Book - A text-book of histology arranged upon an embryological basis (1913) 1-1
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Lewis FT. and Stöhr P. A Text-book of Histology Arranged upon an Embryological Basis. (1913) P. Blakiston’s Son and Co., 539 pp., 495 figs.
|Histology with Embryological Basis (1913): Part I. 1.1. Cytology | 1.2. General Histology | 1.3. Special Histology|
Part II. 2.1. The Preparation of Microscopical Specimens | 2.2. The Examination of Microscopical Specimens
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- 1 I. Cytology
Since 1838 it has been known that all plants and animals are composed of small structural elements called cells (Latin, cellula; Greek, *vros). The lowest forms of animals and of plants are alike in being single cells throughout life. The more complex organisms are groups of cells, which have been derived by process of repeated division from a single cell, the fertilized ovum. Thus the human body, which begins as one cell, becomes in the adult an aggregation of cells variously modified and adapted to perform special functions. Since the liver is a mass of essentially similar cells, the problems of its functional activity are the problems of the functions of a single one of its cells. The diseases of the liver are the result of changes occurring in these cells, which must be restored to a normal condition to effect a cure. As this is equally true of other organs, it is evident that cytology, the science of cells, is a basis for both physiology and pathology.
A cell may be defined as a structural element of limited dimensions, which under certain conditions can react to external stimuli and perform the functions of assimilation, growth, and reproduction. Because of these possibilites a cell may be considered an elementary organism. It is described as a mass of protoplasm containing a nucleus. A third element, the centrosome, is found in the cells of animals, but it is doubtful whether it exists in the cells of the higher plants. It becomes prominent when a cell is about to divide. Some authorities regard the centrosome as a temporary structure, which forms shortly before division begins and disappears after it is completed. Others consider it as a permanent and essential part of a cell, which accordingly consists of protoplasm, nucleus, and centrosome.
Protoplasm is the living substance of which cells are composed. More specifically the term is applied to this living substance exclusive of the nucleus, or to the corresponding dead material, provided that death has not changed its physical properties. It has been proposed to substitute the name cytoplasm for protoplasm in the restricted and earlier sense of the term, to call the nuclear substance karyoplasm, and to consider both cytoplasm and karyoplasm as varieties of protoplasm. Although these names are often employed, the cell substance apart from the nucleus is ordinarily called protoplasm.
Protoplasm is a heterogeneous mixture of substances forming a soft viscid mass of slightly alkaline or neutral reaction. ("The terms may be used interchangeably for an alkalinity which is so slight" Henderson.) It is ordinarily more than three-fourths water, and the remainder consists of salts and organic substances, some in solution and some in a colloidal state. The organic bodies are classed as proteins, glycogen or some allied carbohydrates, and lipoid (fat-like) bodies. Protoplasm may exist in a numberless variety of forms.
FIG. r. DIAGRAM OF A CELL.
In the four quadrants different types of protoplasmic structure are represented namely, homogeneous, granular, foam-like, and fibrillar.
On microscopic examination, even with lenses of the highest power, the protoplasm of certain living cells appears homogeneous and structureless. But most of the cells which the histologist examines are not living.
They have been killed by various reagents, selected as causing the most rapid fixation possible. The protoplasm of such cells usually exhibits granules, fibrils, or networks with closed or open meshes. Whether these structures are wholly due to precipitation and coagulation is difficult to determine, but indications that they preexist have been observed in certain living cells. In any case, the various forms of coagulation occur with such constancy that their study is of the utmost importance to the histologist.
Even the ground substance of protoplasm, in which the fibrils or granules are imbedded, is not necessarily homogeneous. According to Biitschli's interpretation it has the structure oj foam or of an emulsion that is, it consists of minute droplets of one substance completely surrounded by walls of another substance. In these walls, granules and filaments may be lodged, as seen at the margins of the upper right quadrant of Fig. i. The complex chemical activities of a cell are said to be manifestly impossible in any homogeneous mass; but in such a heterogeneous medium as an emulsion, they are conceivable (Alsberg). In other words, the vital qualities of protoplasm may not depend so much on hypothetical complex and unstable living molecules, as upon the interaction of various substances, made possible by their arrangement in droplets and investing films.
The various structures commonly observed in protoplasm may be grouped as follows:
i. Granules. Ultra-microscopic granules doubtless exist in protoplasm, since the smallest of those observed approach the limit of visibility. The minute granules, if abundant, give the Nissl's bodies.
protoplasm a dark color. Often they are absent from the peripheral layer of protoplasm, or exoplasm, which is then clear, somewhat firmer, and chemically different from the inner endoplasm (Fig. i). In addition to minute granules such as may be found in most preserved protoplasm, certain cells contain larger granules, which are important secretory products elaborated by the cell. In active gland cells these granules are well defined and abundant, and they diminish as the cell becomes exhausted.
FIG . 2 . CLUMPS OF~GRANULES (NissL's BODIES) IN A NERVE
Various forms of white blood corpuscles may be distinguished by the size and staining reaction of the granules imbedded in their protoplasm. In certain nerve cells (Fig. 2) granules occur in large groups, known as Nissl's bodies. As Crile has shown, these become disorganized as a result of surgical shock or muscular fatigue. It is evident, therefore, that the careful observation of protoplasmic granules is of very great importance.
FIG. 3. 'FIBRILS IN A NERVE CELL.
FIG. 4. VACUOLES IN A YOUNG FAT CELL.
FIG. 5. CANALS (TROPHOSPONG IUM) IN A NERVE CELL.
2. Fibrils. Protoplasm may be permeated with a delicate meshwork of fibrils, which collectively constitute the spongioplasm, or filar mass. This is imbedded in the clear hyaloplasm, or interfilar mass (Fig. i). In certain cells there are filaments, known as mitochondria, which are formed by the coalescence of rows of granules. The relation between these structures and the reagents used is discussed by Kingsbury (Anat. Rec., 1912, vol. 6, pp. 39-52). The spongioplasm may form an irregular network, or its constituent fibrils may be parallel, passing from one end of the cell to the other. In oblique and transverse sections of such cells, the filaments are cut across, so that they appear as short rods, or even as granules. Fibrils may be extremely slender, as in the case of those which radiate through the protoplasm at the time when the cell divides; or they may be quite coarse, like the permanent fibrils characteristic of certain muscle and nerve cells
FIG.. 6. RETICULAR NETWORK (Fig. 3).
3. Vacuoles. Protoplasm often contains large or small drops of clear fluid, fat, or some other substance less highly organized than the surrounding material (Fig. 4). In preserved cells the spaces which were occupied by these droplets appear clear and empty, and are known as vacuoles.
tiey vary greatly in size, and one or several of them may be found in a single cell.
4. Canals. The protoplasm of certain cells is said to contain fine tubes or clefts which communicate with lymphatic spaces outside of the cell (Fig. 5). Prolongations from the surrounding capsule-cells have been described as entering these canals and as performing, together with the lymph, a nutritive function. Hence the network of canals has been called trophospongium. But it has not been shown conclusively that these canals open to the exterior of the cell. They may be similar to the closed networks or "reticular apparatus" lying wholly within the protoplasm, shown in Fig. 6. Such networks have been described in nerve cells, cartilage cells and gland cells. The network is said to be of a thick fluid consistency. In certain gland cells there are canals within the protoplasm, which convey the secretion to the free surface of the cell. These may be simple, branched, or arranged in a network. Like the other forms of intracellular canals, they can be studied only in special preparations.
5. Inclusions. Various foreign bodies, such as other cells or bacteria, which may have been ingested by the protoplasm, are grouped as inclusions. This term is applied also to crystalloid substances formed within the protoplasm (Fig. 7), and to coarse masses of pigment granules which appear extraneous.
The nucleus (Latin, nucleus, "the kernel of a nut"; Greek, Kdpvov, "a nut") is typically a well-defined round body, situated near the center of the cell, appearing denser or more coarsely granular than the surrounding protoplasm (Fig. i). There are characteristic variations in the shapes of nuclei, in their position within the cells and in their structure.
