Book - Text-Book of Embryology 2
It was stated in the preceding chapter that among the vertebrates the essential condition for the production of a new individual was the union of two sexually different cells. Since the number of chromosomes is constant for all the cells of a species, such a union would cause a doubling of chromosomes unless the latter were reduced to one-half of their normal number. Such a reduction actually takes place, .and forms the essential part of the maturation processes of the germ cells.
Spermatogenesis - Maturation of the Sperm
The spermatozoa arise from the germinal epithelium of the testis. In the mammal this epithelium consists of two kinds of cells: (i) the supporting cells (of Sertoli) and (2) the spermatogenic cells in various stages of development (Fig. 6). Of the latter the basal layer consists of small round or oval cells which are known as spermatogonia. Internal to these are the larger spermatocytes having large vesicular nuclei with densely staining chromatin. Between these and the lumen of the seminiferous tubule are several layers of small round or oval cells, the spermatids. The spermatids have the reduced number of chromosomes, and by direct transformation give rise to the mature spermatozoa which may either lie free in the lumen of the tubule or have their heads embedded in the supporting cells.
The way in which the maturation or reduction divisions take place in the higher animals, such as mammals, is difficult to demonstrate on account of the small size of the cells. The following account is based on data obtained from the study of lower forms (amphibia, fishes, insects, Ascaris) whose maturation processes have been demonstrated with great accuracy. Ascaris and some of the insects show the later stages with remarkable clearness. It is reasonable to suppose that the maturation processes of the mammalian germ cells agree essentially with those of lower forms.
The spermatogonia divide by ordinary mitosis, each daughter cell receiving the full or diploid number of chromosomes. After several generations some of the spermatogonia pass through a period of growth and are then known as primary spermatocytes. During this period important changes take place in the nucleus. The chromatin granules become concentrated into a dense mass in which very little structure is made out. After the period of growth the nucleus assumes again the reticular appearance. Then when the spireme is formed and segmentation occurs, previous to division, only the haploid or one-half the normal number of chromosomes appears. This seems to be due to an actual fusion of chromosomes by pairs, such fusion occurring during the period of growth and being known as synapsis of chromosomes. In some cases the double nature of the chromosomes is still visible while in other cases the fusion is complete.
The fused chromosomes now prepare for division. However, instead of dividing longitudinally into two parts, a double splitting occurs and each chromosome is divided into four elements. Such a quadruple chromosome is termed a tetrad. Since each tetrad represents a double chromosome, the number of tetrads in any species will be equal to onehalf its normal number of chromosomes (Fig. 7, D). The tetrads arrange themselves in the equatorial plane of the spindle and cell division begins (Fig. 7, E, F, G) . Each tetrad is separated into two dyads, and then one dyad from each tetrad goes to each of the two resulting daughter cells or secondary spermatocytes (Fig. 7, H). A new spindle is formed in each of the secondary spermatocytes and the cells divide again, without the return of the nucleus to the resting stage. The dyads go to the equatorial plane (Fig. 7, I, J, K). Each dyad is separated into two monads, each daughter cell or spermatid receiving one monad from each dyad (Fig. 7, L). A primary spermatocyte gives rise therefore to four spermatids in which the number of chromosomes is reduced to one-half the normal.
Fig. 7. Reduction of chromosomes in spermatogenesis in Ascaris megalocephala (bivalens). Brauer, Wilson. A G,
- Successive stages in the division of the primary spermatocyte. The original reticulum undergoes a very early division of the chromatin granules which then form a doubly split spireme (B). This becomes shorter (C), and then breaks in two to form the 2 tetrads (D, in profile, E, on end). F, G, H, First division to form 2 secondary spermatocytes, each receiving 2 dyads. I, Secondary spermatocyte. J, K, The same dividing. L, Two resulting spermatids, each containing 2 single chromosomes.
