Jenkinson, 1913; Kellicott, 1913).
