Difference between revisions of "American Journal of Anatomy 23 (1918)"

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{{Amer. J Anat. Volumes}}
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=THE AMERICAN JOURNAL OF ANATOMY=
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Charles R. Bardeen
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University of Wisponsin
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Henry H. Donaldson
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The Wistar Institute
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Simon H. Gage
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Cornell University
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EDITORIAL BOARD G. Carl Huber
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University of Michigan
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George S. Huntington
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Columbia University
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Henry McE. Knower,
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Secretary University of Cincinnati
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Franklin P. Mall
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Johns Hopkins University
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J. Playfair McMurrich
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University of Toronto
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George A. Piersol
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University of Pennsylvania
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VOLUME 23 1918
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THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY PHILADELPHIA, PA.
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==CONTENTS==
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N0. I. JANUARY
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Raymond Pearl and Alice M. Boring. Sox studies. X. The corpus luteum in the ovary of the chicken. Six text figures and nine plates 1
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Eliot R. Clark. Studies on the growth of blood-vessels in the tail of the frog larva — by observation and experiment on the living animal. Sixteen figures 37
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J. A. Badektscher. The fate of the ultimobranchial bodies in the pig (Sus scrofa). Four plates 89
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J. DuESBERG. Chondriosomes in the testicle-cells of Fundulus. Twenty-one figures (two plates) 133
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.\dolf H. Schultz. The position of the insertion of the pectoralis major and deltoid muscles on the humerus of man. Three figures 155
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\V. B. Chapman. The effect of the heart-beat upon the development of the vascular system in the chick. Seventeen figures 175
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Edward A. Boyden. Vestigial gill filaments in chick embryos with a note on similar structures in reptiles. Three text figures and four plates 205
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No. 2. MARCH
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Alice Thing. The formation and structure of the zona pellucida in the ovarian eggs of turtles. Twelve figures 237
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Adolf H. Schultz. The fontanella metopica and its remnants in an adult skull. Five figures 259
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Franklin Paradise Johnson. The isolation, shape, size, and number of the lobules of the pig's liver. Twelve figures (two plates) 273
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Abram T. Kerr. The brachial plexus of nerves in man. The variations in its formation and branches. Twenty-nine figures 285
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Fr.\nklin p. Mall. On the age of Human Embryos. Two figures 397
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C. R. Bardeen. Determination of the size of the heart by means of the x-rays. One figure 423
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ACITHOns' ABSTRACT OF THIS PAPER ISSUED HY THB BIBLIOGRAPHIC SERVICK, DECEMBER I5
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==Sox studies. X. THE CORPUS LUTEUM IN THE OVARY OF THE DOMESTIC FOWL==
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RAYMOND PEARL AND ALICE M. BORING
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SIX TEXT FIGURES AND NINE PLATES
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I. INTRODUCTION
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The corpus luteum is one of the clearly recognized sources of an internal secretion in the mammal. Various functions have been ascribed to it. Its function in connection with secondary sex characters has been discussed by Pearl and Surface ('15), with one piece of clear cut evidence. The case was that of a cow which developed cystic ovaries and took on male secondary sex characters. The ovaries were compared histologically with those of a normal cow and the two were found to resemble each other in all respects except that the cystic ovaries had no corpora lutea. The interstitial cells were the same in both so that the difference in secondary sex characters could not be attributed to them. The implication of the facts is that the corpus luteum has an inhibitory influence in the female which prevents maleness from developing and that when no corpus luteum is formed, male characters appear.
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The chief difficulty with such a view has been that its application is very limited, as the corpus luteum has been supposed to be a structure occurring only among mammals. The secondary sex characters of birds are particularly pronounced and the results of ovariotomy experiments, such as those of Goodale, ('16) show the possibility of changing these characters experimentally. Also the many cases of hermaphrodite birds (to be
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' Papers from the Biological Laboratory of the Maine Agricultural Experiment Station, No. 115.
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1
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THE AMERICAN JOURNAL OF ANATOMY, VOL. 23, NO. 1 JANUARY, 1918
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2 RAYMOND PEARL AND ALICE M. BORING
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considered in Study XI of this series), with varying degrees of maleness and femaleness indicate the presence of some sex regulating substance in birds. Is this substance entirely different from the corpus luteum probably connected with it in mammals, or is there a corpus luteum or its homologue in birds? An investigation of this question has been undertaken in this study. We consider that we have successfully demonstrated the presence of the corpus luteum in the domestic chicken. Further discussion of the bearing of this fact on the whole question of secondary sex characters will be deferred until a later paper of this series, which will unfortunately probably be delayed for some time, as one of the authors (R. P.) has been called upon by the government to turn his attention to practical problems during the war.
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A careful examination of the ovary of a bird which has been actively laying shows three kinds of structures: the yolks of various .sizes indicating different stages of development, the discharged follicles in various stages of regression, and the atretic follicles or degenerating eggs of different sizes. These are all easy to identify when they are large enough to protrude far from the surface of the ovary, that is, when they are larger than 2 or 3 mm. in diameter. Under this size, it is impossible to distinguish the discharged follicle from the atretic. Both of them show a yellow or orange spot in the center. The question naturally arises whether these yellow spots are homologous in structure and origin with the mammalian corpus luteum. They never develop into a large mass like the corpus luteum of the mammal. They have the color of the spots on the cow ovary which indicate remains of old corpora lutea. In order to interpret these yellow spots, a study has been undertaken of the progressive and regressive changes in the cell structure of egg follicles in different conditions, undischarged, discharged and atretic.
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The material used came chiefly from four birds, an actively laying Bantam, a Barred Plymouth Rock in the same condition, an old Compine past the laying condition, and a guinea-hen with a large ovary containing several large yolks. Material
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CORPUS LUTEUM IN OVARY OF THE CHICKEN 6
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from a number of other Ijirds was used in the study of special points. These are some of the same birds used in Study IX. The ovaries were fixed in Gilson and McC'lendon. In the Barred Plymouth Rock ovary the different discharged follicles were sectioned separately and arranged in a series, according to size and consequent order of age since ovulation. After the study of this series, it was easy to judge of the condition of various follicles in pieces of the other ovaries cut at random. Various stains were tried, iron haematoxylin and Delafield's haematoxyhn for general histology and Mallory's and Mann's stains for secretion granule tests.
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II. UNDISCHARGED FOLLICLES OF THE HEN'S OVARY
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A study of the follicles of large undischarged oocytes shows them to consist of an epithelial layer, the granulosa, and two connective tissue layers, the inner and the outer theca folliculi (fig. 1). In the inner theca are located groups or nests of epithehal cells {I, figs. 1 and 2). They have been described by many authors, notably Ganfini, Sonnenbrodt and Poll, but have been called interstitial cells. Poll calls them Kornzellen at first, describes their collection into the internal theca and then implies their function by saying that the biological role of the theca interna in the formation of the corpus luteum still needs to be worked out. That he also confuses them with interstitial cells is shown by his statement that the theca interna fills up the atretic follicle with groups of Kornzellen, which is the same thing as an interstitial gland. These nests of cells in the bird are not anything like the usual glandular interstitial cells of the ovary in structure. They are about three times as large (compare fig. A and C). The nucleus is bigger and plumper, the cytoplasm is usually clear and vacuolated in appearance, only occasionally containing a few acidophile granules which stain with the fuchsin in Mallory's stain or the eosin in Mann's stain; while the real interstitial cells are crowded with granules. These large clear cells are seldom found alone, but are usually grouped into nests of various shapes, as already mentioned. The cytoplasm of these cells usually will not take
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4 RAYMOND PEARL AND ALICE M. BORING
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up an acid stain. They remain strikingly clear, when the connective tissue all around them is highly colored. So great is the contrast that they show distinctly even at low magnification in a section such as figure 1. Furthermore, they are found in