Ordinarily the karyoplasm, or nuclear substance, is sharply marked off from the cytoplasm by the nuclear membrane. Sometimes, in preserved tissues, the cytoplasm has shrunken away from the nuclear membrane, so as to leave a narrow space partially encircling it; and in certain living cells, the nucleus migrates through cytoplasm, as if it were an independent body. But there are phases of cell-development in which the nuclear membrane disappears and no line can be drawn between karyoplasm and cytoplasm. At all times they have a common structural basis. The ground substance of the nucleus, corresponding with the hyaloplasm, is the nuclear sap; and it contains, for spongioplasm, a meshwork of delicate linin fibrils. These help to form the nuclear membrane, in which they terminate. The nuclear membrane, nuclear sap, and linin reticulum do not stain deeply, and are therefore grouped together as the achromatic constituents of the nucleus.
The principal chromatic constituent of the nucleus is known as chromatin. It stains deeply, since it contains a large amount of nucleic acid, which has a marked affinity for basic stains. Chromatin occurs in the form of granules, which are bound together in strands or masses by the lining fibers (Fig. i). The masses, known as chromatin knots, occur especially at the points of intersection in the linin meshwork. Sometimes they are attached to the nuclear membrane, or so distributed over its surface that it appears to consist of chromatin. It forms morphologically the most important part of the nucleus.
Certain nuclei contain one or more round bodies, which belong with the chromatic elements because of their deep staining, but which are chemically different from chromatin. These bodice, known as nucleoli, are stained with acid or neutral dyes. They are said to be composed of paranuclein, whereas chromatin is composed of nuclein. In distilled water the structures formed of nuclein disappear, but those consisting of paranuclein remain. The nuclei of nerve cells contain typical nucleoli (Figs. 3 and 5). Sometimes a nucleolus, lodged in the nuclear reticulum, is more or less covered with chromatin (Fig. 9, A), but the term should not be applied to irregular knots of chromatin, even when most of the chromatic material within a nucleus is gathered into one or two such bodies. These are the so-called false nucleoli (pseudonucleoli).
Every nucleus, therefore, consists of ground substance or nuclear sap, a network of linin, and granules and masses of chromatin. Usually it is surrounded by a membrane, and sometimes it contains a nucleolus. Most cells contain a single nucleus; but occasionally a single cell contains two nuclei, as is frequent in the liver, or even several nuclei, as in certain cells associated with bone. Non-nucleated bodies, like the mammalian red blood corpuscles, and the dead outer cells of the skin, have lost their nuclei in the course of development.
Functionally the nucleus is regarded as a center for chemical activities necessary for the life of the cell. It is believed to produce substances which pass out into the cytoplasm, where they may be further elaborated. Evidences of nuclear extrusions into the cytoplasm have been frequently recorded. But the interactions between nucleus and cytoplasm, of such nature that they cannot be observed under the microscope, are presumably of far greater biological importance.
The centrosome is typically a minute granule in the center of a small sphere of differentiated protoplasm. Often the term is applied to this entire structure, but it refers particularly to the central granule; the enveloping sphere is known as the attraction sphere, and it is composed of archoplasm^ When a cell is about to divide, delicate fibrils, either rearranged from the protoplasmic reticulum or formed anew, radiate from the archoplasm toward the periphery of the cell. The central granule becomes subdivided into two, which then move apart. In resting cells, or those which are not undergoing division, the centrosome may already have divided into a double body or diplosome preparatory to the next division of the cell (Fig. i).
Centrosomes have been detected in many forms of resting cells, and it is assumed by some authorities that the centrosome is an invariable constituent of the cells of the higher vertebrates. According to this opinion the centrosome may become inconspicuous but it never loses its identity. Often they are found very close to the nuclear membrane, which may be indented to accommodate them; and rarely, as in certain cancer cells and in one form of the worm Ascaris, they have been reported as within the nucleus. They may occur near the free surface of certain cells, usually in the form of diplosomes, as shown in cell a, Fig. 8. Just above the diplosome, such cells may send out contractile projections of protoplasm (pseudopodia), with the activity of which the! diplosome may be in some way associated.j Pseudopodia, with an underlying diplosome, have been observed in the columnar cells of the human large intestine. In cell b of Fig. 8 there are four diplosomes, one of which lies beneath the protoplasmic projections. It is believed that the diplosomes may multiply by fission, and that thus they may give rise to the numerous motile hairs, or cilia, which project from certain cells. Of these they form the basal bodies (Fig. 8, c). In many gland cells the centrosome lies in the midst of the protoplasm where the secretion accumulates. The discharge of the secretion is accomplished by the contraction of the protoplasmic strands in which the centrosome is lodged. In all these relations the centrosome appears to be a center for motor activities, and it is described as the kinetic or dynamic center of the cell.
FIG. 8. CELLS OF THE EFFERENT DUCTS OF THE TESTIS OF A MOUSE. (After Puchs.)
To show diplosomes, and (in c) cilia with basal bodies.
The protoplasm at the surface of certain cells floating in the blood or lymph forms a thin pellicle, apparently as a result of protoplasmic concentration, or other reaction to the surrounding medium. Cells which line the greater part of the digestive tube, and have only one surface directed toward the intestinal contents, are provided with a thick wall on the exposed surface. Such a wall is called a cuticular border, or cuticula. On the other sides of these cells, the membrane is much thinner, and on the basal surface it is sometimes lacking. In such cases the protoplasm appears to be continuous with that of the underlying cells. In other cases the entire cell is devoid of any membrane. The cell membrane, therefore, is not an essential part of a cell; if present it ranges from a thin pellicle, on the border line of visibility, to a well-defined wall, which may be formed as a secretion of the underlying protoplasm. If the several surfaces of the cell are in relation to different environments, there is often a corresponding difference in the structure of their walls.
In examining a group of cells, it will be important to determine whether they are merely in contact, or actually continuous. Sometimes cells are so completely fused that their nuclei are irregularly distributed through a single mass of protoplasm. Such a formation is a syncytium in which the position of the nuclei is the only means of estimating the territory of a single cell. A syncytium may arise from the fusion of cells, or, as in striated muscle fibers, it may be due to the multiplication of nuclei in an undivided mass of protoplasm. Instead of being completely fused, cells are often joined to one another by protoplasmic processes of varying length and width, thus forming cellular networks. Fibrils within such a syncytium may pass continuously from the protoplasm of one cell into that of another.
Although cell membranes are often inconspicuous in animal cells, they cannot be overlooked in plants. Thus cork is a mass of dead cells from which nuclei and protoplasm have disappeared, leaving only the cell walls. In describing cork, Robert Hooke introduced the name "cell," in 1664. He wrote: "I took a good clear piece of Cork and with a Pen-knife sharpen'd as keen as a Razor, I cut a piece of it off, and thereby left the surface of it exceeding smooth, then examining it very diligently with a Microscope, me thought I could perceive it to appear a little porous. . . . These pores, or cells, were not very deep, but consisted of a great many little Boxes ."
In this way one of the briefest and most important of scientific terms was introduced.
Form and Size of Cells
Cells are regarded as primarily spherical in form. Spherical cells are comparatively numerous in the embryo, and in the adult the resting white blood corpuscles, which float freely in the body fluids, assume this shape. Such cells are circular in cross section. When spherical cells are subjected to the pressure of similar neighboring cells, they become polyhedral and usually appear six-sided in cross section. Such cells, as a whole, may be cuboidal, columnar, or flat. Certain cells become fusiform (spindle-shaped) or are further elongated so as to form fibers; others send out radiating processes and are called stellate. Thus the form of cells is extremely varied. The shape of the nucleus tends to correspond with that of its cell. It is usually an elliptical body in elongated cells, and spherical in round or cuboidal cells. In stellate cells it is either spherical or somewhat elongated. Crescentic nuclei, and others more deeply and irregularly lobed, are found in some of the white blood corpuscles and in giant cells.
The size of cells ranges from that of the yolks of birds' eggs which are single cells, at least shortly before being laid down to microscopic structures four thousandths of a millimeter in diameter. The thousandth of a millimeter is the unit employed in microscopic measurements. It is called a micron, and its symbol is the Greek letter /*. The small cells referred to are therefore four microns (4 ju) in diameter. The size of any structure in a section of human tissue may be roughly estimated by comparing its dimensions with the diameter of a red blood corpuscle found in the same section. These red corpuscles are quite uniformly 7.5 p. in diameter.
Cytomorphosis is a comprehensive term for the structural modifications which cells, or successive generations of cells, undergo from their origin to their final dissolution. 1 In the course of their transformation, cells divide repeatedly, but the new cells begin development where the parent cells left off. Cell division, therefore, is an unimportant incident in cytomorphosis.