After the last spermatocyte division and the resulting formation of the spermatid, the nucleus of the latter acquires a membrane and intranuclear network, thus passing into the resting condition. Without further division the spermatid now becomes transformed into a spermatozoon (Fig. 8). This is accomplished by rearrangement and modification of its component structures. The centrosome either divides completely, forming two centrosomes, or partially, forming a dumbbell-shaped body between the nucleus and the surface of the cell. The nucleus passes to one end of the cell and becomes oval in shape. Its chromatin becomes very compact and finally condensed in the homogeneous chromatin mass which forms the greater part of the head of the spermatozoon. Both centrosomes apparently take part in the formation of the middle piece. The one lying nearer the center becomes disk-shaped and attaches itself to the posterior surface of the bead. The more peripheral centrosome also becomes disk-shaped and from the side directed away from the head a long delicate thread grows out the axial filament. The central portion of the outer centrosome next becomes detached and in mammals forms a knob-like thickening end knob at the central end of the axial filament. In amphibians this part of the outer centrosome appears tq pass forward and to attach itself to the inner centrosome. In both cases the rest of the outer centrosome in the shape of a ring passes to the posterior limit of the cytoplasm. As the two parts of the posterior centrosome separate, the cytoplasm between them becomes reduced in amount, at the same time giving rise to a delicate spiral thread the spiral filament which winds around the axial filament of the middle piece. Meanwhile the axial filament has been growing in length and part of it projects beyond the limit of the cell. The cytoplasm remaining attached to the anterior part of the filament surrounds it as the sheath of the middle piece. In mammals there appears to be more cytoplasm than is needed for the formation of the sheath of the middle piece, and a large part of it degenerates and is cast aside. The sheath which surrounds the main part of the axial filament appears in some cases at any rate to develop from the filament itself. The galea capitis or delicate film of cytoplasm which covers the head is also a derivative of the cytoplasm of the spermatid.
Fig. 8. Transformation of a spermatid into a spermatozoon (human). Meves, Bonnet. Schematic.
Fig. 9. Three stages in spermatogenesis in man (negro). Wieman.
- In a is shown a nucleus of a primary spermatocyte during the growth period; p, plasmosome; x and y, accessory chromosomes. In b is shown the metaphase in a primary spermatocyte in which there are 1 2 bivalent chromosomes that have resulted from synapsis of the 24 in the spermatogonium, the x and y uniting with each other. In c is shown a later stage of spermatocyte division in which the xy pair has divided longitudinally, the daughter chromosomes passing toward the poles of the spindle ahead of the main group.
The developing spermatozoa lie with their heads directed toward the basement membrane, and attached, probably for purposes of nutrition, to the free ends of the Sertoli cells (Fig. 6) . Their tails often extend out into the lumen of the tubule. When fully developed they become detached from the Sertoli cells and lie free in the lumen of the tubule.
The work done within the past decade on spermatogenesis in the human has established the relation of chromosome behavior here to that in the lower animals, showing some interesting coincidences. In the last of several studies by different investigators, Wieman has critically observed conditions in both the white and the negro. In division of the spermatogonium 24 chromosomes appear, two of which are designated idiochromosomes (XY pair). During the period of growth to a primary spermatocyte the XY pair persists as a deeply staining bipartite body (Fig. 9, a). In the prophase of primary spermatocyte division pairing or synapsis results in 12 bivalent chromosomes, the XY pair retaining its identity (Fig. 9, V). When metakinesis occurs the XY element divides lengthwise, but whether the other 11 divide lengthwise or transversely has not been determined (Fig. 9, c). In division of the secondary spermatocyte the n chromosomes divide, each giving one-half of itself to a spermatid; but the XY element gives X to one spermatid and Y to the other. The result of this chromosomal behavior is, therefore, that the usual reduction in number is accomplished but that the spermatids, and hence the spermatozoa, are of two classes differing as to the X and Y chromatin content.
Maturation of the Ovum
The female germ cell, before it is fertilized, goes through a process of maturation similar to that of the male germ cell. The result is essentially the same: the mature ovum contains a reduced number of chromosomes. There is this difference, however, that while the chromatin elements are distributed equally during the reduction divisions, one cell alone retains practically all the cytoplasm and deutoplasm present in the primary oocyte. This cell becomes the functional ovum while the other cells are pinched off as minute bodies, containing but little of the cytoplasm, which are known as polar bodies and eventually degenerate and disappear.
