Book - A Laboratory Text-Book of Embryology 6 (1903)

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Minot CS. A Laboratory Text-Book Of Embryology. (1903) Philadelphia:P. Blakiston's Son & Co.

A Laboratory Text-Book of Embryology: 1. General Conceptions | 2. Early Development of Mammals | 3. Human Embryo | 4. Pig Embryos | 5. Chick Embryos | 6. Blastodermic Vesicle and Ovum Segmentation | 7. Uterus and the Foetal Appendages in Man | 8. Methods | Figures | Second edition | Category:Charles Minot
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This historic 1903 embryology textbook by Minot describes human development.

This textbook was republished in a second edition 1917: Minot CS. A Laboratory Text-Book Of Embryology. (1917) Philadelphia:P. Blakiston's Son & Co.

See also his earlier 1897 textbook; Minot CS. Human Embryology. (1897) London: The Macmillan Company.

Minot Links: Harvard Collection | 1889 Uterus And Embryo - Rabbit | 1905 Harvard Embryological Collection |1897 Human Embryology | 1903 A Laboratory Text-Book of Embryology | 1905 Normal Plates of Rabbit Embryo Development | Category:Charles Minot

See also: Historic Embryology Textbooks

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Chapter VI. Study Of The Blastodermic Vesicle And The Segmentation Of The Ovum

Method of Obtaining Blastodermic Vesicles from the Rabbit

The does should be allowed to become pregnant and be isolated until they have littered ; the date of littering should be noted, and thirty days thereafter the buck be admitted and the exact time of the covering recorded. At the proper number of days thereafter the animal should be killed and the uterus removed at once. It may be opened with two pairs of forceps used to split the outer muscular walls of the organ, beginning the operation at the lower end of the uterus. With a little care this can be done without rupturing the mucous membrane, which is to be afterward also opened in a similar manner with the forceps and the blastodermic vesicles exposed. They are small bodies of rounded form and with a brilliant pearly luster, and are easily observed. During the earlier stages, which occur in the Fallopian tubes, the ova are very small and difficult to find, but by the time the ovum has reached the uterus it has become a blastodermic vesicle measuring about 0.6 mm. in diameter, and, therefore, easily seen by the naked eye. From the fourth day after coitus until the beginning of the seventh day the vesicles lie free in the uterus. Usually early in the seventh day the vesicles, which then measure about 4.5 by 3.5 mm., begin to attach themselves to the wall of the uterus, and thereafter are much more difficult to remove. At the beginning of the fifth day the ova measure about 0.6 to 0.9 mm. in diameter, but vary greatly in size, and are found more or less near together in the upper portion of the oviduct. By the end of the sixth day they measure about 4.0 mm. and are distributed throughout the entire length of the uterus.

The most useful stages are the vesicles from the beginning of the sixth and seventh days. To preserve the vesicles they must be gently removed from the uterus, great care being necessary not to injure them, and dropped into Zenker's or Hermann's fluid. In either of these they may be left for about an hour and then washed and preserved in the usual manner. Specimens should be examined in the fresh state, just after the}* have been preserved, and after they have been stained before they are imbedded. For staining, alum cochineal or borax carmine is recommended. Finally, the specimens are to be imbedded in paraffin and cut in series in the usual manner ; sections of from 6 to 8 ft are desirable. Unfortunately no method has yet been devised by which these delicate vesicles may be imbedded without distortion of their form, so that, when the sections are finally obtained, the blastodermic walls are wrinkled and more or less out of shape. But fortunately, owing apparently to its greater thickness, the embryonic area usually escapes distortion and appears in the sections of normal form, or nearly so.

Study of Rabbit Blastodermic Vesicles in Alcohol

All of the most important points in the structure of the blastodermic vesicles of the rabbit from the fourth to the seventh day may be fairly well observed by examining the hardened vesicles in alcohol under the microscope. For such examinations the so-called live-box, such as was formerly much used by microscopists for the study of living creatures, will be found very convenient. Care must be taken to have plenty of alcohol around the specimen and not to lower the cover so much as to exert any pressure upon the vesicle. It is not difficult to place the vesicles so that any part of their surface may be examined with a No. 7 objective. In the uncolored specimen the nuclei, and even many of the boundaries of the cells, can be clearly made out.