Cytomorphosis is a continuous advance in which four successive stages are recognized first, the stage in which the cells are undifferentiated ;_ second, the stage of specialization or differentiation: third, the stage of degeneration; and fourth, the stage in which the cells die and are removed. These may be considered in turn.
Undifferentiated cells, as can be seen in sections of young embryos, are characterized by large nuclei and little protoplasm. They multiply rapidly, but the rate of division declines with the gradual increase of the protoplasm and the consequent functional differentiation of the cell. In the adult, relatively undifferentiated cells are found in many situations, as, for example, in the deepest layer of the epidermis. As the cells at the surface die and are cast off, new ones come up from below to take their places. But since the basal cells can produce only epidermal cells, they are themselves partly differentiated. From this point of view the fertilized ovum, which can produce all kinds of cells, must be regarded, in spite of its size and great mass of yolk-laden protoplasm, as the least differentiated cell.
Differentiated cells may preserve a round or cuboidal form, but usually they are elongated, flattened, or stellate. The cytoplasm usually contains coarse granules, fibrils, masses of secretion or other special formations. As a result of their own protoplasmic activity, the cells of many tissues become surrounded by intercellular substances, which may far exceed in bulk the cells which produced them. Intercellular substances may be solid or fluid. When present in small amount they form thin layers of cement substance between closely adjacent cells; in large amount these substances constitute a ground work in which the cells are imbedded, as, for example, in cartilage and bone.
- 1 The term cytomorphosis was introduced by C. S. Minot in 1901 in a lecture entitled "The Embryological Basis of Pathology" (Science, 1901, vol. 13, p. 494). Cytomorphosis is further discussed by Professor Minot in "The Problem of Age, Growth, and Death," published by G. P. Putnam's Sons, 1908.
Although the differentiation of cells is chiefly cytoplasmic, there is some evidence of corresponding nuclear changes. Thus while the muscle
cells of the salamander are elaborating complex fibrils, the nuclei become modified as shown in Fig. 9. The significance of the nuclear changes is unknown.
Degeneration is the manifestation of the approaching death of the cell. In nerve cells this process normally takes place very slowly. These cells remain active throughout life, and if destroyed, they can never be replaced. In many glands, in the blood and in the skin, however, the cells are constantly dying and new ones are being differentiated. In a few organs the cells perish, but no new ones form, so that the organ to which they belong atrophies. Thus a large part of the mesonephros (Wolffian body) disappears during embryonic life; the thymus becomes vestigial in the
adult; and the ovary in later years loses its chief function through the degeneration of its cells.
The optical effects of degeneration cannot at present be properly classified. In a characteristic form, known as "cloudy swelling," the cell enlarges, becoming pale and opaque. In another form the cell shrinks and stains deeply, becoming either irregularly granular or homogeneous and hyaline. The nucleus may disappear as if in solution (karyolysis, chromatolysis) ; or it may become densely shrunken or pycnotic, and finally break into fragments and be scattered through the protoplasm (karyorhexis). If the process of degeneration is slow, the cell may divide by amitosis. It may be able to receive nutriment which it cannot assimilate, and thus its protoplasm may be infiltrated with fat and appear vacuolated. It may form abnormal intercellular substances, for example, amyloid; or the existing intercellular substances may become changed to mucoid masses, or have lime salts deposited in them. Thus an impairment or perversion of function is often associated with optical changes in the cell substance.
FIG. 9. NUCLEI OF STRIATED MUSCLE FIBERS FROM YOUNG SALAMANDERS (NECTURUS). (Eycleshymer.)
A, From a 7 mm. embryo; B, from one of 26 mm.; ch, chroma tin knot; g. s, ground substance; 1, linin fibril; n, nucleolus; n.m, nuclear membrane.
The removal of dead cells is accomplished in several ways. Those near the external or internal surfaces of the body are usually shed or desquamated, and such cells may be found in the saliva and urine. Those which are within the body may be dissolved by chemical action or devoured by phagocytes.
Every specimen of human tissue exhibits some phase of cytomorphosis. In some sections a series of cells may be observed from those but slightly differentiated, to those which are dead and in process of removal. Because of the similarity and possible identity of this normal "physiological" regression, with that found in diseased tissues, such specimens should be studied with particular care.
The vital properties of cells are fully treated in text-books of physiology. They include the phenomena of irritability, metabolism, contractility, conductivity, and reproduction. Under irritability may be grouped the response of cells to stimuli of various sorts, such as heat, light, electricity, chemical reagents, the nervous impulse, or mechanical interference. Metabolism, in a wide sense, includes the ingestion and assimilation of food, the elaboration and secretion of desirable products, together with the elimination of waste products. Contractility may be changes in form observed during ten minutes;
FIG. 10. LEUCOCYTES OF A PROG. * au.
, at the beginning of the observation;
manifest m the locomotion Of the J.a half minute later, etc.
entire cell, in the vibratile action of
slender hair-like processes, the cilia, or in contraction of the cell body. Conductivity is the power of conveying impulses from one part of the cell to another. Reproduction is seen in the process of cell division. Many phases of these activities are observed in microscopic sections and as such they will be referred to in later chapters. A few which are of general occurrence will be described presently.
The unicellular animal, Amoeba, exhibits a type of motility known as amoeboid, which has been observed in many sorts of cells in the vertebrate body. In marked cases, as in certain white blood corpuscles (the leucocytes), the cell protoplasm sends out fine or coarse processes which divide or fuse with one another, causing the cell to assume a great variety of forms. The processes may be retracted, or they may become attached somewhere and draw the remainder of the cell body after them, the result of which is locomotion or the so-called wandering of the cell. Such wandering cells play an important part in the economy of the animal body. Their processes can flow around granules or cells and thus enclose them in protoplasm. Some of these ingested bodies may be assimilated by the cell as a result of complex chemical and osmotic reactions. Cells which feed on foreign particles and can alter or digest them are known as phagocytes. Amoeboid movements take place very slowly. In preparations from warm-blooded animals they may be accelerated by gently heating the object.
Another form of motion is that which occurs within the protoplasm of fresh cells, whether living or dead, and consists in a rapid oscillation of minute granules, due to diffusion currents. Although these movements were first observed within protoplasm, it was soon shown that they occurred when various inert particles were suspended in a liquid. Robert Brown described the motion in 1828, in an essay entitled "On the General Existence of Active Molecules in Organic and Inorganic Bodies," and the phenomenon is called the molecular or Brownian movement. It may often be seen in salivary corpuscles.
Formation and Reproduction of Cells
In the past, two sorts of cell formation have been recognized, namely the spontaneous generation of cells, and the origin of cells through the division of pre-existing cells. According to the theory of spontaneous generation it was once thought that animals as highly organized as intestinal worms came into existence from the fermentation of the intestinal contents. After this had been disproved, it was still thought that unicellular animals arose spontaneously and that cells might be formed directly from a suitable fluid, the cytoblastema. Something of the sort may have occurred when life began, and it is the expectation of certain investigators that conditions may yet be produced which shall lead to the formation of organic bodies capable of growth and reproduction. At present, however, only one source of cells is recognized the division of existing cells. "Omnis cellula e cellula." A nucleus likewise can arise only by the division of an existing nucleus; it cannot be formed from nonnucleated protoplasm.
The simplest form of cell division is one which rarely occurs. Ordinarily the division of the cell is accompanied with the production of protoplasmic filaments, and the process is therefore called mitosis (Greek, /uVos, a thread). But in direct division or amitosis these filaments are not developed. The nucleus merely becomes increasingly constricted at the middle until divided in two; or it may be bisected by a deep cleft or fissure. Preceding the division of the nucleus, the nucleolus, if present, may subdivide and supply each half of the nucleus with a nucleolus (Fig. n). Cells which divide by this method are usually degenerating, and the process may terminate with the multiplication of nuclei. If carried to completion, the protoplasm also divides, and a cell membrane develops between the daughter nuclei. The role of the centrosome in amitosis has not been determined. Maximow finds it in a passive condition between the two halves of the nucleus, or beside the stalk connecting these halves if the division is not complete (Anat. Anz., 1908, vol. 33, p. 89). He states that certain mesenchymal cells which divide by amitosis in the rabbit embryo are not degenerating, but may later divide by mitosis, and thus he confirms Patterson's similar conclusion in regard to certain cells in the pigeon's egg. These instances are regarded as exceptional. In the human body the detachment of a portion of the lobate nucleus of certain leucocytes has been described as amitotic division, but the superficial cells of the bladder furnish more typical examples. E. F. Clark has found many cells dividing by amitosis hi the degenerating parts of a human cancer. The occurrence of two nuclei within one cell by no means indicates this form of division. Associated with such cells, others containing nuclei of the dumb-bell shape, or those partially bisected by clefts must be found, in order to prove that amitotic division is taking place.