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Fig. A Part of follicle of wall of medium sized oocyte in hen ovary. (X i)50.) Compare figure 2.
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Fig. B Part of thoca interna of sixth discharged follicle in hen ovary, showing many vacuolated lutear cells. (X 9.50.) Compare figure 0.
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CORPUS LUTEUM IN OVARY OF THE CHICKEN 5
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different parts of the ovary, mostly in the thcca interna, while the interstitial cells he in the general stroma, and especially on the periphery.
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Figure 3 shows several very young oocytes from the same ovary as figure 1. In these, the follicle consists only of a single layer of epithelial or granulosa cells (g). The connective tissue layers are not yet formed. But there are nests of clear cells (I) in the stroma nearby. Presumably these are included with the connective tissue when the theca interna is formed.
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III. DISCHARGED FOLLICLES OF THE HEN'S OVARY
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In the largest follicles before ovulation, the three layers are stretched out very thin by the pressure of the large yolk within them. After o^oilation, there is a shrinkage of the follicle walls, probably due to the elasticity of the connective tissue recoiling at the sudden release of pressure from inside. On the Barred Plymouth Rock ovary, the ripe yolk measured about 40 mm. in diameter, and the last discharged follicle measured 20 mm. in length from base to tip, while the next to last was 12 mm., and the fourth in the series was 7 mm. As this shrinkage in length takes place, the walls thicken until finally a small oval mass results having no resemblance to a hollow follicle. The ruptured place through which ovulation took place, becomes gradually closed up, by the growing together of the edges, and the filling of cells into the cavity. Sometimes this mass of cells proti-udes shghtly from the cavity at the old place of rupture, thus somewhat more resembling a miniature mammalian corpus luteum. Yellow pigment forms in the puckered edges of the follicle and also in the central mass.
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The microscopic study of sections through discharged follicles of various ages shows that the increase of thickness of walls is due chiefly to a thickening of the theca interna. Figure 4 is a section of the last discharged follicle of the Barred Plymouth Rock ovary. It shows the thickened theca interna (i) and in addition the remnants of the granulosa (g). The latter seems to loosen from the follicle after ovulation, and the cells collect in masses in the cavity and degenerate.
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6 RAYMOND PEARL AND ALICE M. BORING
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The first subsequent discharged folHcle in the series to show any new microscopic features is the sixth (fig. 5), where there appears a marked increase in the number of nests of vacuolated cells in the theca interna (l). They are concentrated toward the cavity. The closeness of nests together may be partlj^ due to the shrinkage of the cavity after discharge of the egg. But as this does not seem sufficient to account entirely for the increase, the number must be added to either by di\dsion or migration. The fact that division plays some part in the process is proven by the observation of several mitotic spindles. The character of these cells shows better in greater magnification, as in figure 6 and figure B.
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The further progress of the increase of vacuolated cells in the theca interna is shown in figure 7, a section of a discharged follicle too small to have been placed in the series as to time of discharge. Here the whole internal theca looks full of holes, due to vacuolated cells (I). The central ca\dty is nearly obliterated, almost as though the edges had been pulled up by a gathering string. There are, however, a few cells in the central cavity (p). These get in there by migi-ation from the internal theca.
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Figure 13 shows the process in an atretic follicle where it is more conspicuous, but it is true to a more limited extent in the discharged follicles. The cells concerned have a speckled appearance in figiue 13 (d). They are abundant in the follicle wall, some are scattered among the yolk spheres in the central cavity and some are on the border line between the follicle wall and the cavity, indicating that the cells actually migrate into the cavity. Occasionally a very large central plug is formed which protrudes from the spot of rupture. Figure 7 shows a small plug of this kind (p).
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The cavity usually becomes finally obhterated by the thickening of the internal theca and the formation of large masses of vacuolated cells from the original nests. In figure 8, the chief tissue consists of the masses in the internal theca (i). The line between the theca interna and externa is marked by the irregular spaces and blood vessels. The connective tissue in the
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CORPUS LUTEUM IN OVARY OF THE CHICKEN 7
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center (c) shows where the edges of the internal theca have drawn together and obhterated the cavity.
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We have traced thus far the general histological changes involved in the shrinking and lilling up of the discharged follicle. We must consider next in more detail, the cytology of these particular cells involved. Figure 2 and figure A show them in their original condition from a large undischarged follicle. We have earlier in this paper pointed out their especial characteristics in distinction to the interstitial cells. By the time they are close enough together to cause the vacuolated appearance of the whole inner part of the theca interna, the nuclei are somewhat shrunken and pushed to the side of the cell, suggesting active elaboration of secretion material (fig. 6 and fig. B). By the time the closing in of the follicle has neared completion (figs. 8 and 9), the character of the cells is decidedly modified (fig. C). The cell boundaries in any one small mass of cells are indistinguishable. The cells seem to have melted together so that the outlines of the vacuoles are the evidently visible lines rather than the cell outlines. The vacuoles also are much larger than previously. The nuclei are smaller and less regular in outline, they stain darker, in fact, they look shrunken. These figures show nicely the contrast between the cells which fill up this discharged follicle and the interstitial cells. The interstitial cells lie in the connective tissue of the external theca and of the internal theca in between the masses of transformed epithelial nest cells. They are entirely unchanged from their usual appearance. They show clearly because the granules with which they are packed stain vividly with acid stains. A homologous mass of cells from an older solidly filled follicle (fig. 10) is shown in figure 11 and figure D. Here the nuclei show still further signs of degeneration and the general network of the cytoplasm contains clumps that look like secretion material. These secretion particles are yellow in color. They look amorphous in character, and they vary greatly in size (fig. 20). They can not be fatty, for they have not dissolved in the clearing oils. They cannot be of the protein nature of the secretion granules of the interstitial cells, as they retain their distinct yellow color
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8
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EAYMOND PEARL AND ALICE M. BORING
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no matter how the preparation may be stained. They make a fine contrast with iron haematoxyhn, acid fuchsin, eosin, methyl blue, and still show their own characteristic yellow even with
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^ <^-nyr>:.m^^
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Fig. C Masses of lutear cells from older discharged follicle, with interstitial cells lying in connective tissue between masses. (X 950.) Compare figure 9.
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l>!S'-»v
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^■■■y'
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^<M
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WV^'^
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Fig. D Mass of lutear cells from discharged hen follicle, with pigment particles developed in the network. (X 950.) Compare figure 11 and figure 20.
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CORPUS LUTEUM IN OVARY OF THE CHICKEN 9
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orange (1. The cell masses finally become nearly filled with this yellow material, some of it collecting in clumps several times larger than the degenerated nuclei.
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Further tests of the character of the cell contents in these cell masses were made with Sudan III. Hand sections were made of material in McClendon's fluid. Although these could not be cut very thin, they showed that the inner lining of the early discharged follicles contains fatty material. In an old follicle with central yellow mass the cells of the yellow mass take the red of the Sudan III, but the yellow amorphous particles show in the mid^t of the red. They can be squeezed out of broken cells and isolated from the red fatty background, showing they are still yellow, unaffected by the Sudan III, and therefore not of a fatty nature. The fatty substance indicated by the Sudan III reaction in both young and old folhcles is probably contained in the vacuoles so conspicuous in paraffin sections. The xylol would have dissolved out all the fat lea\dng the vacuoles in which it had been contained.
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IV. DEGENERATION OF CORPUS LUTEUM IN COW OVARY
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In order to show the significance of the yellow mass formed in the center of discharged follicles in the hen ovary, we have made a brief study of the degeneration of the corpus luteum in the cow ovary for comparison. There is an extensive literature on mammalian corpus luteum, but this deals chiefly ^vith the development and early involution. Now the bird quite evidently has no structure similar to the large corpus luteum which fills up half the ovary of a cow at its full development. The small yellow spot on the bird ovary resembles the small yellow spots on the cow ovary which mark the old remains of former corpora lutea. Ovulation in the cow alternates between the two ovaries. So by studying the two largest corpora lutea on both ovaries we can arrange a series of four involution stages. Beyond that, they all seem equally shrunken and therefore can not be arranged in a further series. Such a series of four involution stages has been studied for two cows, and in addition several older corpus luteum remains.
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10
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RAYMOND PEARL AND ALICE M. BORING