In the following descriptions ages have been chosen at which the important characteristics can usually be observed. The variation is so great in range during early stages that the development described below for a given age is often found in older or younger specimens, and specimens of a given age may exhibit a less or a more advanced stage of the embryonic formation than is here put down for that age. In general the correspondence of the stage of development to the size of the vesicle is more exact than to its age.

Vesicles at Five Days (5 X 24 hours). — At this age the vesicles are always found in the upper portion of the uterus. Sometimes all of those in one uterus are quite close together, at other times somewhat scattered and lying singly. The vesicles are extremely variable in size, for they measure from 0.6 to 0.9 mm. They are spherical or nearly so, and are surrounded by a thin membrane, which in reality corresponds to both the zona pellucida and the outer albuminous envelope, which in the rabbit ovum during segmentation is very thick and conspicuous, but which is always extremely thin when the stage of the blastodermic vesicle is reached. Upon the outside of this really double membrane appear a certain number of small villus-like projections, which are highly refringent. They are probably identical in character with the villi which have been observed upon the ovum of the dog (pages 59, and 60), but are smaller in all of their dimensions. Immediately underneath the external membrane there is a continuous layer of cells belonging to the ectoderm and extending completely around the ovum. The layer is sometimes designated specifically as the "outer layer" or as the " subzonal layer." It also extends over the embryonic shield ; the portion upon the shield is often termed Rauber's layer, it having been first observed by that investigator. The cells of the outer layer are quite large and their boundaries are easily recognizable in surface views. Their sides may number four, five, or six, six being perhaps the more usual number, and are variously disposed, so that the cells differ in shape and size. During the next two days of development the cells become, if anything, more irregular in outline and somewhat smaller. The boundaries between the cells are very fine lines ; the nuclei are rather large and oval in form, and contain from three to four or five highly refringent granules. Each nucleus is surrounded by a denser court of protoplasm in which there are many granules, some of which are highly refringent. The peripheral portion of the cell is of a loosely reticulate structure with comparatively wide meshes between the threads of the protoplasm. Occasionally there appear in the protoplasm of these cells narrow, elongated, highly refringent bodies somewhat resembling bacilli in appearance, and therefore they are termed the bacillijorm bodies. Their nature is unknown ; they are more apt to be found in older vesicles. The outer or subzonal layer can be made out over the embryonic shield only by very careful observation. In the shield the cells are several layers thick. The inner cells are very much smaller in size than the cells of the outer layer, are more granular, and contain smaller nuclei which take up a relatively large place in the cell in proportion to its apparent area. Closer observation, utilizing the fine adjustment of the microscope, will show that there are two kinds of cells in the inner part : first, those which, like the cells of the subzonal layer, belong to the ectoderm; and, second, an inner layer of cells, which apparently belongs entirely to the entoderm. In the region of the embryonic shield the ectoderm is, therefore, made up of two distinct layers of cells. The outer or subzonal (Rauber's layer) disappears during the sixth day of development as a distinct layer. The cells of the entoderm form a very thin continuous layer on the under side of the embryonic shield. They may be recognized by the very granular, and therefore dark,* appearance of their protoplasm, and by the rounded form and small size of their nuclei. Similar cells may be observed also extending beyond the limits of the embryonic shield, though not there forming a continuous layer, except perhaps for a very short distance, but rather lying scattered about in patches or isolated. As the cuboidal cells of the ectoderm are confined to the region of the embryonic shield, the cells of the entoderm outside of the shield lie close against the subzonal layer. Here they may be more easily studied than in the shield itself. They are very much smaller than the cells of the outer layer and contain each a nucleus with highly refringent granules, which are now numerous and smaller than the somewhat similar granules in the overlying nuclei of the ectoderm. The further away we proceed from the edge of the embryonic shield, the fewer we find the entodermal cells. The extent of their distribution varies greatly, and apparently more or less in relation to the size of the blastodermic vesicle, since in the smallest vesicles of this age we find the cells only a short distance beyond the edge of the shield, yet in the largest vesicles they have expanded even past the equator.