FIG. ii. AMITOSIS ix EPITHELIAL CELLS FROM THE BLADDER OF A MOUSE. Xs6o. Such preparations as that shown in the figure are made by pressing the lining of a freshly obtained piece of the bladder against a clean cover-glass. Certain of the superficial cells adhere to it, and they are then fixed and stained.
Mitosis, also called indirect division and karyokinesis, is the ordinary mode of cell division. Although it is a continuous process, it has been conveniently divided into four successive phases the prophase, metaphase, anaphase, and telo phase. During the prophase the chromatic material of the nucleus prepares for division and collects in the center of the cell. It is divided in halves in the metaphase, and the two halves move apart during the anaphase. The chromatic material becomes reconstructed into resting nuclei during the telophase. The various patterns which the chromatic material and protoplasmic fibrils present during these phases are known as mitotic figures.
Mitotic figures are found hi all rapidly growing tissues, but especially favorable for preliminary study are the large cells in the root tips of plants. In longitudinal sections of root tips, the cells are cut at right angles to the plane of cell division, which is desirable; and often in a single section 5 mm. long, all the fundamental stages may be quickly located. The following general description of mitosis is based upon such easily obtained preparations, and the plant selected is the spiderwort (Tradescantia virginiana). 1 They may be satisfactorily stained with saffranin, or with iron haematoxylin and a counter stain such as orange G. There are many descriptions of mitosis in root tips, among them the following:
Rosen, (Hyacinthus oriental/is) Beitr. zur Biol. der Pflanzen, 1895, vol. 7, pp. 225-312; Nemec, (Allium cepa} Sitz.-ber. kon. Ges. der Wiss. Prag, 1897, No. 33, pp. 25-26, and Jahrb. fur wiss. Bot., 1899, vol. 33, pp. 313-336; Schaffner, (Allium cepa) Bot. Gaz., 1898, vol. 26, pp. 225-238; Hof, (Ephedra major) Bot. Centralbl., 1898, vol. 76, pp. 63-69, 113-118, 166-171, 221-226; Gregoire and Wygaerts, (Trillium grandiflorum) La Cellule, 1904, vol. 21, pp. 1-76; Farmer and Shove, (Tradescentia mrginiana) Quart. Journ. Micr. Sci., 1905, vol. 48, pp. 559-569; Richards, (Podophyllum peltatum] Kansas Univ. Sci. Bull., 1909, vol. 5, p. 87-93.
The cells to be described are found in the interior of the root tip, just back of the protecting cap of cells which covers its extremity. They are oblong in shape and their long axis corresponds with that of the root. The walls are very distinct, and the cells consist of granular vacuolated protoplasm, which in preserved specimens is generally irregularly shrunken.
The resting cells (Fig. 12, A) contain large round nuclei in which the chromatin is in the form of fine granules evenly distributed throughout the nucleus. A nucleus usually contains from two to five round nucleoli, each of which, when in focus, is seen to be surrounded by a clear zone. The nuclear membrane is distinct.
- 1 Good specimens may be obtained from any rapidly growing root tip. Those starting from hyacinth bulbs placed in water are very favorable. Onion root tips have been extensively used, and also those of bean and corn seedlings. The pointed ends are snipped off and dropped into Flemming's stronger solution
Prophase. The first indication of approaching division is a change in the chromatin, which becomes gathered into fewer and coarser granules and takes a deeper stain. Portions of the linin network break down, so that the chromatin granules come to be arranged in long convoluted threads. Such threads are developing in the cell, Fig. 12, B, but are more perfectly formed in C. It is possible that at a certain stage the nucleus contains only a single continuous thread, but this condition cannot be demonstrated in Tradescantia. The stage of nuclear division in which the chromatic material appears to be arranged in a coiled thread or skein is called a spireme. The "close spireme" (B) is succeeded by the "loose spireme" (C). Successive stages in the development of the spireme in animal cells are seen in Fig. 20, D, E, and F.
As the spireme develops, the nuclear membrane becomes less distinct, and the clear zones disappear from around the nucleoli. The nucleoli become apparently less regular in outline, and forms which suggest that two of them have fused (Fig. 12, B) are perhaps more frequently seen than in resting cells. Usually it is stated that the nucleoli break up into smaller bodies toward the time of their dissolution, and that some of these escape into the cytoplasm after the disappearance of the nuclear membrane. Farmer and Shove believe that the nucleoli contribute to the chromatin; Richards regards them as a store of food material for the rest of the cell; and others believe that they form the achromatic "spindle" which will be described presently. Their function in animal cells is equally uncertain.
In the stage shown in Fig. 12, D, which may be regarded as the end of the prophase, the nuclear membrane and the nucleoli have disappeared, and the spireme thread has become divided into a number of segments or chromosomes. These are straight or curved rods of different lengths. Sometimes they appear as bent V-shaped bodies, but these often represent two chromosomes with their ends together. J-shaped forms, with one long and one short arm, have been described in various plants. The chromosomes become so arranged that one end of the rods, or the apices of the V's, are situated in the equatorial plane, which extends transversely across the middle of the cell. Often it is temporarily tilted (as in D and E) as if the mitotic apparatus had shifted to a position in which it obtained more space. It may do this mechanically if the contents of the cell are under pressure. When the chromosomes are gathered at or in the equatorial plane, they constitute collectively the equatorial plate. Because of their stellate arrangement at this stage, which is best seen in transverse sections of the cell, this mitotic figure is known as the aster.
The manner in which the chromosomes are formed from the spireme thread is difficult to determine. According to Gregoire and Wygaerts, the linin and chromatin, which have often been regarded as closely related
substances, are identical, and linin is merely a name for slender filaments of chromatin. Accordingly the chromatin simply draws together to
FIG. 12. MITOTIC CELL DIVISION IN THE ROOT TIP OF Tradescanliavirginiana. Xias diam. A, resting cell; B, C, D, prophase; E, metaphase; F, anaphase; G, H, I, telophase.
form chromosomes, and the beaded appearance of the spireme thread is due to alternate enlargements and constrictions of one substance. Others consider that a different substance connects the granules of chromatin
with one another; and Rosen states that each chromatin granule is completely imbedded in a broad strand of linin. Davis similarly interprets the spireme shown in Fig. 20, F. Whatever the actual structure may be, the chromatin granules in the spireme thread early divide in two, so that the thread appears double. When the thread shortens and condenses to form the chromosomes, the rows of granules may coalesce so as to produce a rod already divided lengthwise, although its halves are in close apposition. Occasionally the ends of the chromosomes are seen to be slightly separated.
Metaphase. In the metaphase (Fig. 12, E), the two longitudinal halves of each chromosome are being drawn apart toward the opposite poles of the cell. If the chromosome is V-shaped, the separation of the two halves begins at the apex of the V.
At this stage an achromatic figure, known as the spindle, is evident in plant cells, but it is more sharply defined in those of animals. As seen in the diagram (Fig. 13), it consists of fibrils which pass from the equatoria
Polar radiation. Nuclear spindle.
FIG, 13. EARLY METAPHASE.
FIG. 14. LATE METAPHASE.
plate toward either pole, where, in animal cells, there is a well-defined granule, the centrosome. Around each centrosome there are radiating protoplasmic fibrils, forming the polar radiation (Figs. 13 and 14). The polar radiation is also called an aster, and the two asters connected by the spindle are known as the amphiaster. Some of the spindle fibers are attached to the chromosomes and appear to pull their halves apart; others pass from pole to pole without connecting with the chromosomes. In animal cells the spindle arises as the two centrosomes, lying beside the nucleus, move apart (Fig. 20, A). As they pass to the opposite poles of the nucleus, the spindle forms between them, either from the nuclear reticulum, or the cytoplasmic reticulum, or hi part from both. These conditions appear to vary in different animals.