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The last formed corpus luteum is of a salmon pink color, due to a combination of the blood color and the lutein color. Sections show it composed of large plump cells with rounded nuclei, as described by Corner. These luteum cells are scattered in the midst of an areolar connective tissue groundwork (fig. 18 and fig. E). In dehydrating for embedding, the absolute alcohol and xylol become very yellow, indicating that the cells contain something soluble in these reagents. This is of course one chemical character of lutein.
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Fig. E Cells from youngest corpus luteum of cow. (X 950.) Compare figure 18.
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Fig. F Cells from older corpus luteum of cow, showing pigment developed in cells. (X 950.) Compare figure 19 and figure 21.
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The next to last corpus luteum is much reduced in size. Its color has lost the pinkish shade and it appears a solid bright yellow. This is also soluble in absolute alcohol and xylol as in the first stage. The cells and nuclei both look a little shrunken. In one cow, this second corpus luteum contained a few amorphous yellow particles like those described foi the hen.
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CORPUS LUTEUM IN OVARY OF THE CHICKEN 11
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111 the third oldest corpus luteum, the tissue is shrunken so that a mere speck shows on the surface. This is the stage resembhng the yellow spots of the hen's ovary. Dissection shows that it is reduced in all diameters. That part of this decrease in size is due to cell shrinkage is well demonstrated by comparison of figures E and F, which are drawn to the same scale. Not only the nucleus but the cell body is at least halved in size. The color now is darker, being a brick red. This is not due to blood vessels, as sections do not show any more than formerly. It is due to the development of a dark yellow pigment, the same substance which appeared in small quantity in the younger corpus luteum and in large quantity in the hen ovary. In this stage of involution it is developed in large quantities, practically fiUing up many of the lutear cells (figs. 19 and 21 and fig. F). In unstained sections it gives a yellow color to most of the section.
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In the fourth oldest corpus luteum of the two series and in the scattered older ones sectioned, the structure is similar to that in the third oldest, the yellow amorphous masses being possibly larger and more distinct.
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This yellow material certainly looks the same as that in the hen ovary. The chief structural difference is that it is all confined within cells with distinct cell walls in the cow, while in the bird, the cells forming it, lose their boundaries and the particles are formed in a vacuolated network with scattered shrunken nuclei (cf. figs. 20 and 21).
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Sudan III reacts similarly with hand sections of formalin material from both cow and hen ovary. All four stages in the cow series take the red color showing the presence of a fatty substance in the cell. This corroborates the evidence from the solvent action of absolute alcohol and xylol. But in the third and fom-th stages, yellow amorphous pigment particles can be seen glistening in the red background. The pigment is not of fatty nature in the cow, any more than it is in the hen. In fact, this substance is so similar in the two animals, that we shall from now on speak of a corpus luteum in the hen, and call the cells forming this pigment, lutear cells.
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12 RAYMOND PEARL AND ALICE M. BORING
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This development of a non-fatty pigment in the mammalian lutear cells has been already described by Mulon as occuiTing in atretic follicles. He speaks of the lipocholesterine as changing over to an indelible pigment. This same substance certainly forms in the involution of the corpus luteum of a discharged follicle as shown in this present work.
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It is of especial interest to find that Blair Bell's description of the corpus luteum in Ornithorhynchus, a primitive oviparous mammal, shows it very much like that in the hen. It often remains hollow, it never becomes very large. It consists chiefly of a thickened theca interna. Sometimes it becomes a solid fibrous mass. One of Bell's figures almost exactly resembles figure 4 of this paper. One would like to know whether the yellow pigment is found in Ornithorhynchus thus making its resemblance to the bird even more striking.
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V. BIOCHEMICAL CHARACTER OF PIGAIEx\T OF CORPUS LUTEUM
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The identity of this yellow amorphous pigment in the corpus luteum remains in the ovary of the hen and of the cow has been put to chemical tests as well as morphological; first of a microchemical nature, as already partially described, and secondly by various special chemical solvents. The work of Escher and of Palmer and Eckles on animal pigments has been consulted in selecting the reagents to use.
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The microchemical tests have been discussed in previous sections, but will be summarized here. Microscopical technique processes have shown the identical beha\'ior of the pigment in hen and cow. It does not dissolve in alcohol or oils. It will not stain with basic nuclear stains such as haematoxylin and Kresylviolet, or with acid counterstains, such as eosin, methyl blue, anihn blue, orange G, or with such a stain as iron haematoxyhn. Neither does it stain with the fat stain, Sudan III, although there may be much fatty material in the cell in which it lies. As normal secretion granules of a protein nature take acid stains and secretion granules of a fatty nature take Sudan III, this pigment is neither protein nor fat in composition.
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CORPUS LUTEUM IN OVARY OF THE CHICKEN 13
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A further test of its chemical nature was made by ti-ying some of the various solvents used by Escher and by Palmer. Sections were cut in paraffine and mounted on slides and then the paraffine removed b}^ xylol and the sections treated with different chemicals. This pigment is not the carotin described by Palmer, but we could not reach any conclusion as to its chemical nature, as nothing could be found to dissolve it. But the fact of the identity of this pigment in the hen and cow is proven beyond a doubt. Concentrated HCl, HNO3 and H2SO4 were tried and had no effect except that the H2SO4 turned the particles dark brown and made them even more distinct than before. For an alkali solvent, strong KOH was used; it turned the pigment bright orange but did not ehssolve it. In adeUtion to these various other solvents were tried after consultation with the chemistry department, petroleum ether, sulphuric ether, acetone, carbon bisulphide, and carbon tetrachloride, but none of these had the slightest solvent effect on the pigment. Acetone cleared the background and this made the particles stand out more sharply. Carbon bilsulphide was allowed to act for several hours, but the preparations still contained the pigment at the enei of that time in undiminished degree. We conclude that any two substances which can withstand the action of as many well known solvents of as many different properties as this list includes must be of very similar chemical nature. This gives us one more proof that the yellow particles in the hen ovary are the same as those in involuted mammalian corpora lute a.
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VI. CHANGES IX ATRETIC FOLLICLES IN THE HEN'S OVARY
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Among the developing yolks and discharged follicles of the hen ovary are many degenerating eggs. They can be distinguish d from developing eggs by the shrunken appearance as though the contents did not ejuite fill out the foUicle. Eggs may start to degenerate at different stages. Tbe largest one on the Barred Plymouth Rock ovary was 12 mm. in diameter. Many of them show dark spots which are masses of coagulated blood. Mostly they are smaller than this when involution begins. The
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14 RAYMOND PEARL AND ALICE M. BORING
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degree of shrinkage shows whether the involution process had recently begun or not. When these degenerating eggs are cut open, the contents is found to be in a more or less fluid state. When these atretic follicles have become reduced in size to 2 or 3 mm., it is no longer possible to distinguish them externally from the discharged follicles; the same kind of a yellow pigment appears in the center.
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Studied microscopically, the chief difference between atretic and discharged follicles is that the former have a more distinct cavity which becomes obliterated chiefly by migration of lutear cells into it instead of by shrinkage of the walls. The granulosa is shed similarly. There must frequently be hemorrhage as corpuscles are often found ia the cavity. The varying quantity of yolk spheres is one indication of the degree of involution, also the number of lutear cells in the cavity. Figure 12 is an atretic follicle with considerable yolk still unabsorbed. A few lutear cells have filled in to the cavity (fig. 13, 1). It is particularly clear here that the cells inside of the inner mar^n of the theca interna are the same in structure as those of epithelial nature in the interna theca. This is just as Benthin describes it for the atretic mammalian follicles. Figures 14 and
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15 show a later stage where the yolk is almost all absorbed and the ca\dty is filled with lutear cells.
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Not until the cavity is filled with lutear cells does the yellow pigment already described in discharged follicles, make its appearance. It forms in the lutear cells of atretic follicles in a similar way to that in the discharged follicle. The cell boundaries are possibly not obliterated so completely, so that the morphological resemblance to the cow corpus luteum remains is even more striking than in the case of the discharged follicles. Figure 16 is part of an atretic follicle where the cells are filled with pigment. The amorphous character of this matedal shows in figure 17 a part of figure 16 under higher magnification.
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It is of interest to notice that the lutear cells in the hen in both discharged and atretic follicles originate entirely from the theca interna. In mammals the origin of the lutear cells is a mooted question. Some authors, as Niskoubina, hold that they
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CORPUS LUTEUM IN OVARY OF THE CHICKEN 15
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 +
have a double origin, from granulosa and theca interna, while others such as B(>nthin and Hegar, claim that they all come from the theca interna. This point is perfectly clear in birds due to the ease with which one can distinguish these peculiar cells in the internal theca of undischarged follicles and follow them to the thickened mass in the center of the discharged follicles, and see them migrating out into the cavity of the atretic follicles.
 +
 