  • As seen by transmitted light.

Vesicles at Six Days. — At this age the vesicles are found more or less scattered and isolated in position from one another through the upper half of the uterus. They measure from i .o to 1.6 mm. , their walls are very transparent, and the somewhat more opaque, round or oval embryonic shield can be readily distin guished with a hand lens. Its size varies with the diameter of the vesicle, being larger in the larger vesicles; but the proportions are not exact, for a vesicle of given diameter may have an embryonic shield of either larger or smaller dimensions than other vesicles of the same size. Hence, vesicles of different sizes may have embryonic shields of similar dimensions. The actual diameter of the shield is between 0.2 and 0.35 mm. The general structure of the vesicles is the same as at five days, but certain differences may be noted. In preserved specimens the external membrane is very apt to be wrinkled. The subzonal layer has very much the same appearance as before, though the cells are somewhat smaller and it lias almost disappeared over the region of the embryonic shield. The manner of its disappearance has not been definitely settled. There is no evidence that the cells degenerate or are cast off, hence one inclines to the hypothesis that the cells of the subzonal layer become incorporated in the inner layer of the" cuboidal ectodermal cells, for in sections shown at this stage the ectoderm is one-layered in the region of the shield. Kntodermal cells also have essentially the same appearance as at five days, but they extend considerably further around the vesicle, are more numerous, and form a more continuous layer. Sections show that the subzonal finer outside of the shield is very thin, but its outer surface is fitted to the inner surface of the zona pellucida. The center of each cell is somewhat thicker, projecting toward the interior of the vesicle. It is in this thicker projecting portion that the nucleus is situated. Along the borders of the cells the layer is of course thinner, and it is under these thinner parts that the thicker nucleated portions of the entodermal cells are lodged. Hence, in surface views.

the nuclei of the two layers are seen to alternate more or less with one another. This characteristic disposition is not kept everywhere, but is subject to considerable variations. In the very most advanced ova of six days a small spot sometimes can be observed in the embryonic shield which is noticeable from its greater opacity. This spot corresponds to Hensen's knot, but it does not usually show itself distinctly until considerably later.

Vesicles at Seven Days. — Vesicles at this age vary greatly in size, and the stage of development varies with the size — how exactly, we do not yet know. Preliminarily we may fix on the normal size as being that of vesicles, the greatest diameter of which is about 4 mm. Such vesicles are somewhat oval in shape and slightly flattened on the side bearing the embryonic shield. The membrane enclosing them is very thin; the albuminoid layer can scarcely be distinguished, but the zona pellucida is very distinct. The shield (Pig. 175, Sh) is somewhat elongated and distinctly pear-shaped. Its long axis is parallel with that of the vesicle.

It varies greatly in its dimensions. Shields 1 mm. wide, / _ 5A and from 1.3 to 1.4 mm. long, are not uncommon. The student will be likely to encounter other dimensions. The most striking addition is the appearance of a darker area, W~~m ts .

vies, at the posterior or pointed end of the shield. This darker area is also somewhat pear-shaped, but its pointed FlG 5 —blastodermic end is near the center of the shield, its rounded end a little vesicle of a Rabbit distance behind the point of the shield. The darker area at Seven Days.