In the cells of root tips, a condensation of protoplasm forms a cap at the poles of the nucleus at the time when the nuclear membrane and nucleoli are disappearing. From the "polar cap," spindle fibers develop
which invade the nucleus, and also radiations which have been traced even to the cell walls. But as Rosen states, sun-like figures, such as certain botanists have pictured, do not occur. Schaffner has described a distinct centrosome or central granule in the root tip of the onion, but Richards finds that in Podophyllum there is no such structure, and the weight of evidence appears to be against the existence of a definite centrosome in the higher plants.
Anaphase. In the anaphase the halves of each chromosome move to the opposite poles (Fig. 12, F). The figure thus produced is known as a double star or diaster. Since each chromosome has divided into two, the original number of chromosomes is preserved, and an equal number of rods will be found in either star. They cannot all be brought into focus together, and because of overlapping, they are hard to count. Sometimes one chromosome, longer than the others, remains for a time as a continuous bar from one aster to the other. Between the asters there are always straight spindle fibers, but they vary in distinctness. (The anaphase in an animal cell is well shown in Fig. 21, D.)
Telophase. After the chromosomes have reached the opposite poles, they form two dense masses. They are generally said to unite end to end, thus forming a spireme thread. But in the root tips of Trillium, Gregoire and Wygaerts state that they come into contact with one another laterally; and as they separate, transverse connections are retained, which, with the vacuolization of the chromosomes, restore the nuclear reticulum. This may not be the correct interpretation, but immediately after the anaphase the chromosomes form a very compact mass, easily overstained so that it appears solid. Subsequently the mass enlarges (Fig. 12, H), and the chromosomes become coarsely granular, taking the form of wide bands. Nucleoli reappear, and according to Richards, "it is a general rule that they arise on the side of the nucleus nearest the new cell wall." This accords with Nemec's statement that they form from the outer fibers of the spindle. Nemec and Rosen agree that they first appear outside of the nucleus, which they enter before the nuclear membrane develops. These are details which require confirmation.
The new cell wall arises in plants as a series of thickenings of the interzonal spindle fibers, which at this stage form a barrel-shaped bundle (Fig. 12, G). The thickenings coalesce to form a membrane which does not at first reach the sides of the cell. While this wall is developing the nuclei are in a condition resembling the spireme stage of the prophase. The entire mitotic figure is therefore called the double spireme or dispireme. The cell wall is soon completed and the nuclei return to the resting condition (Fig. 12, I).
The time required for mitotic cell division varies from half an hour (in man) to five hours (in amphibia). After death, if the tissues are not
INDIRECT DIVISION 19
hardened by cold or reagents, it is thought that mitoses go on to completion. Forty-eight hours may elapse before they entirely disappear from the human body.
Pluri-polar mitosis. Under abnormal conditions, as in the cancer cells shown in Fig. 15, spindles may develop simultaneously in connection with three or four centrosomes. Similar pluri-polar spindles have been produced experimentally, by treating cells with various poisonous solutions. An unequal distribution of chromatin may occur under such conditions, and this may happen also with bipolar spindles, as shown in Fig. 15, a.
Number and individuality of the chromosomes. It is now generally believed that every species of plant or animal has a fixed and characteristic number of chromosomes, which regularly recurs in the division of all
FIG. 15. MITOSES IN HUMAN CANCER CELLS. (From Wilson, after- Galeotti.) a, Asymmetrical mitosis with unequal distribution of chromatin; b, tripolar mitosis; c, quadripolar" mitosis.
its cells, with the exception of the germ cells, in which the number is reduced. In certain species, however, the two sexes regularly differ from one another in the number of their chromosomes, and one sex may contain an odd number. Usually the number of chromosomes is believed to be even.
There is considerable difficulty in counting the chromosomes. Generally it is possible that some have been cut away in the process of sectioning, so that, if the number is believed to be invariable, the highest number found in any cell is assumed to occur regularly. Another source of error lies in the fact that a bent chromosome may be counted as two, or rods with their ends overlapping may appear as one. Farmer and Shove have ventured to state that the number in Tradescantia "varies from about twenty-six to thirty- three." Nemec found that twelve chromosomes occur regularly in young tissues of the onion, but that in older tissues the number diminishes even to four. Sixteen have been recorded in the onion by other botanists. Podophyllum is said to have sixteen (Mottier), but Richards records counts of fourteen. In man the number has been placed at 16 and 32, but it is now believed to be 24. Gutherz, with particularly favorable material, emphasizes the difficulty of counting
the chromosomes in man. He found only two cells in which a count could be made, in neither case with absolute certainty. But he agrees with Duesberg that the reduced number is twelve, according to which the whole number should be twenty-four. Recently, however, Wieman has found cells in the brain of a 9-mm. human embryo which contained 33 chromosomes. Some cells in the nasal epithelium and mesenchyma of this specimen contained 34, and others 38. Thus Wieman concludes that the number in man is certainly greater than 24 and is perhaps variable (Amer. Journ. Anat., 1913, vol. 14, pp. 416-471).
In the grasshoppers, which are among the most favorable objects for the study of mitosis, not only is the number of chromosomes for a given species believed to be constant, but each cell appears to contain a definite series of chromosomes, the members of which vary somewhat in shape and size. Recent studies of such cells favor Rabl's hypothesis of the individuality oj the chromosomes, according to which the chromosomes persist in the resting nucleus, although disguised by their lateral branches and diffuse granular form. If this hypothesis is correct, when a nucleus prepares for division the same chromosomes which entered it will reappear. Sometimes in the prophase the bands of chromatin are arranged hi a polar field such as is seen in the telophase (Fig. 12, H). This arrangement has been observed by Farmer and Shove in the prophase of Tradescantia, and by others in various plants and animals. It is regarded as evidence that the chromosomes are "independent and continuously perpetuated organs of the cell." Nevertheless it is generally true that in resting nuclei no trace of individual chromosomes can be made out. The great importance of accurate knowledge of the chromosomes is shown by the following considerations.
As a result of mitotic cell division, it is evident that every new cell regularly receives one-half of each chromosome found in the parent cell, and thus the number of chromosomes remains constant. But in the germ cells the number is invariably reduced, and hi some animals it becomes exactly one-half of the number found elsewhere in the body. In such a case, when the male sexual cell, or spermatozoon, unites with the female sexual cell, or mature ovum, in the process of fertilization, the original number is restored. Each parent thus contributes one-half of the chromosomes found in the cell which gives rise to a new individual; and since each of these divides with every subsequent cell division, it is evident that one-half of the chromatin in every cell of the adult body is of maternal origin and one-half of paternal origin. The process by which the sexual cells acquire the reduced number of chromosomes and become ready for fertilization is known as maturation. The production of the sexual cells in the male is called spermatogenesis and in the female oogenesis.
FIG. 16. DORSAL (a) AND LATERAL (b) VIEWS OF THE ABDOMEN OF GRASSHOPPERS. (After Hyatt and Scudder.)
In its essential features, the process of spermatogenesis in insects corresponds with that in mammals, and very favorable material can be obtained in abundance from grasshoppers of various genera.
The males may be distinguished from the females by the shape of the abdomen. In males it is more rounded (Fig. 16) with various appendages directed dorsally. The abdomen of the female is pointed, terminating in the ovipositor, the parts of which as seen from the side may be together, or widely separated dorso-ventrally. The genital glands can be readily removed by dissecting as follows: Male grasshoppers, which have been chloroformed, are opened by a mid-ventral incision. The abdominal walls are pinned out on a wax plate under normal salt solution (0.6 per cent.). The intestinal tube, which is usually black or green, is taken out with forceps, and the yellow or orange testes are seen close together at the upper end of the abdomen, attached to the back. Each testis consists of a number of separate cylindrical lobes, and it should be worked loose from the surrounding tissue with forceps in such a way that these lobes remain together. The tissue may be preserved in Flemming's strong solution or in Hermann's fluid, and stained with iron haematoxylin.