 +
The formation of a corpus luteum in atretic as well as discharged follicles makes it possible to identify ovarian tissue in ovaries too abnormal to have ovulated any eggs. Most of the literature of the mammalian ovary considers the involution of the atretic follicle as something distinct from that of the discharged follicle. The mass forming in the atretic follicle is called the corpus atreticum or fibrosum in contradistinction to the corpus luteum. However, Hegar says that it is hard to tell one from the other. They are practically identical in the hen,
 +
 
 +
VII. SUMMARY
 +
 
 +
We are now in a position to sum up the points proving the homology of the corpus luteum in the hen and in the cow. There has been much discussion about the origin of the corpus luteum in mammals. In the hen there is no question but that the origin is simply from the theca interna.
 +
 
 +
The course of development in the hen corpus luteum is an abbreviation or fore-shortening of that in the cow. It corresponds directly to the late involution stages of the cow corpus luteum. They both contain a yellow fatty substance, as shown by the Sudan III, absolute alcohol and xylol reactions. There develops in both a yellow amorphous pigment in the cells containing the fatty substance. This pigment is similar chemically in that it will not stain with basic or acid stains; also in that it will not dissolve in any of the usual solvents, acid alkali or oil.
 +
 
 +
In the hen, a corpus luteum forms m both discharged and atretic follicles. «
 +
 
 +
 
 +
 
 +
16 RAYMOND PEARL AND ALICE M. BORING
 +
 
 +
VIII. LITERATURE CITED
 +
 
 +
Bell, W. Blaik 1917 The sex complex. Wood and Company, New York. Bknthin, W. 1910 Ueber Follikelatresie in kindlichen Ovarien. Arch. f.
 +
 
 +
Gyniikologie, Bd. 91, p. 2.
 +
 
 +
1911 Ueber Follikelatresie in Siiugetier Ovarien. Arch. f. Gynakol ogie, Bd. 94, p. 599. BouiNT ET Ancel 1912 Sur la nature lipoidienne, d'une substance active se cretee par le corps jaune des mammifere. C. R., T. 151, p. 1391. Corner, G. W. 1915 Corpus luteum of pregnancy as it is in swine. Carnegie
 +
 
 +
Inst. Washington, 222, p. 69. DuBAissoN, H. 1906 Contribution a I'etude du vitellus. Arch, de Zool. Exp.
 +
 
 +
et Gen. T. 4. Series 5, p. 153. EscHER, H. H. 1913 Ueber den Farbstoff des Corpus Luteum. Zeitschr.
 +
 
 +
Physiol. Chem., Bd. 83, p. 198. Fraenkel, L. 1910 Neue Experimente zur Function des Corpus Luteum.
 +
 
 +
Arch. f. Gynakologie, T. 91, p. 705. Ganfini, C. 1908 Sulla strutturo e sviluppo delle cellule interstiziali dell'
 +
 
 +
ovajo. Arch, di Anat. e di Emb., S. 7, p. 373. GooDALE, H. D. 1916 Gonadectomjr in relation to the secondary sex characters of some domestic birds. Carnegie Inst. Washington, 243. Hegar, K. 1910 Studien zur Histogenese des Corpus luteum und seiner Riick bildungsproducte. Arch. f. Gynakologie, Bd. 91, p. 530. Henneguy, L. F. 1894 Recherches sur I'atresie des foUicules de Graaf chez
 +
 
 +
les mammiferes et quelques autres vertebres. Jour, de I'Anat. et
 +
 
 +
Phys., T. 30, p. 1. Miller, J. W. 1910 Die Riickbildung des Corpus luteum. Arch. f. Gynakologie, Bd. 91, p. 263. MuLON AND Jong 1913 Corps jaunes atresiques de la femme. Leur pigmentation. C. R. Soc. Biol., T. 74. NiSKOUBiNA 1909 Recherches sur la morphologie et la fonction du corps
 +
 
 +
jaune de la grossesse. Dissert, de la facultc de med. de Nancy. Palmer, L. S. and Eckles, C. H. 1914 Carotin. The principal natural yellow
 +
 
 +
pigment of milk fat. I, II, III, IV. Research Bui., nos. 9, 10, 11, 12,
 +
 
 +
Univ. of Mo., Agr. Exp. Stat. Pearl, R. and Surface, F. M. -1915 Sex Studies. VII. On the assumption
 +
 
 +
of male secondary characters by a cow with cystic degeneration of
 +
 
 +
the ovaries. Ann. Rept. Me. Agr. Expt. Stat., 1915, p. 65. Poll, H. 1911 Mischlingstudien VI: Eierstock und Ei bei fruchtbaren u.
 +
 
 +
unfruchtbaren Mischlingcn. Arch. f. Mikr. Anat., Bd. 78, II, p. 63. Sonnenbrodt 1908 Die Wachstuuisperiode der Oocyte des Huhnes. Arch.
 +
 
 +
f. Mikr. Anat., Bd. 72, p. 415.
 +
 
 +
 
 +
 
 +
PLATES
 +
 
 +
 
 +
 
 +
17
 +
 
 +
THE AMERICAN JOURNAL OF ANATOMY, VOL. 23, NO. 1
 +
 
 +
 
 +
 
 +
DESCRIPTION OF PLATES
 +
 
 +
We wish to take this occasion to acknowledge our indebtedness to Mr. Royden Hammond for all the photomicrographs, and to Mrs. Maud DeWitt Pearl for the paintings on plate 9.
 +
 
 +
PLATE 1
 +
 
 +
EXPLANATION OF FIGURES
 +
 
 +
1 Medium sized oocyte in hen ovary (X 40), showing three layers to the follicle, the granulosa {g), theca interna ({), and theca externa (e), with nests of lutear cells in the theca interna {I).
 +
 
 +
2 Part of follicle wall in figure 1 at greater magnification (X 352). Labels the same as in figure 1.
 +
 
 +
 
 +
 
 +
18
 +
 
 +
 
 +
 
 +
CORPUS LUTRUM TM OVARY OF THE CHICKEN
 +
 
 +
liAVMOND PEAIili AN'I) AI.UK M. IIORINO
 +
 
 +
 
 +
 
 +
PLATE 1
 +
 
 +
 
 +
 
 +
 
 +
^'
 +
 
 +
 
 +
 
 +
 
 +
l'.»
 +
 
 +
 
 +
 
 +
PLATE 2
 +
 
 +
 
 +
 
 +
EXPLANATION OF FIGURES
 +
 
 +
 
 +
 
 +
3 Young oocytes in hen ovary (X 352), with follicles consisting of a single layer of granulosa (g). Nests of lutear cells in the stroma nearby (l).
 +
 