The early maturation stages of the female sex cell are very similar to those of the male. The oogonia contain the diploid number of chromosomes and divide by ordinary mitosis. After several generations they pass through a period of growth and are then known as primary oocytes. During the growth period there occurs a condensation of the chromatin, and synapsis of the chromosomes probably takes place at this time. The nucleus then resumes its reticular structure. Following this the spireme is formed, preparatory to division, and segments into the haploid number of chromosomes. From this stage the process varies somewhat in different animals.
Fig. 10. Maturation of the ovum of Ascaris megalocephala (bivalens). Boveri, Wilson.
- A, The ovum with the spermatozoon just entering at x" ', the egg nucleus contains 2 tetrads (one not clearly shown), the somatic number of chromosomes being 4. B, Tetrads in profile. C, Tetrads on end. D, E, first spindle forming. F, Tetrads dividing. G, First polar body formed, containing 2 dyads; 2 dyads left in the ovum. H, I, Dyads rotating in preparation for next division. J, Dyads dividing. K, Each dyad divided into 2 single chromosomes, thus completing the reduction.
In Ascaris, whose diploid number of chromosomes is four, both maturation divisions occur after the sperm has entered the egg and lies embedded there as the male pronucleus. An achromatic spindle forms near the surface of the ovum and the two tetrads go to the equatorial plane (Fig. 10, E). Each tetrad separates into two dyads, and one dyad from each tetrad passes into a small mass of cytoplasm which becomes detached from the egg cell as the first polar body (Fig. 10, F, G). A new spindle forms without the return of the nucleus to the resting stage, and each dyad divides into two monads. The second polar body is now given off in the same manner as the first (Fig. 10, H, I, J, K). One monad from each dyad passes into a small mass of cytoplasm and is separated from the egg cell. The maturation is now complete. The nucleus of the mature ovum contains the haploid number of chromosomes and is ready for union with the male pronucleus.
Fig. 11. From sections of ova of the mouse, showing stages in the maturation process. Sobotta.
- A, Ovum showing prophase of maturation division. f, fat; z.p., zona pellucida.
- B, Ovum showing maturation spindle with chromatin segments undivided.
- C, Ovum showing diaster stage of maturation division, formation of ist polar body (p.b.), and sperm nucleus (male pronucleus, m.pn.) just after its entrance.
- D, Ovum showing polar body (p.b.) and male (m.pn.) and female (f.pn.) pronuclei.
- E, Ovum showing both polar bodies (p.b.) and pronuclei.
- F, Ovum showing pronuclei preparing to unite.
The maturation of the mouse ovum, described by Mark and Long, may be taken as an example of mammalian maturation. The diploid number of chromosomes is twenty, but when the growth of the primary oocyte is completed and the cell prepares for division only ten chromosomes are present. Each chromosome is V-shaped and shows the structure of a tetrad. While still in the Graafian follicle the first polar body is given off and lies as a small globule beneath the zona pellucida. The egg cell and the first polar body constitute secondary oocytes, comparable with the secondary spermatocytes of the male. The egg now leaves the ovary and reaches the oviduct. If a sperm enters the ovum, another spindle forms and a second polar body is given off. The nucleus of the mature ovum or female pronucleus, with the haploid number of chromosomes, is now ready for union with the male pronucleus. (See Fig. 11.)
Comparing maturation in the male and female sex cells, it is to be noted that the spermatogonia and oogonia proliferate by ordinary mitosis, maintaining the somatic or diploid number of chromosomes up to a certain period in their life history. They then enter upon a period of growth in size, resulting in primary spermatocytes and primary oocytes. When these prepare for division the nuclear reticulum in each case resolves itself into the haploid number of chromosomes. During division this reduced number is given to each resulting secondary spermatocyte or oocyte.
There is, however, this marked peculiarity about the division of the primary oocyte, that while the division of the nuclear material is equal the division of the cytoplasm is very unequal, most of the latter remaining in one cell, the secondary oocyte proper. The other cell, very small owing to its lack of cytoplasm, is extruded from the oocyte proper as the first polar body. The same condition obtains in the next division. One cell, the mature ovum, retains most of the cytoplasm, the other being detached as the second polar body. In some cases the first polar body also divides. Thus the primary oocyte gives rise to three or four cells, each of which has the reduced number of chromosomes. One of them becomes the mature ovum, the others are cast off as apparently useless cells and eventually disappear. The primary spermatocyte, on the other hand, gives rise to four functioning cells which are equal in cytoplasmic content. (See Fig. 12.)