Among the many publications upon spermatogenesis in the grasshoppers, the following may be cited: McClung, C. E., The accessory chromosome sex determinant? Biol. Bull., 1902, vol. 3, pp. 43-84; Sutton, W. S., On the morphology of the chromosome group in Brachystola magna, Biol. Bull., 1902, vol. 4, pp. 24-39; McClung, C. E., The chromosome complex of orthopteran spermatocytes, Biol. Bull., 1905, vol. 9, pp. 304-340; Robertson, W. R. B., The chromosome'complex of Syrbula admirabilis, Kansas Univ. Sci. Bull., 1908, vol. 4, pp. 273-305; Davis, H. S., Spermatogenesis in Acrididae and Locustidae, Bull. Mus. Comp. Zool., 1908, vol. 53> PP- 57-IS7; Wilson, E. B., The sex chromosomes, Arch, fur mikr. Anat., 1911, vol. 77, pp. 249-371.
As seen in sections, each lobe of the testis of the grasshopper contains a considerable number of closed sacs or cysts, which are filled with sexual cells; and all the cells within a cyst are in approximately the same stage of development. The cysts are shown in Fig. 17, which represents a longitudinal section of a single lobe. Usually the testis is sectioned as a whole, and the specimen consists of a group of lobes cut transversely or obliquely. Cross sections from the apical portion, furthest from the outlet, will contain younger stages than the sections lower down in the lobe, since the cysts form at the apex and gradually move downward. At the apex, according to Davis, there is an apical cell which is surrounded by young sexual cells known as spermatogonia (Fig. 17, a). The spermatogonia move away from the apical cell, and each becomes enclosed in a cyst-wall derived from the surrounding tissue. Within the cysts thus formed, the spermatogonia multiply, and the cysts in the upper part of the lobe are filled with spermatogonia (Fig. 17, b). After repeated divisions the spermatogonia pass through a period of growth, accompanied by a rearrangement of their nuclear contents. The large cells with characteristic nuclei which are thus produced, are known as primary spermatocytes. They fill the cysts further down in the lobe (Fig. 17, c). Each primary spermatocyte divides into two secondary spermatocytes, and each of these divides into two spermatids, after which no further cell division is possible until fertilization takes place. But each spermatid becomes transformed from a round cell into a linear body with a whip-like tail, and is then capable of independent motion. Since in this form these cells were once thought to be parasitic animals living in the spermatic fluid, they received the name spermatozoa, which they still retain. 1 Cysts containing spermatozoa occur near the outlet of the lobe, or if the grasshoppers are collected late in the season, they may be found throughout most of the testis. Specimens from young grasshoppers, in which the spermatocyte divisions are abundant, are more desirable, even though no spermatozoa have become fully developed.
FIG. 17. LOBE OF THE TESTIS OF A GRASSHOPPER. Xso. (After Davis.)
a, apical cell.
d, spermatocytes dividing.
The succession of cell divisions described in the preceding paragraph is shown in tabular form in Fig. 18. Except for the number of chromosomes within the various cells, this diagram is quite as applicable to man as to the grasshopper. In this figure, however, only two spermatogonial divisions have been included. The number of times which the spermatogonia may divide before becoming spermatocytes is considerable and
1 It has been proposed to substitute the term spermium for spermatozoon; and consequently spermiocyte, spermid, etc., for spermatocyte and spermatid. The secondary spermatocytes are sometimes called praespermatids or praespermids; but these changes in names are of questionable value
FIG. 1 8. DIAGRAM OF THE CELL DIVISIONS IN SPERMATOGENESIS. The figures indicate the number of chromosomes found in the cells of certain grasshoppers.
presumably indefinite. As seen in sections, the spermatogonia, spermatocytes, and spermatids may be described as follows, using for illustrations Davis's figures of a common grasshopper Dissosteira Carolina.
Spermatogonia. The nucleus of each spermatogonium contains the full number of chromosomes, which in most of the grasshoppers (Acrididee) is 23. With every spermatogonial division, each chromosome is split lengthwise. In this and other respects the mitotic figures are quite like those occurring elsewhere in the body. They are shown in Fig. 20, A, B, and C. When the twenty- three chromosomes have formed the equatorial plate, it is sometimes possible to see all of them in a single transverse section of the cell (Fig. 19, A). It then appears, as found by Montgomery (1901) in certain Hemiptera, and a year later by Button in grasshoppers, that the chromosomes vary in size, but the "gradations in volume are not between individual chromosomes but between pairs, the two members of which are of approximately equal size." In Fig. 19, A, twelve forms of chromosomes have been identified by Davis; and all of these are paired except the one numbered 4. The members of a pair are often, but by no means invariably, side by side. In some cases, owing to foreshortening, their resemblance in size is not apparent in the drawing. The behavior of the odd or accessory chromosome is of special interest, since according to McClung's hypothesis, now well established, this accessory chromosome is the bearer of those qualities which determine sex.
Primary spermatocytes. After the last spermatogonial division, the cells begin their "growth period." At this time the chromatin tends to collect on one side of the nucleus, in a condition known as synapsis (or more recently as synizesis). This distribution of the chromatin has been frequently observed, but it has not been shown to be of special significance. In the primary spermatocytes drawn in Fig. 20, D, E, and F, the chromatin is evenly distributed. All of the chromosomes, except the accessory chromosome, have become filamentous, but the accessory chromosome remains as a compact, darkly staining body close to the nuclear membrane. It resembles a nucleolus, for which in fact it has been mistaken. True nucleoli may occur in these cells, together with the accessory chromosome, but they stain differently.
As the primary spermatocytes prepare for the next division, the spireme becomes resolved into eleven loops, each of which represents the two members of a pair of chromosomes joined end to end. The granules imbedded in the linin thread divide as usual, so that each loop contains a double row of granules (Fig. 20, F). These loops contract to form eleven chromosomes, which, because of their four parts, are known as tetrads. The structure of the tetrads is shown in Fig. 19, B-G. The filaments seen in the upper row of drawings contract into corresponding solid forms of chromosomes seen in the lower row, in which the place of attachment to the spindle fibers has been indicated.
Each tetrad represents two chromosomes joined end to end and split lengthwise. The simplest forms are shown in Fig. 19, B and C, which illustrate respectively two ways in which the tetrad may later divide. The two component chromosomes may simply be pulled apart, as indicated in Fig, 19, B, in which the spindle fibers are attached to the ends of the rod. If this takes place, each secondary spermatocyte will receive one member of every pair of chromosomes which occurred in the spermatogonium, but no part of the other member. Such a division, which eliminates one-half of the chromosomes from the daughter cell, is known as a reductional division. The other form of chromosome division is known as equational. When it takes place, every chromosome divides lengthwise, and the daughter cells receive one-half of every chromosome in the parent cell. This occurs in ordinary cell division, and also in the division of the tetrads provided that the spindle fibers are attached to the place where the two component chromosomes come together (Fig. 19, C).
FIG. 19. A, POLAR VIEW OF THE METAPHASE OF A SPERMATOGENIAL DIVISION IN Dissosteira Carolina. X 1450 (After Davis.) The pairs of chromosomes have been numbered. B-G, various forms of tetrads, rom primary spermatocytes. (After Davis and Robertson.)
As a stage in the separation of the two halves of a rod-shaped tetrad, crossshaped forms are produced (Fig. 19, D). If the separation is almost complete, such shapes are seen as in Fig. 19, E. The arms of the tetrad which are not attached to the spindle fibers may bend toward one another and unite, so as to form rings (F), or they may twist about like a figure 8, as shown in G. If the spindle fibers are attached to the points xx in the upper figure in G, the division will be equational; if as shown in the lower figure it will be reductional.
Usually it is considered that the division of the tetrads into double bodies or dyads, is equational, and that the division of the dyads, which takes place when the secondary spermatocytes divide, is reductional. According to Davis, however, the first division of the tetrads is reductional and the second division is equational. In either case the end-result is the same. Each spermatid will contain one of the four parts of each tetrad, and thus one member of every pair of chromosomes will be eliminated from any given spermatid.
Since in the testis tetrads occur only in the primary spermatocytes, the cells shown in Fig. 20, G-J, are easily identified. These are success ive stages in the division of the primary spermatocyte. In G the accessory chromosome is seen as a rod-shaped body above and to the right; in H it is below and to the right. In J it is obliquely placed just above the equatorial plate and in K it is passing to the upper pole of the spindle. In the spermatogonial divisions the accessory chromosome always divides with the others; but in the division of the primary spermatocyte it passes undivided into one of the daughter cells. Thus one secondary spermatocyte will receive eleven chromosomes (dyads) and the other will receive twelve (eleven dyads and the accessory chromosome) . In the late anaphase shown in Fig. 20, L, the accessory chromosome cannot be recognized.