 +
4 Portion of last discharged follicle in hen with thickened theca interna(0 and granulosa (g) being sloughed off into the cavity. (X 40.)
 +
 
 +
 
 +
 
 +
20
 +
 
 +
 
 +
 
 +
CORPUS LUTEUM IN OVARY OF THE CHICKEN
 +
 
 +
nAYMOND PKARL AND ALICE M. BOHING
 +
 
 +
 
 +
 
 +
PLATE 2
 +
 
 +
 
 +
 
 +
 
 +
 
 +
!
 +
 
 +
 
 +
 
 +
 
 +
!^H
 +
 
 +
 
 +
" ^t
 +
 
 +
 
 +
 
 +
A^
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
<i
 +
 
 +
 
 +
V " '
 +
 
 +
 
 +
y^
 +
 
 +
 
 +
 
 +
 
 +
 
 +
QKFW
 +
 
 +
 
 +
 
 +
 
 +
'^'^' ^^^^~'^"
 +
 
 +
 
 +
 
 +
^^.
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
^v^^'
 +
 
 +
 
 +
 
 +
<^»B^-,;:\^%.^
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
>i*- :*v.->75 .17
 +
 
 +
 
 +
 
 +
21
 +
 
 +
 
 +
 
 +
PLATE 3
 +
 
 +
 
 +
 
 +
EXPLANATION OF FIGURES
 +
 
 +
 
 +
 
 +
5 Portion of sixth from last discharged follicle, showing large number of lutear cells (Z) in the theca interna (X 40).
 +
 
 +
6 Part of figure 5 enlarged (X 176).
 +
 
 +
7 Small discharged follicle with cavity nearly obliterated. Small plug of cells (p), filling in the cavity. Theca interna filled with masses of lutear cells H). (X 40.)
 +
 
 +
 
 +
 
 +
22
 +
 
 +
 
 +
 
 +
CORPUS LUTEUM IN OVARY OF Till'; CHICKEN
 +
 
 +
KAYMOND PEAHL AND AMCH M. liOIUXG
 +
 
 +
 
 +
 
 +
PLATE 3
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
23
 +
 
 +
 
 +
 
 +
PLATE 4
 +
 
 +
 
 +
 
 +
EXPLANATION OF FIGURES
 +
 
 +
 
 +
 
 +
8 Discharged follicle with cavity completely obliterated. The chief component is masses of lutear cells (l). The connective tissue center represents original location of cavity (c). X 40.
 +
 
 +
9 Part of figure 8 at greater magnification (X 176), showing interstitial cells (i.e.) in connective tissue between lutear masses.
 +
 
 +
 
 +
 
 +
24
 +
 
 +
 
 +
 
 +
CORPUS LUTEUM IN OVARY OF THE CHICKEN
 +
 
 +
RAYMOND PEARL AND ALICE M. UOIUNO
 +
 
 +
 
 +
 
 +
PLATE 4
 +
 
 +
 
 +
 
 +
 
 +
 
 +
s
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
"«3i>i&;
 +
 
 +
 
 +
 
 +
 
 +
 
 +
t
 +
 
 +
 
 +
-'■AfA
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
t
 +
 
 +
 
 +
 
 +
9
 +
 
 +
 
 +
 
 +
 
 +
M
 +
 
 +
 
 +
 
 +
PLATE 5
 +
 
 +
EXPLANATION OF FIGURES
 +
 
 +
10 Later stage of solid discharged follicle, showing large development of yellow pigment in lutear masses (X 40).
 +
 
 +
11 Part of figure 10 at greater magnification (X 176), showing pigment particles.
 +
 
 +
12 Atretic follicles in hen ovary, with yolk spheres in central cavity (X -10).
 +
 
 +
13 Part of figure 12 at greater magnification (X 176). Lutear cells (/) show in theca interna and also among yolk spheres in the cavity.
 +
 
 +
 
 +
 
 +
26
 +
 
 +
 
 +
 
 +
CORPUS LUTEUM IN OVARY OF TIIi: CIHC^KEX
 +
 
 +
RAYMOND PEARL AND ALICK M. IIOUIVC
 +
 
 +
 
 +
 
 +
PI, ATI; 5
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
-^-^fis. ^•' '■^/:t;i^.^^l^'<^SP^
 +
 
 +
 
 +
 
 +
L>-,>:
 +
 
 +
 
 +
 
 +
 
 +
27
 +
 
 +
 
 +
 
 +
PLATE 6
 +
 
 +
EXPLAXATIOX OF FIGURES
 +
 
 +
14 Later stage of atretic follicle (X 40). Only a few yolk spheres remain in cavity. Cavity is practically filled with lutear cells.
 +
 
 +
15 Part of figure 14 at greater magnification (X 176), showing lutear cells in theca interna {i), as well as the central cavity.
 +
 
 +
 
 +
 
 +
28
 +
 
 +
 
 +
 
 +
CORPUS LUTEUM IN OVARY OF THE CHICKEN
 +
 
 +
RAYMOND PEAUL AND ALICE M. DOUINfi
 +
 
 +
 
 +
 
 +
PLATE (i
 +
 
 +
 
 +
 
 +
 
 +
29
 +
 
 +
 
 +
 
 +
PLATE 7
 +
 
 +
EXPLANATION OF FKiURES
 +
 
 +
16 Atretic follicle in which the pigment particles have developed in the lutear cells (X 40).
 +
 
 +
17 Part of figure l(i (X 176).
 +
 
 +
 
 +
 
 +
31)
 +
 
 +
 
 +
 
 +
CORPUS LUTKL'.M IN 0\AI{V OF Till'; ClirCKKN
 +
 
 +
KAVMOMl TKAHL AND AI.ICIO M. HOUINC
 +
 
 +
 
 +
 
 +
PLATE 7
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
Orrf
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
31
 +
 
 +
 
 +
 
 +
PLATE 8
 +
 
 +
EXPLANATION OF FIGURES
 +
 
 +
18 Section of youngest corpus luteum of cow (X 176).
 +
 
 +
19 Section of older corpus luteum of cow (X 176), showing cells filled with pigment particles.
 +
 
 +
 
 +
 
 +
32
 +
 
 +
 
 +
 
 +
CORPUS LUTEUM IN OVARY OF THE CHICKEN
 +
 
 +
RAYMOND PEAUL AND ALICE M. BORING
 +
 
 +
 
 +
 
 +
PLATE 8
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
i9kJt.MMLl .MM
 +
 
 +
 
 +
 
 +
 
 +
33
 +
 
 +
 
 +
 
 +
PLATE 9
 +
 
 +
EXPLAXATIOX OF FIGURES
 +
 
 +
20 Section of discharged follicle of hen ovary, stained in Alallory's stain. Connective tissue = blue. Corpuscle = red. Interstitial cells = purple. Lutear pigment = yellow.
 +
 
 +
21 Section of older corpus luteum of cow, stained in Mallory's stain. Tissues colored as in figure 20.
 +
 