owes its formation to the appearance of a new layer of -S*. Embryonic shield, mes, cells between the ectoderm and entoderm. This layer J grammatic. )

consists of loosely connected cells with rounded nuclei easily distinguishable in surface views from those of the subzonal layer. The greater part of these cells are certainly mesodermic, but a portion of them share in the formation of the primitive streak and notochordal canal, and perhaps do not belong to the mesoderm. In the region outside the embryonic shield the outer layer is easily distinguished ; its cells have marked outlines, but are of smaller dimensions than in earlier stages, their nuclei are large, for the most part oval, and contain several highly refringent and conspicuous granules. The number of granules varies ; when there are only two or three, they are apt to be elongated as if several small granules had united. The entodermal cells have spread well past the equator of the vesicle and present, for the most part, a distinctly epithelial arrangement, although at the edge of the expanding layer the cells are still more or less scattered. The entodermal cells are easily distinguished by changing the focus of the microscope, when their darker protoplasm and smaller size, together with their smaller darker nuclei, make them readily recognizable. The granules in the entodermic nuclei are smaller and more numerous than in the overlying ectodermal nuclei.

During the next few hours further changes ensue. At the apex of the pearshaped mesodermic area there appears a small spot, which is known as Hensen's knot. At first; Hensen's knot consists of a little thickening accompanied by a union of the cells of the middle layer with those of the overlying ectoderm. Next occurs the development of the primitive streak, which runs from Hensen's knot backward toward the apex and embryonic shield, and very soon thereafter along the line of the primitive streak there develops the external and shallow primitive groove. At Hensen's knot the three layers now are found to be intimately united, so that, though they may everywhere else, when fresh, be separated from one another, the germ-layers at this point cannot be separated, except by tearing. Finally, in the next stage there grows out in front of Hensen's knot the so-called head process, an axial band of cells in which the notochordal canal develops.

File:Minot1897 fig176.jpg

Fig. 176. Rahbit Embryo of Seven Days. Transverse Series 12, Section. 216, through the Anterior Portion of the Embryonic Shield.

Ec, Ectoderm. Eiit, Entoderm. >: 300 diams.

The structure of the embryonic shield at seven days is further illustrated by the two sections represented in figures 176 and 177, the former passing across the anterior portion of the shield, where it is two-layered; and the latter across the posterior portion, in which the middle layer has appeared. Figure 176 shows the middle portion of a section. It consists merely of the outer, thicker, ectodermal layer, Ec, and the very thin entodermal layer, Ent. Both surfaces of the ectoderm are quite sharply defined. The nuclei are rather large and show several large, deeply stained nucleoli in each. The outline of the nucleus is sharp, and, in addition to the larger granules, there are many smaller ones less deeply stained scattered through the nucleus. The nuclei vary considerably in size, shape, and position. The protoplasm of the ectodermal cells is lightly stained, and granular in appearance. The boundaries between adjacent cells are indicated by delicate lines, which extend through the entire thickness of the ectoderm, which is now but a single layer of cells. The original outer ( Rauber's) layer has disappeared. The entoderm is very thin, but is thickened a little where each nucleus is lodged. The nuclei are smaller than those of the ectoderm, more darkly stained, and the granules in them less coarse than those in the nuclei of the ectoderm. Between the two layers is a narrow space; whether an artefact or not is difficult to say. Figure 177 represents a transverse section through the posterior part of the embryonic shield; the position of the median plane is approximately indicated by the vertical line M. About this plane there is a considerable accumulation of cells which merges without boundary into the superficial cells of the shield. A short distance from the median line the outer layer of the shield becomes a distinct epithelium, Ec, consisting of a single layer of cells. The edge of the shield is marked by a rather abrupt transition to the thin outer layer of the extraembryonic region. On the under side of the section extends the thin entoderm as a continuous layer, which is only loosely connected with the central mass of cells overlying it near the median plane. Finally, from the median mass of cells extends laterally the sheet of mesoderm, Mes, between the outer and inner germ-layers. The mesodermie cells are somewhat loosely distributed, and have round nuclei with distinct chromatin granules and wellmarked protoplasmic bodies, which give off strands by which the cells are united to one another. The middle germ-layer is the least compact of the three.