The apparent difference between maturation of the male and female sex cells the single functional cell in the female as contrasted with four in the male loses some of its character when one notes that in some forms the polar bodies are not so rudimentary as is generally the case. Thus in certain forms one or more of the polar bodies may develop into cells very similar to the mature egg cell, may be penetrated by spermatozoa, and may even be fertilized and proceed a short distance in segmentation. There is perhaps warrant for considering the polar bodies as rudimentary or abortive ova.
The time of formation of the polar bodies varies in different animals. In a few (echinoderms) they are formed before the sperm enters the egg. In Ascaris they are both formed after the entrance of the sperm. In other forms, like the mouse, the first polar body is formed while the egg is still in the Graafian follicle, the second one after the entrance of the sperm. The only recorded observations on maturation of the human ovum are those of Thomson's. In an extensive series of ovaries he has observed both polar bodies and the spindles preceding extrusion. Both maturation divisions occur before the Graafian follicle ruptures and discharges the ovum, the time of formation of the second polar body therefore differing from that in other mammals.
From the data in the above description it is evident that the phenomena of maturation are essentially similar in the male and female sex cells. In the female two or three of the cells are indeed abortive, probably in order to insure a large amount of food material to the functioning ovum; but the result, the reduction of the number of chromosomes in the mature sex cell to one-half the number characteristic of other cells of the species, is always the same.
Fig. 12. Diagram representing the histogenesis of (a) the female sex cells and (6) the male sex cells. Modified from Boveri.
Significance of Mitosis and Maturation
The earlier investigators regarded maturation merely as a means of reducing the number of chromosomes in the mature germ cells, so as to prevent a doubling of chromatin material at the subsequent fertilization. This, however, seems to be but a minor object of maturation. As a matter of fact, the reduction of the chromatin mass is not one-half but three-quarters and even more. It is also well known that the chromatin mass increases or diminishes under certain conditions during the life history of a cell.
The chief significance of maturation is to be considered rather from the standpoint of heredity. Modern biologists are convinced that the chromatin particles constitute the inheritance substance of the cell. During mitosis the chromatin granules arrange themselves in a continuous thread, the spireme, which differs qualitatively in different regions. The chromosomes, which are only segments of the spireme, likewise differ from end to end. In ordinary mitosis these chromosomes split longitudinally, half of each chromosome going to each of the resulting daughter cells. This is an equational division in which the chromatin material is exactly halved.
In maturation, however, a synapsis of the chromosomes takes place, the latter fusing in pairs. The chromosomes of each pair are probably separated again in one of the subsequent maturation divisions, the reduction division. If the chromosomes are qualitatively different, then the mature germ cells resulting from this division will be of two different kinds, varying more or less in their content of hereditary factors. Experimental evidence confirms this interpretation of maturation.
There is another interesting point to be considered. The recent work of cytologists leads to the assumption that the fusion of chromosomes during synapsis is not a matter of chance, but takes place in a definite manner. The chromosomes in the primordial germ cells seem to form a series of homologous pairs the members of which fuse during synapsis. The individual pairs can often be distinguished from other pairs by differences in shape 01 size. There is much evidence to support the belief that each pair consists oi one paternal and one maternal chromosome, which had been brought together at the antecedent fertilization. This seems to indicate also that the chromosomes retain their identity even when resolved into the chromatic reticulum of the resting nucleus. The reduction division will separate the fused chromosomes, and the resulting mature germ cells will be either paternal or maternal in their chromatic constitution. The maturation processes therefore produce a segregation of the paternal and maternal chromosomes.
The cytological data described above, which support and in turn are supported by a great mass of experimental evidence, illustrate Mendel's law of segregation. This law is that the units contributed by two parents separate in the germ cells without having had any influence upon each other. For instance, when a mouse with gray coat color is mated with a mouse with black coat color, one parent contributes a unit for gray and the other a unit for black. These units will separate during the maturation of the germ cells, and the resulting spermatozoa and ova will again recover the pure paternal or maternal units.