 +
 
 +
 
 +
34
 +
 
 +
 
 +
 
 +
CORPUS LUTEUM IN OVARY OF TIIK CllICKKN
 +
 
 +
RAYMOND PEAUI, AND AI.ICK M. BOUINC
 +
 
 +
 
 +
 
 +
PLATE 9
 +
 
 +
 
 +
 
 +
.***^% '
 +
 
 +
5?.^
 +
 
 +
.>••-'
 +
 
 +
 
 +
 
 +
»
 +
 
 +
 
 +
 
 +
••1.
 +
 
 +
 
 +
 
 +
%J>
 +
 
 +
 
 +
 
 +
20
 +
 
 +
 
 +
 
 +
^^*
 +
 
 +
.«>'^^
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
^\^
 +
 
 +
 
 +
 
 +
^
 +
 
 +
 
 +
 
 +
>3i
 +
 
 +
 
 +
 
 +
<^
 +
 
 +
 
 +
 
 +
35
 +
 
 +
 
 +
 
 +
AITIIOH s AMSlUAtT OK THIS PAPEI! ISSUED UV THE lUHI.HXIHAPHIC SEBVICE, DECEMBER 29
 +
 
 +
 
 +
 
 +
STUDIES ON THE GROWTH OF BLOOD-VESSELS IN THE TAIL OF THE FROG LARVA— BY OBSERVATION AND EXPERIMENT ON THE LIVING ANIMAL
 +
 
 +
ELIOT R. CLARK
 +
 
 +
■' Department of Anatomy, University of Missouri
 +
 
 +
SIXTEEN FIGURES
 +
 
 +
These studies were begun and part of them were made in the laboratory and under the inspiration of my beloved teacher and master, the late Professor Franklin P. Mall, and it is with a sense of the deepest gratitude and reverence that I acknowledge the immeasurable debt which I owe to him.
 +
 
 +
INTRODUCTION
 +
 
 +
The development of the vascular system falls broadly into two stages: (1) the stage of primary differentiation, or histogenesis, and (2) the stage of extension and elaboration of arteries, veins, and capillaries. The exact^ manner and place, in which the primary differentiation occurs is an unsettled problem, and is, at the present time, the subject of spirited controversy. It has not been satisfactorily decided whether blood-vessel endothelium differentiates from entoderm, or mesoderm — and if from mesoderm, whether from mesenchyme generally or from the mesothelial lining of the coelom. Nor has it been determined whether this primary differentiation occurs on the walls of the yolk sac alone, or in the embryo proper, or whether it may take place both on the yolk sac and in the embryo. Another unsettled point is the extent of time over which the primary differentiation takes place. Recent discussions and observations supporting one or another of these views may be found in the following: Minot ('12), Evans ('12), Ruckert and Mollier ('06), Schulte ('14), Bremer ('14), Stockard ('15 A),Reagan ('17), Sabin ('17).
 +
 
 +
The second stage of vascular development includes the further
 +
 
 +
37
 +
 
 +
THE AMERICAN JOURNAL OF ANATOMY, VOL. 23, NO 1 ,
 +
 
 +
 
 +
 
 +
38 ELIOT R. CLARK
 +
 
 +
extension and development of the system after the primarydifferentiation has taken place and after the circulation has been established, and it is with this second stage that the studies here reported are concerned. In this stage, which continues throughout life, the vascular endothelium spreads through the growing organism, arteries and veins develop, until the extensive and complicated vascular system of the adult is perfected. It is principally characterized by the formation of new vessels by the sending out of sprouts from the vessels already present, instead of by the transformation of mesenchyme, of other undifferentiated cells, as in the first stage, and by the action on the vessels of the mechanical and chemical factors concerned with the circulation of blood and interchange of substances through the wall.
 +
 
 +
In spite of the abundant evidence in favor of this mode of spreading of the vascular endothelium, after its primary differentiation, there ■ are observers who adhere to the view that at any time throughout life, mesenchyme (or other undifferentiated ceUs) may be transformed into vascular endothelium. This view is held by Maxinow, Weidenreich, and Mollier (cf. discussion in Schulte, '14,) who believe that, not only may reticulum cells and leucocytes be transformed into blood-vessel endothelium, but that the reverse transformation may take place — in brief, that vascular endotheUum is not a specific tissue, but is interchangeable with the other tissues mentioned. The evidence for this view has in no case been conclusive. It is clear, however, that the manifestation of the property of sprouting does not form a sharp boundary line in time of development between stages, for apparently sprouting commences before the differentiation of endothelium is everywhere complete (cf. Stockard '15, B and Sabin '17). It is probable that the period of overlapping is very short.
 +
 
 +
It is also clear, particularly from the studies of Miss Sabin ('17), that the development of arteries and veins takes place to some extent before the circulation is established. In chick embryos she found that, before circulation starts, part of the aortae, the two vitelline veins next the heart, parts of the cardinal veins and the duct of Cuvier are clearly present as definite vessels.
 +
 
 +
 
 +
 
 +
GROWTH OF BLOOD-VESSELS IN FROG LARVAE 39
 +
 
 +
That there is a secondary stage in the development of the blood-vascular endothelium, in which the endothelium spreads by sprouting, instead of by the transformation of indifferent cells, has been proven by direct observation. In the transparent fin expansion of the tail of the tad-pole, this process has been watched during life by several observers, especially Golubew ('69), J. Arnold ('71) and Rouget ('73), who have seen blood capillaries send out sprouts, which extended until they met and anastomosed with other sprouts or capillaries and into which a lumen advanced — all without the interposition of outside cells. This view is supported also, among others, by J. Meyer ('53), Bobritzky ('85), His ('69), KoUiker ('86), R. Thoma ('93), Marchand ('01), Ziegler ('05) and Evans ('09 A). While this mode of growth has not been proven by direct observation for all vessels in all animals, and while the existence of other modes of extension is perhaps not necessarily excluded, it is a fair hypothesis that this is the universal mode of spreading of the vascular endothelium, once it has differentiated, and cannot be abandoned until more convincing objections are brought than have been produced up to the present time.
 +
 
 +
It is not the primary purpose of the present study to enter either into the problem as to the time, in embryonic development, at which the second stage begins, nor the problem whether growth by sprouting is the universal mode of spreading during this period. It is rather to consider the problem as to how, in a region where, and at a time in development when growth by sprouting has been repeatedly verified, and after the circulation has become established, the capillaries are transformed into arteries and veins; to study the modes of action and reaction of endothelium — the laws which regulate its growth.
 +
 
 +
Such a study is by no means new, for it has been, through many years of fruitful investigations, the object of W. Roux and particularly of R. Thoma and numerous coworkers to discover the factors which regulate the growth of vessels, while many others, including Nothnagel ('84), Mall ('03), Evans ('09, A and B, '12) have studied the same problem less extensively.
 +
 
 +
 
 +
 
 +
40 ELIOT R. CLARK
 +
 
 +
The initial stimulus to this study was given by W. Roux ('79), in his Inaugural Dissertation, in which he studied the '"angle of branching" in relation to the relative size of the branch, and the shape of vessels in the neighborhood of a branch. He found that this angle, which lies between a line continuing the axis of the main stem and the axis of the branch, varies with the relative size of the branch — that, in general, the larger (relatively) the branch, the smaller the angle, and the smaller the branch, the larger the angle. He also found that the lumen of an artery shows a widening with subsequent narrowing immediately after branching, and that the opening of the branch is oval rather than round. By experiments with openings made in vessels and in tubes and with the use of malleable substances such as lard placed in such openings and on the interior of tubes, he found that the direction taken and the shape found is, in the case of the artery, practically the same as the shape and direction of the stream of fluid emerging from openings in vessels and tubes. He concluded that the shape and direction of arteries at the place of branching are determined by the action of hemodynamical factors; that the blood-vessel wall responds by taking the shape which allows a minimum of fric-. tion. The general and important conclusion was that the size and shape of arteries and veins, in the growing and adult animal, are regulated, not by heredity, but by the action of mechanical factors.
 +
 
 +
Thoma's conclusions were based mainly on studies made on the extra-embryonic yolk sac vessels of chick embryos. From a series of injections he found that there is formed, first, an indifferent plexus of capillaries, interposed between the aorta and the venous end of the heart, and that out of this plexus, those vessels which are so placed as to have the greatest amount of blood flowing through them enlarge to become arteries and veins, while others remain capillaries, or atrophy.
 +
 