The Maturation, Fertilization, and Segmentation of the Ovum in White Mice

These animals are selected for the practical study of the earliest stages of development for two reasons: first, because the processes have been more thoroughly studied in them than in any other mammals; and, second, because they are easily kept and breed freely, so that abundant material may be secured with comparatively little trouble. Those desiring further information are referred to Sobotta's original memoir.* Heat occurs twenty-one days after littering, a fact which may be taken advantage of to secure ova of the desired age. Coitus can take place only during heat, for it is then only that the vagina is found open; at other times its epithelium concresces to a solid mass. The spermatozoa do not penetrate into the tube until some time after the coitus. After the discharge of the semen, the contents of the large seminal vesicle are ejaculated into the vagina, completely filling it and hardening into a white plug {bouchon vaginal), as in guinea-pigs. From twenty to thirty hours later the plug softens and falls out.

The Fallopian tubes are narrow, much contorted canals. The fimbriate opening of the tube penetrates the connective tissue about the ovum so that the fimbriae lie in the periovarial space. There is ciliated epithelium in the proximal region of the tube only, none in the distal parts or in the uterus itself. During heat the periovarial space is filled with an abundant clear fluid. This also distends the proximal part of the tube, forming, as it were, a special sac, with a distended epithelial lining. At the time of coitus ovulation has generally taken place; the ovum, still surrounded by the cells of the corona radiata, is found in the fluid of the distended proximal section of the tube. It is probable that the ova are carried from the periovarial space not only by the currents created by the cilia of the fimbriate opening, but also by a sort of pumping action of the tube itself. For at the beginning of the period of heat we find that the periovarial space contains much fluid, but later, when the ova are in the tube, this space is empty and the tube contains fluid. The ovum of the mouse measures only 59 " in diameter, and is, therefore, the smallest known mammalian .ovum. It is surrounded by a very thin zona pellucida measuring about 1.2 fi, and contains only a few yolk-grains, a portion of which may be blackened by osmic acid. These ova offer the further special peculiarity that they sometimes form two and sometimes (from 80 to 90 per cent, of the cases) only a single polar globule. When two are formed, the first appears while the cell is still in the ovary, the second after it has been transferred to the Fallopian tube; when only one formed , it is developed in the ovum as it lies in the tube. When two globules are formed, the nuclear spindles of the first and second differ somewhat in appearance ; when only one is formed, it resembles that of the second globule. Hence we are led to surmise that there may have been in these cases really a first polar globule formed, but that it appeared so much earlier in the history of the cell that it escaped observation.

  • "Die Befruchtung und Furchung des Eies der Maus," " Arcliiv f. mikroskopische Anatomie," vol. pp. 15-93, Pis- II-IV (1895).

The First Polar Globule. — The first polar globule is formed, as stated, while the ovum is still in the unruptured Graafian follicle of the ovary. The nucleus moves toward one side of the ovum and is there transformed into a mitotic spindle, the axis of which is more or less nearly at right angles to the radius of the ovum (Fig. 178). The spindle itself is large, pointed at the ends, with curving achromatic threads. The chromosomes, which are probably twelve in number, gather themselves into an equatorial plate. They are elongated, pointed at the ends, with irregular sides, and are very large. No trace of the centrosome has been observed at the end of the spindle and there are no astral rays extending from the ends of the spindle into the protoplasm. The chromosomes are somewhat V-shaped. They divide by a transverse separation at the apex of the V. Chromosome halves migrate toward the end of the spindle. These stages occur probably about twenty-four hours before the rupture of the follicle. The actual extrusion of the first globule has not yet been described. It takes place before ovulation, for the first polar globule is always found while the ovum is still in the ovary. In the mouse it is remarkable, as is also the second polar globule, for its large size. It is usually oval or spherical in form, and may measure say 16 or 17 // in length by 9 ft in diameter. It has a distinct cell membrane, a protoplasm which resembles that of the ovum, and may even contain granules of yolk. Soon after its separation from the ovum its nucleus becomes well developed and membranate. Except, therefore, that the number of chromosomes which enter into its formation is half the normal number, we might say that it differs little from an ordinary cell.