In the great bulk of cytological and experimental studies of recent years there is abundant evidence for the belief that certain chromosomes play an important part in the determination of sex. In the grasshopper (Stenobothrus viridulus) the somatic number of chromosomes in the male is seventeen and in the female eighteen. Owing to the odd number there is an unusual complication in the maturation of the male germ cell. When synapsis occurs eight pairs of chromosomes are formed but the odd chromosome, which can usually be distinguished by its appearance, is left without a mate (Fig. 13, 4). At the first maturation division this univalent chromosome does not divide but passes as a whole to one of the resulting cells, thus giving two kinds of secondary spermatocytes (Fig. 13, 5). When the secondary spermatocytes divide, however, the odd chromosome in one of them also divides like the other chromosomes, each of the resulting spermatids receiving one-half (Fig. 13, 8). Thus two kinds of sperms are formed in equal numbers, containing respectively eight and nine chromosomes. The odd chromosome is also known as the accessory or X-chromosome.
Fig. 13. Stages in the spermatogenesis of a grasshopper (Stenobothrus viridulus). Meek,
- 1, Spermatogonium in process of division, having 17 chromosomes (8 pairs and one odd). 2, Representing growth period of spermatogonium. 3-6, Division of the primary spermatocytes sixteen of the chromosomes are paired while the "accessory" has no mate and passes as a whole to one of the two secondary spermatocytes. 7-8, Division of the secondary spermatocyte with the odd chromosome, the latter splitting and giving one-half to each resulting spermatid. x, "Accessory" chromosome.
In the ovum no such complication arises, there being two accessory chromosomes which unite in synapsis. All the mature ova will therefore contain nine chromosomes. As a result, there are two combinations possible when the male and female sex cells unite: an ovum may be fertilized by a sperm containing either eight or nine chromosomes. In the first case the somatic number in the fertilized egg will be seventeen and, the egg will develop into a male. In the second case the somatic number will be eighteen and the resulting individual will be a female. In the example given, therefore, the presence or absence of the accessory or odd chromosome will determine the sex.
The presence of accessory chromosomes has been demonstrated in many invertebrates, especially insects. They have also been described in several vertebrates such as the rat, fowl, guinea-pig, and even man. In many cases the accessory chromosome of the male germ cell has a mate which differs, however, in some way (size, appearance, etc.) and is designated the Y chromosome. An ovum fertilized by a spermatozoon containing the Ychromosome will give rise to a male; if fertilized by one containing the X-chromosome the egg will develop into a female.
There are many cases, particularly among parthenogenetic forms, where sex cycles arise, which cannot be explained by chromosomal behavior. In these cases nutrition seems to play an important part in determining the : sex of the individual. But as to the great majority of forms investigated, the weight of evidence supports the view that the chromosomes are the chief agents in sex determination.
Ovulation is the discharge of the ovum from the ovary, whether in the human female or any of the lower animals. Our attention will here be confined to the phenomenon as it occurs in mammals.
Before the ovum escapes from the ovary it is contained in a structure known as the Graafian follicle, which consists of a wall of epithelium, the granular layer, enclosing a space filled with a viscid fluid, the follicular fluid. Surrounding the follicle is a special layer of connective tissue, the theca folliculi, which is a part of the ovarian stroma and contains many small blood vessels. The egg cell is situated within a thickened portion of the epithelial wall, the germ hill. The growth of the follicle itself will be described in the chapter on the geni to-urinary system.