FIG. 30. SPERMATOGENESIS IN Dissosleira Carolina A-FXI4SO; G-LX966. (Davis.)
A, B, C, prophase. metaphase. and telophase of a spennatogonial division. D-L, successive stages in the
.division of a primary spermatocyte into secondary spermatocytes.
Secondary spermatocytes. The secondary spermatocytes pass rapidly from the condition shown in Fig. 20, L, to that of Fig. 21, A. A nuclear membrane has developed, and the dyads have become somewhat filamentous. Without passing through a complete resting stage they proceed to divide as shown in Fig. 21, B-F. The dyads separate into their component halves. In those secondary spermatocytes which received the accessory chromosome, that body will be seen dividing with the dyads, and each spermatid will receive one-half of it. It has been questioned whether the division of the accessory chromosome is longitudinal and therefore equational, or transverse and reductional. Many cytologists consider that if a chromosome splits lengthwise, all of its parts will be represented in the resulting halves, but if it divides transversely, essential elements will be lost. This conception lends importance to the question of transverse or longitudinal division of the accessory chromosome. By the division of this chromosome it comes about that one-half of the spermatids contain twelve chromosomes, and one-half contain eleven, as indicated in the diagram, Fig. 18. The spermatids shown in Fig. 21, F, contain the accessory chromosome.
Spermatids and Spermatozoa. In forming spermatozoa, the spermatids become elongated, passing from the condition shown in Fig. 21, F, to that of Fig. 21, G. The chromatin within the nucleus is distributed in fine granules throughout the linin reticulum. Close to the nuclear membrane a small dark body has appeared, from which a slender filament has grown out. This body is usually described as the centrosome. A condensation within the cytoplasm, seen also in F, is known as the paranucleus. It is of uncertain origin, but may proceed from the cytoplasmic structure called mitochondrium. The paranucleus forms a sheath about the axial filament.
Successively later stages are shown in Fig. 21, H, I, and J. The chromatin within the nucleus becomes homogeneous and very dense; at the same time the nucleus elongates and forms the head of the spermatozoon. This is enveloped by the cell membrane, but there is no appreciable layer of protoplasm around it. The centrosome elongates and forms the middle piece of the spermatozoon; and the axial filament, with a covering derived from the paranucleus and cytoplasm, constitutes the tail. Only a portion of the tail is included in the figure. The human spermatozoon likewise consists of a head, which is essentially the nucleus, a middle piece containing the centrosome, and a tail; but the form of the head is very different from
FIG. 21. SPERMATOGENESIS IN Dissosteira Carolina. Xi4So. (Davis.)
A-F, successive stages in the division of a secondary spermatocyte into spermatids. G-J, successive stages in the transformation of spermatids into spermatozoa.
that in the grasshopper. It will be described in a later chapter
Although the spermatozoa of the grasshopper appear alike, it has been shown that one-half of them contain eleven chromosomes, and one-half contain twelve. The mature ova all contain twelve chromosomes. If a spermatozoon with eleven chromosomes unites with an ovum with twelve, a male animal will be produced, in every cell of which there will be twentythree chromosomes. But if the spermatozoon contains twelve chromosomes, a female animal is formed, containing twenty-four chromosomes in every cell. Thus sex appears to be determined by the presence or absence of a chromosome within the spermatozoon.
In some cases, as in several Hemiptera described by Wilson, the accessory chromosome is paired, but its mate is of small size. Thus the spermatozoa all have the same number of chromosomes; but half of them contain the large member of the pair and will produce females, and the other half contain the small member and will produce males. The mature ova all contain the large member. In tfyese insects, therefore, both sexes contain the same number of chromosomes, but the cells of the male contain a small chromosome, whereas the corresponding one in the female is large. From these observations it is reasonable to conclude that sex may be determined by a difference in the nature of certain chromosomes in those animals in which there are no appreciable differences in size or number.
In man, a difference in the number of chromosomes in the sexes has been reported, but the observations have not been confirmed. It is supposed that the spermatogonia contain twenty-four chromosomes, but it has not been shown that they exist as pairs. The spermatocytes, spermatids and spermatozoa apparently contain twelve. As the principal constituents of the spermatozoon, the chromosomes are believed to be the essential agents in the transmission of all qualities inherited from the male parent, and certain of them may determine sex.
Mature ova result from a succession of cell divisions closely comparable with those which produce spermatozoa. The primitive female sexual cells correspond with the spermatogonia, and are called oogonia. They are provided with the full number of chromosomes, and divide an indefinite number of times. After a period of growth they become primary oocytes, in which the number of chromosomes is reduced one-half. The primary oocytes divide to form secondary oocytes; and these again divide to produce the mature ova, which are incapable of further division unless fertilization takes place. (The term ovum is ordinarily loosely applied, so that it includes not only the mature cells, but also oocytes, and the clusters of cells resulting from the division of the fertilized ovum.)
Although the mature ovum and the spermatozoon are closely similar in their nuclear constitution, they differ radically as to form, size, and cytoplasmic structure. The ova are very large cells, stored with nutriment for the embryo which each one may later produce. In the higher vertebrates they are formed in relatively small numbers. According to Hensen's estimate, about two hundred, ready for fertilization, are produced by the human female in a life-time. But the male, according to Lode, produces 340 billion spermatozoa, or, as stated by Waldeyer, nearly 850 million per ovum. A large number must be produced, since many will fail to traverse the uterus and tube so as to find the ovum at the time of its discharge from the ovary. The ova of lower vertebrates, which are fertilized and develop outside of the body, are discharged in great numbers; in certain fishes from three to four million are produced annually.
The multiplication of oogonia in the human ovary takes place before birth, and about fifty thousand are produced. At birth, or shortly thereafter, all the oogonia have become primary ob'cytes (Keibel). At first the oocytes are small, but they enlarge at varying rates, and the largest are indistinguishable from mature ova except by their nuclear contents. Since some grow more rapidly than others, the ovary in childhood contains primary oocytes of many sizes. Each oocyte becomes enclosed in a cyst or follicle. The way in which these follicles develop, and the. manner in which the oocyte escapes into the uterine tube by the rupture of these follicles, will be described in connection with the ovary. Between the cells of the follicle and the oocyte, there is a broad, radially striated membrane, known as the zona pellucida or zona radiata (Fig. 2 2) . This zona has sometimes been regarded as a cell membrane, but the oocyte divides within it as if enclosed in a capsule. It does not invest the daughter cells like a membrane. The radial striations have been interpreted as slender canals containing processes of the f ollicular cells, and the zona has been considered as a product of these cells. In certain cases a perivitelline space has been described as encircling the oocyte and thus separating it from the zona, but this space has been considered as artificial, or as a refractive line wrongly interpreted as a space.
The cytoplasm of the oocyte becomes charged with yolk granules or spherules. They constitute the deuteroplasm (or deutoplasm), but this term is equally applicable to fat droplets and other secondary products of the protoplasm. In the human oocyte the granules are centrally placed (Fig. 22), and they are so transparent, when fresh, as to cause only a slight opacity. In the eggs of many animals the yolk is more highly developed, and it may be evenly distributed or gathered at one pole. Within the cytoplasm of the developing oocyte, a large dark body of radiate structure is sometimes conspicuous. It is inappropriately known as the yolk nucleus, and is probably a derivative of the centrosome and surrounding archoplasm. Other "vitelline bodies," of uncertain origin and significance, have been described. Some have been considered as nuclear extrusions.
The nucleus of the oocyte is very large and vesicular. The chromatin occurs chiefly along the nuclear membrane and about the nucleolus. The nucleous is also very large, and Nagel stated that in the fresh condition it exhibits amoeboid movements, but this observation has not been verified. The nuclei of the oocytes ordinarily show no signs of mitosis, and they may remain in the resting condition for thirty years or more and then divide. Many of them, however, will degenerate without division.
FIG. 22. THE OVUM AS DISCHARGED FROM A VESICULAR FOLLICLE OF AN EXCISED OVARY OF A WOMAN THIRTY YEARS OF AGE. Examined fresh in the liquor folliculi. (Nagel.)
c. i., Corona radiata composed of cells of the follicle; n., nucleus; p., granular protoplasm; p. s., perivitelline space; y., yolk; z. p., zona pellucida. (From McMurrich's "Embryology.")