The Second Polar Globule. — After the formation of the first polar globule we do not find the nucleus of the ovum entering into a condition of repose, but it at once transforms itself, as in other animals, into the second polar spindle. The form and changes through which this passes are similar to those of the spindle in the case in which only a single globule is found, to the description of which we now pass.

File:Minot1897 fig178.jpg

Fig. 178. Ovum of White Mouse, with the First Polar Spindle in Tangential Position. X 5°° diams. — (Af/er J. Sobotta.)

The Single Polar Globule. — As stated above, when only a single polar globule is formed, it appears in the ovum in the ampulla of the tube. The nucleus moves toward the surface, loses its membrane, and produces a polar spindle, which is much smaller than that previously described. It lies at right angles to the axis of the ovum and quite close to the surface. It contains twelve thick, achromatic fibers, which do not unite at the poles with one another, but end parallel, so that the tip of the spindle is blunted. The chromosomes, when the membrane first disappears, lie irregularly, but shortly after the formation of the spindle they collect together to form an equatorial plate, somewhat as in figure 178. They are irregular and of uneven size, twelve in number, or possibly the number may vary somewhat. The chromosomes then divide transversely, the halves move rapidly toward the ends of the spindle, which during this change passes into the radial position (Fig. 179). The surface of the ovum or the apex of the spindle forms a protuberance, and the spindle moves partly into this protuberance. Division of the achromatic fibers takes place, and there is formed a well-marked cell plate (Fig. 180), and presently the polar globule becomes constricted off. The cell plate appears with unusual distinctness. It is at about this stage that the spermatozoon is found to have entered the ovum (Fig. 179), and to have formed there the male pro-nucleus. During all these stages no centrosome appears at the poles of the spindle, and no astral rays appear in the protoplasm, although in many eggs these astral figures are extremely conspicuous. The nuclear elements in the ovum proper appear at first as a dense cluster of chromatin granules. These fuse apparently into a compact mass, which grows rapidly in size, presumably by the absorption of fluid from the yolk, and, as it enlarges, acquires a distinct membrane, and presently shows a network structure in its interior (Fig. 182), with irregular chromatin masses. It continues to grow more and more, and develops at the same time a series of nucleoli more or less uniform in size. This stage may be regarded as that of the completed female pro-nucleus.

Fig. 179.— Ovum of White Mouse, dividing to Produce the Polar Globule. X S°° diams. — (After J. Sobotta.) The elongated male pro-nucleus lies in the inferior protuberance of the ovum.

Fig. 1S0. — Ovum of White Mouse, Showing the Metaphase of the Division Producing the First Polar Globule. X 'S 00 diams. — \ After J. Sobottu.) ■

Fig. 181. — Ovum of White Mouse, after the Formation ok the Polar Globule. Both Pro-nuclei are Present. X 5°° diams. — {After J. Sobotta.)

Fig. 182. — Ovum of White Mouse, with Two Well-developed Pro-nuclei. X 5°° diams. — {After J. Sobotta.)

Fertilization. — As the general account of the fertilization or impregnation of the mammalian ovum is based on the process in the white mouse, it is necessary only to refer to descriptions and figures, pages 49 to 54.

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Pages where the terms "Historic" (textbooks, papers, people, recommendations) appear on this site, and sections within pages where this disclaimer appears, indicate that the content and scientific understanding are specific to the time of publication. This means that while some scientific descriptions are still accurate, the terminology and interpretation of the developmental mechanisms reflect the understanding at the time of original publication and those of the preceding periods, these terms, interpretations and recommendations may not reflect our current scientific understanding.     (More? Embryology History | Historic Embryology Papers)
A Laboratory Text-Book of Embryology: 1. General Conceptions | 2. Early Development of Mammals | 3. Human Embryo | 4. Pig Embryos | 5. Chick Embryos | 6. Blastodermic Vesicle and Ovum Segmentation | 7. Uterus and the Foetal Appendages in Man | 8. Methods | Figures | Second edition | Category:Charles Minot

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