When the Graafian follicle is mature, having reached its maximum size, it produces a bulge on the ovary; and there is only a thin membrane, composed of the granular layer, the theca and the germinal epithelium of the ovary, between the follicular cavity and the exterior of the ovary (Fig. 14). At a certain time this membrane breaks and the follicular fluid gushes out, carrying with it the ovum and some of the cells of the germ hill. The ovum is then free in the abdominal cavity whence normally it passes into the open end of the oviduct, or Fallopian tube. The cause of the rupture of the follicle has not been ascertained; but there are certain facts which throw light upon it. In the dog ovulation occurs during oestrus, or the period of "heat," independently of approach of the male. In the mouse, the rat and the guinea-pig ovulation also occurs spontaneously during oestrus. In the rabbit ovulation occurs about ten hours after coitus, and it has been shown experimentally that the follicle does not rupture after any stimulus except coitus. The sheep ovulates spontaneously during the earlier "heat "periods of the breeding season, but in the later periods coitus seems necessary to bring about the rupture of the follicle. In the bat, however, there are peculiar circumstances: Copulation takes place in the autumn, the spermatozoa remaining alive in the uterus until the following spring, and then ovulation occurs apparently in response to seasonal temperature changes without even a "heat" period. These are only a few instances out of a great number of observations, but they show that in general ovulation occurs during the oestrus or period of "heat" in the female, sometimes coincident with copulation. Just prior to the oestrus period there is a marked increase of blood flow to the generative organs, during a pro-cestrual period or pro-cestrus. During oestrus the increased blood flow is maintained and may be accentuated at the approach of the male, and it has been suggested that an increase in blood pressure in the ovary is at least one of the factors in causing the rupture of the Graafian follicle. Another contributing factor may be an increase in the quantity of fluid within the follicle thereby increasing the intrafollicular pressure.
Fig. 14. From section of human ovary, showing mature Graafian follicle ready to rupture. Kollmann's Atlas.
In monkeys there is a slight menstrual flow which may occur periodically the year round, but there seems to be a limited season for ovulation and conception. Menstruation and ovulation therefore do not necessarily coincide. In the human the menstrual flow is a pronounced feature during the years of reproductive activity of the female, recurring at average intervals of 28 days except during pregnancy and usually during lactation. It is generally admitted that the time of menstrual flow corresponds to the pro-cestrual period of the lower mammals, that is, the period immediately preceding the oestrus or rutting time. It would be expected that in the human female the period of sexual desire would follow menstruation. It seems, however, that conditions of modern society have disturbed the natural cycle of physiological activities, although there is reason to believe that in primitive man there was at least an approximation to conditions in the lower mammals. In highly civilized man there appears to be no particular period of sexual desire, and there is considerable evidence that ovulatiqn is not always associated with menstruation but may occur at any time during the intermenstrual period. With the disappearance of a fixed oestrus in the human female the definite relation between ovulation and the oestrus has broken down, although biologically the most favorable condition for conception is ensemination just after the menstrual flow.
Earlier in this chapter it was stated that the number of ova in the two ovaries approximated 70,000. Allowing one ovum to each ovulation, not more than about 400 of these attain maturity during the years of a woman's reproductive activity, the others along with their follicles probably degenerating within the ovaries. The general concensus of opinion is that in the great majority of cases only one ovum escapes at ovulation either from one ovary or the other. One possible exception to this occurs in the case of twin offspring where the twins are not identical. There is good evidence that identical twins arise from a single ovum, and it is not impossible even that ordinary twins develop from the same ovum.
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References for Further Study
BUCHNER, P.: Praktikum der Zellenlehre. Teil I. 1915.
CONKLIN, E. G.: Heredity and Environment in the Development of Men. 3d Ed., 1920.
CRAGIN, E. B.: Text-book of Obstetrics. 1915.
HERTWIG, R.: Eireife und Befruchtung. In Hertwig's Handbuch der vergleichenden und experimentellen Entwickelungslehre der Wirbeltiere. Bd. I, Teil I, Kap. II, 1903. Contains extensive bibliography.
KELLICOTT, W. E.: Text-book of General Embryology. Chap. IV, 1913.
MARSHALL, F. H. A.: The Physiology of Reproduction. 1910.
MORGAN, T. H.: Heredity and Sex. 1913.
MORGAN, T. H.: The Physical Basis of Heredity. 1919.
THOMSON, A.: The Maturation of the Human Ovum. Journal of Anatomy, Vol. 53, 1919.
WIEMAN, H. L.: The Chromosomes of Human Spermatocytes. American Journal of Anatomy, Vol. 21, 1917.
WILSON, E. B.: The Cell in Development and Inheritance. 1900.
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