The cell divisions which give rise to the secondary oocyte and the mature ovum respectively, have never been observed in man. Some of the cells within the ovary may be secondary oocytes and the cell shown in Fig. 22 may be of this sort, or possibly a mature ovum, but this cannot be determined. From what is known of other mammals, however, it may confidently be assumed that the cell divisions take place as shown in the diagram, Fig. 23.
When the primary oocyte divides, the chromosomes, reduced in number, also divide and are equally distributed to the daughter cells; but the great mass of cytoplasm remains with one of these cells, namely, the secondary oocyte. The other cell, which is relatively very small, is known as the first polar body, or polar cell. It has the same nuclear contents as the secondary oocyte, and may divide into two other polar bodies, equivalent to mature ova. More often it degenerates without division. When the secondary oocyte divides, it likewise produces one large cell, the mature ovum, and one small cell, the second polar body. The latter is said to be capable of fertilization, but to what extent it may develop is unknown. Functionally the production of polar bodies serves to prevent the subdivision and distribution of the nutritive material elaborated within the primary oocyte. One mature ovum with abundant yolk is provided at the expense of three ova (polar bodies) which degenerate.
Although the maturation of the ovum has not been observed in man, nor even the presence of definite polar bodies, the entire process has been carefully studied in other mammals, notably in the mouse. 1 It has been shown that the maturation of the ovum of the mouse takes place rapidly, both of the oocyte divisions being accomplished within from four to fifteen hours. The first polar body usually forms before the oocyte is discharged from the ovarian follicle in other words, before ovulation takes place. The second polar body is usually formed in the uterine tube, after the spermatozoon has entered the oocyte. Long and Mark have found that the chromosomes of the primary oocyte are tetrads, or bodies showing transverse and longitudinal divisions; and that those of the secondary oocyte are dyads. They believe that the first division is transverse or reductional, and that the second is equational.
FIG. 23. DIAGRAM OF THE CELL DIVISIONS IN OOGENESIS. (Compare with Fig. 18.)
- 1 Among the most important papers are: Sobotta, J., Die Befruchtung und Furchung des Eies der Maus. Arch. mikr. Anat., 1895, vol. 45, pp. 15-91. Long, J. A., and Mark, E. L. The maturation of the egg of the mouse. Carnegie Inst. Publ. No. 142, 1911, pp. 1-72.
The difficulty of counting chromosomes is apparent from the varying numbers which have been reported in the mouse After reduction the number has been placed at 8, 12, 16, 18 and 20 by different observers.
The polar bodies in the mouse are relatively large. In the upper part of Fig. 24, A, a polar body is about to be formed, and it is completely cut off from the oocyte in Fig. 24, C. In D and G, two polar bodies are shown.
In the mouse, from six to ten hours after coitus, spermatozoa have made their way to the distal end of the uterine tube, where fertilization takes place. According to Long and Mark, the maturation of ova usually occurs at some time during the period from "13! to 28^ hours " after the mouse has given birth to a litter; and during the process of their maturation, the oocytes are discharged from the ovary and enter the distal end of the tube. Here, if fertilization takes place, a single spermatozoon penetrates the zona pellucida. In a section obtained by Sobotta, the entrance of the spermatozoon has been partially accomplished (Fig. 24, B). Its tail lies outside of the zona, and appears to have become thickened. In another specimen Sobotta found the head, middle piece and a part of the tail within the cytoplasm of the oocyte. The tail had broken as it crossed the zona, and the portion remaining outside had drawn together and was disintegrating. In some animals it is said that the entire spermatozoon enters the ovum, but in others only the head and middle piece. In any case the tail appears to be a propelling apparatus which becomes functionless after the head and middle piece have passed through the zona. It has entirely disappeared in the stage shown in Fig. 24, A, in which the head of the spermatozoon is seen within the oocyte on the right side of the figure. Meanwhile the oocyte is becoming a mature ovum by undergoing divisions and producing the second polar body; and the anaphase of this division is shown in Fig. 24, A. Sobotta stated that no centrosomes occur in connection with the spindles of the maturation divisions, and Long and Mark have likewise failed to find any "typical centrosomes."
In Fig. 24, C, the second polar body has become a separate cell. The chromosomes of the ovum, which is now mature, have formed a compact mass. They next become resolved into a chromatic reticulum, and a resting nucleus is produced, provided with a nuclear wall and distinct nucleoli (Fig. 24, D and E). This nucleus, which becomes large and moves toward the center of the cell, is known as the female pronucleus. Meanwhile the head of the spermatozoon has enlarged and formed the male pronucleus, as shown in Fig. 24, C, D and E.
The two pronuclei, which are very similar, develop rapidly, "probably within a few minutes after the entrance of the spermatozoon." Simultaneously they prepare for division, and the chromatic reticulum of each becomes resolved into the reduced number of chromosomes which it received during maturation (Fig. 24, F). A centrosome with astral radiations is now seen between the two groups. In Fig. 24, G, it has divided in two, and the spindle has developed. There has been much discussion as to the origin of these centrosomes. Since in this case they arise by the division of a single body, the possibility that one comes from the spermatozoon and one from the ovum has been eliminated. Moreover in the mouse they cannot be derived from the surviving centrosome of the last maturation division of the ovum, for that division takes place without centrosomes. Therefore the centrosome must either be brought in by the spermatozoon as a constituent of its middle piece, or it must be a new formation. Sobotta accepted the former alternative, and he observed a centrosome in connection with the head of the spermatozoon in certain stages (Fig. 24, C) but not in all. It is probable, according to Conklin, that "the source of the cleavage centrosomes may differ in different animals, or even in the same animal under different conditions."
FIG. 34. MATURATION AND FERTILIZATION OF THE OVUM OF THB MOUSE .A,C-J,Xsoo; 8X750.
A-C, entrance of the spermatozoon and formation of the second polar body. _ D-E, development of the pronuclei. F-J, successive stages in the first division of the fertilized ovum.
Later stages in the division or "cleavage" of the fertilized ovum into two cells are shown in Fig. 24, H-J. The two groups of chromosomes come together upon the spindle so that the full number, characteristic of the species, is restored. Each chromosome then divides lengthwise, and thus each daughter cell receives one-half of its chromosomes from the male parent and one-half from the female parent. This is strikingly evident when the eggs of the fish Fundulus, which have long rod-shaped chromosomes, are fertilized with the sperm of Menidia, which has shorter rods. Moenkhaus, who performed this experiment (Amer. Journ Anat., 1904, vol. 3, pp. 29-64), states that the two kinds of chromosomes remain grouped and bilaterally distributed on the spindles during the first and second divisions of the fertilized ovum, but that later they become gradually mingled.
Important information in regard to the nature of fertilization has been obtained by experiments upon unfertilized eggs. Changes in the concentration or composition of the sea water in which the eggs of marine animals have been placed, mechanical agitation, or, in the case of frogs' eggs, puncturing the outer layer with a needle, have led to repeated cell divisions. In this way embryos or larvae have been produced from unfertilized eggs, and, in a few instances, adult animals. Loeb, who has been a foremost investigator in this field, concludes that the spermatozoon causes the development of the egg by carrying a substance into it which liquefies the cortical layer of the egg, and thereby causes the formation of a membrane. "This membrane formation, or rather the modification of the surface of the egg which underlies the membrane formation, starts the development." At the same time there is an acceleration of the oxidations in the egg. "What remains unknown at present is the way in which the destruction of the cortical layer of the egg accelerates the oxidations."
For the physicist and chemist, the production of mitotic figures and the process of fertilization, have been subjects of great interest, and discussions of their significance will be found in various text-books of physiology and biological chemistry. For further morphological details the student is referred to "The Cell in Development and Inheritance," by E. B. Wilson (2nd ed., New York, 1900) and to the chapters on "Die Geschlechtszellen" and "Eireife, Befruchtung und Furchungsprozess," by W. Waldeyer and R. Hertwig respectively, in vol. i of Hertwig's "Handbuch der vergl. u. exp. Entwickelungslehre der Wirbeltiere," (Jena, 1906).
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Reference: Lewis FT. and Stöhr P. A Text-book of Histology Arranged upon an Embryological Basis. (1913) P. Blakiston’s Son and Co., 539 pp., 495 figs.
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