 +
The results of these and other studies by Thoma ('11) may be briefly summarized. He finds that blood-vessels are regulated in their growth by mechanical factors, which he expresses in the form of 'laws' ('Histomechaniche Principien'), as follows:
 +
 
 +
 
 +
 
 +
GROWTH OF BLOOD-VESSELS IN FROG LARVAE 41
 +
 
 +
1. Das Wachstuin ties qucrcn Durchinessers, also des Gefasslichtung ist abliiingig von dcr Geschwindigkeit dcs Blutstromes. Dasselbe l)egmnt, sowie die Stronigeschwindigkcit der nahe an der Gefiisswand stromenden Blutschichten einen Schwellenwert iiberschreitet, den ich mit U bezeichnen will, und i.st innerhalb gewisser Grenzen ein um so raschcres, je so mehr die Stronigeschwindigkeit liber den Schwellenwert U, hinaus zunininit. Dagegcn tritt ein negatives Wachstum, eine Abnanie des Gefiissumfanges ein, wenn die Geschwindigkeit der nahe an der Gefiisswand stromenden Blutschichten kleiner wird als der Schwellenwert v.
 +
 
 +
2. Das Liingenwachstum der Gefiisswand ist abhiingig von den Zugwirkungen der das Gefass umgebenden Gewebe und zwar sowohl von denjenigen Zugwirkungen welche das Langenwachstum der umgebenden Gewebe erzeugt als von denjenigen Zugwirkungen, welche bei "Anderungen der Gelenkstellungen eintreten," etc.
 +
 
 +
3. "Wird das Wachstum der Wanddicke bestimmt durch die Spannung der Gefiisswand." This is determmed by the l)lood pressure and the size of the vessel.
 +
 
 +
4. (proposed as an hypothesis, not yet proven). Die Umbildung von Kapillaren ist abhiingig von dem in den Kapillaren herrschenden Blutdruck und stellt such an denjenigen Stellen der Kapillarbezirke ein, an welchen der zwischen dem Kapillarinhalte und der Gewebsfliissigkeit bestehende Druckunterschied einen gewissen Schwellenwert p iiberschreitet. Dieser Schwellenwert ist jedoch in den verschiedenen Kapillarbezirken je nach den Eigenschaften der die Kapillaren umgebenden Gewebe verschieden gross.
 +
 
 +
Expressed more simply they are:
 +
 
 +
1. Increase or decrease in the size of a vessel is regulated by the rate of the blood flow. 2. Increase or decrease in the length of a vessel is governed by the tension exerted on the vessel wall in a longitudinal direction by tissues and organs outside of the vessel.
 +
 
 +
3. Increase or decrease in the thickness of the vessel w^all is dependent upon the blood pressure.
 +
 
 +
4. New^ formation of capillaries depends upon increase of pressure in the capillary area (proposed as an " hypothesis — not yet proven).
 +
 
 +
The ultimate controlling factor Thoma considers to lie in the metabohsm of the organs ('93, pp. 49-51). It is this which regulates, primarily, the increase or decrease in capillaries, which, in turn, sets in motion the mechanism which results in the increase or decrease in the size of arteries and veins, the increase in strength of heart beat, etc.
 +
 
 +
 
 +
 
 +
42 ELIOT P.. CLARK
 +
 
 +
Roux, in his later writings, discusses, mainly in a theoretical way, the factors involved in the increase in size of vessels, and the new formation of capillaries. His views as to the new formation of capillaries, expressed briefly in 1895, repeated more fully in 1910, and again repeated, in a controversial article in 1911, are perhaps most completely expressed in 1910, p. 88, where he says:
 +
 
 +
1st der Verbrauch in dem Parenchym, welches cine Kapillare umgibt, eiiiige Zeit dauernd derartig gesteigert, dass aiis den vorstehend erorterten Griinden mehr Stoff als normal liindurchtritt, so wird wohl die an der Stelle stiirksten Durchtritts gelegene Wandungszelle diirch die verstarkte Leistimg in der Richtung des Austritts zur Sprossimg angeregt. Dasselbe geschieht natlirlich auch an der denselben grosseren Parenchj'mtheil von der andern Seite der umschliessenden und ernahrenden Kapillare. Diese noch nicht als Kapillarenfungierenden sprossen treffen, Avohl diii'ch chemotropisch vermittelten Cjd^otropism, aiifeinander, also in ahnlicher Weise wie ich es an von mir isolirten Furchungszellen sah, einerlei ob diese Zellen noch freilagen oder schon wieder an etwas anderem (an der Zellen oder am Boden des Gefasses) hafteten. Der vererbte gestaltende Reaktionsmechanismus der Kapillarwand, der zum Hohlwerden und zur weiteren Ausbildung der neuen Kapillaren mit Bildung von Nerven und kontraktilen Elementen fiihrt, wird auf diese Weise aktiviert und so eine neue funktionsfabing KapiUare gebildet.
 +
 
 +
Like Thoma, Roux considers the metabolism of the tissue the primarj^ factor in new growth of capillaries. As for the specific stimulus, however, he disagrees. According to Thoma, increased metabolism causes increase in blood pressure in the capillary area, to which the endothelium is thought to respond by sending out sprouts, while Roux' view is that the new sprout is sent out as a direct response on the part of the endothelial cell to the passage through it of an increased amount of substances. In criticism of Thoma's hypothesis, Roux ('11, p. 201) calls attention to the absence of any noticeable new formation of capillaries in tricuspid or mitral insufficiency, in which conditions there is a rise in blood-pressure in the capillaries.
 +
 
 +
Thoma's first histomechanical law that the size of the vessel is regulated by the rate of blood flow, is criticized by Roux chiefly because he can see no way in which the moving stream can affect the wall, since, as first shown by Helmholtz, there is
 +
 
 +
 
 +
 
 +
GROWTH OF BLOOD-VESSELS IN FROG LARVAE 43
 +
 
 +
a thin layer of fluid next the wall which is immovable. His explanation for growth in size of vessels is that it is brought about through the agency of the vasomotor nervous mechanism; that, following increased metabolism and formation of new capillaries, there is a reflex widening of the arteries and possibly also of the veins of the affected region. This widening, if continued long enough, results in a permanent adaptation of the vessel wall to the increased volume of blood by growth processes.
 +
 
 +
Roux apparently agrees with Thoma's law as to the increase in thickness of the vessel wall.
 +
 
 +
Mall ('00), in an extensive review and discussion of Thoma's histomechanical laws, finds support for Thoma's first law, in his studies on the growth of glands. Like Roux, however, he disagrees with Thoma in his hypothesis that the formation of capillaries is dependent on increase in blood-pressure in the capillary area. In reality," he says (p. 250), we can only state definitely that with the new formation of tissue new blood-vessels may grow into it, for all new tissue does not have bloodvessels." The precise stimulus for the formation of capillaries is unknown. Again (p. 251), he says, "The first and guiding blood-vessel is the capillary, which grows in all directions, forming a plexus. Secondary changes made arteries and veins of them and their laws of growth have been discovered and clearly stated by Thoma."
 +
 
 +
It has been shown by a series of investigators — among them — Erick Miiller ('03, '04), Rabl ('07), Bremer ('12) and H. Smith ('09), and particularly Evans ('09, A and B) that many of the larger arteries and veins in the body of the developing embryo are first formed as capillaries, which grow as irregular plexuses, and out of which certain ones are differentiated to form arteries and veins. Evans, who has made the most extensive studies in this field, has described the caudal portion of the aorta, the chief veins, the pulmonary, subclavian and sciatic arteries as developing in this manner. He concludes that the histomechancal laws of Thoma are the factors which govern the process.
 +
 
 +
A number of investigators have suggested that new capillaries are formed as the result of the action of specific 'chemiotactic'
 +
 
 +
 
 +
 
 +
44 ELIOT R. CLARK
 +
 
 +
(better 'hemangiotactic') substances outside the capillaries. According to Marchand ('01, p. 148), Leber ('88) first suggested this explanation, to which Marchand is slightly inclined. It was suggested again by J. Loeb ('93) as an explanation for the growth of vessels in fish embryos whose heart action was eliminated by the action of chemical substances. Evans makes a similar suggestion. In each case it has been proposed merely as a tentative hypothesis and has not been tested.
 +
 
 +
Over against this group of investigators whose studies have gone to show that blood-vessels are regulated in their growth by the action of mechanical and chemical factors, and some of whom have attempted to define this regulation in terms of specific laws of growth, there are others who have supported the view that mechanical factors play little if any part in determining the formation of arteries and veins, and who attribute it rather to the action of hereditary influences. Possibly the strongest adherent of this view is Hochstetter, who has made so many important studies on the comparative anatomy of the vascular system. His view is probably most concisely presented by his pupil, Elze ('12) in an article criticizing the conclusions of Evans and Thoma. In brief, it is that the primitive form of the" vascular system is not a capillary plexus, but a single artery and vein, such as is formed in the limbs and digits of amphibians, and also in the segmental arteries ; while capillary plexuses are secondary formations.
 +
 
 +
Now it is interesting that support for this view has come in part from the two men who have been most prominent in advocating the regulating action of mechanical factors, namely, Thoma and Roux. Thoma ('93, p. 28) mentions that the aorta is developed as a definite vessel before the heart commences to beat, while Roux emphasizes a first stage in the development of the vascular system, as of other systems, in which differentiation and growth take place as a result of heredity (preformation) — a stage which includes the formation of ' the anlage of the typically laid down chief vascular stems' ('95, pp. 326-7, footnote). Roux bases this conclusion on chance observations made on the area vasculosa of chick embryos, in which the embryo failed to
 +
 
 +
 
 +
 
 +
GROWTH OF BLOOD-VESSELS IN FROG LARVAE 45
 +
 
 +
develop, but in which vessels, including the border vein and some others differentiated in situations corresponding with the normal. That growth of capillaries and larger vessels in embryos is regulated not entirely by the metabolism of the tissues, but in part at least by hereditary influences, is shown, he believes, by the richness of the capillary plexus in the lung and the relatively great size oJf the pulmonary arteries and veins, which, ' according to Wiener,' are, before birth, four to six times as large as the weight of the lung tissue justifies, in comparison with other organs. Wiener studied the proportion between size of artery and weight of organ.
 +
 
 +
Support is lent to this view by the results of studies made on embryos whose heart-beat has been eliminated experimentally either by mechanical removal or by chemical inhibition. Dareste (77) J. Loeb ('93), Patterson ('09), Knower ('07) and Stockard ('15 A) agree in finding certain typical arteries and veins formed in such embryos, in which the mechanical action of the circulation has been eliminated — in fish, frog and chick embryos.
 +
 
 +
The indications are that the truth lies between the two extreme views; that what we are forced to call hereditary factors do play a part, not only in the primary differentiation of bloodvascular endothelium and its capacity for gro^vth by sprouting, but in the formation of some of the main vessels in the embryo (how great a part and how long exerted in embryonic life, has not yet been cleared up, cf. Miss Sabin, '17, previously referred to) that, on the other hand, the vascular system does become, at an early stage, dependent, at least to a very great extent, upon the regulative action of mechanical and chemical forces.
 +
 
 +
Were it found that arteries and veins in latter stages are completely regulated as regards diameter, length, thickness of wall, and position by the action of mechanical and chemical factors, it would be quite compatible with our knowledge of the development of other tissues and organs, to find that a crude pattern of such mechanically controlled structures should reappear in the embryo (Thoma, '93, p. 28).
 +
 
 +
As to the precise nature of the mechanical and chemical factors which regulate the growth of the vascular endothelium,
 +
 
 +
 
 +
 
 +
'46 ELIOT R. CLARK
 +
 
 +
there is, as the foregoing review and discussion shows, difference of opinion sufficient to justify further observation and experiment.
 +
 
 +
METHODS USED IN PRESENT STUDIES
 +
 
 +
Since most of the studies referred to were made on successive stages, usually of injected embryos, in fixed preparations, it seemed that it would be worth while to study the changes in the same vessels of the same living embryo, following certain vessels through the critical stages in their development, keeping records of the circulatory conditions, and of all changes in the size of the vessels, and the direction of the angle of branching, et cetera. For such a study the transparent fin expansion of the tail of frog larvae is admirably adapted, for a larva can, by the use of chloretone as an anesthetic, be kept under observation over a period of weeks, and careful camera lucida records made as frequently as desired. Since the chloretone interferes but little with the heart beat, records can also be kept of the circulatory conditions in each of the vessels which is being watched. (For details of the method used see E. R. Clark ('12).) In the most extensive series of studies made on a single tad-pole, the observations were started when the larva (rana sylvatica) first became transparent enough to enable the course of the vessels in the dorsal fin to be made out, and records were made at daily intervals, at first, when new formation of vessels was most rapid, later, when changes were slower, at considerably longer intervals. During the observations the larva increased in length from 10.5 mm. to 29 mm. There was thus procured a record giving the vascular changes, with notes as to the condition of circulation in each vessel for a considerable section of the fin, from a stage at which the entire system consisted of a few capillary loops, to a stage in which a fairly complicated system of arterioles, capillaries and venules had developed. In addition to this series of studies, numerous shorter studies were made, on larvae of r. sylvatica, r. palustris and r. catesbiana. Brief reference has been made in an earlier paper (E. R. Clark ('09) ) to the bloodvessel changes in the tail of the frog larva, and some of the matter
 +
 
 +
 
 +
 
 +
GROWTH OF BLOOD-VESSELS IN FROG LARVAE 47
 +
 
 +
included in the present study was presented at the meeting of the Am. Ass. of Anat. in 1914 (E. R. Clark ('15), ) where drawings were shown.
 +
 
 +
DESCRIPTION or FINDINGS
 +
 
 +
When the blood-vessels in the dorsal fin of r. sylvatica larvae first become clearly visible, owing to the absorption of some of the yolk and pigment present in young larvae, they form a system of capillary loops, making an irregular meshwork of rather wide vessels, all connected with one another. On the arterial side they are connected with the main caudal artery, and on the venous side with the main caudal vein, which are located ventral to the notocord, and between the two layers of myotomes. The vessels reach the dorsal fin by passing dorsally between the notocord and spinal cord in the center and the layers of myotomes on either side. With the low power of the microscope their course may be easily follow^ed from the main caudal vessels to their emergence ^ from between the myotomes. With the higher power this is more difficult, and in most of the studies made, only the vessels in the dorsal fin proper, after their emergence from between the myotomes, are drawn.
 +
 
 +
While in many of the studies all the vessels iu the dorsal fin have been followed, a small area is selected for closer study, and for reproduction, because any section illustrates the fundamental principles involved in blood-vessel development. In the series which is reproduced an area was chosen which included an arteriole and a venule and the region between the two, as well as a part of the regions on either side. This area is sufficiently large to make it possible to follow the changes introduced by the development of new capillaries on the vessels already present.
 +
 
 +
The changes which occur in such a selected area are shown in figures 1 to 8, and will now be taken up in detail and analyzed.
 +
 
 +
There is present in the first record a very simple type of circulation. An arteriole, or, perhaps better, an arterial capillary is seen toward the left. Two branches are given off from this vessel on the right, and two on the left, through which blood corpuscles are circulating. In addition there is a third branch
 +
 
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 +
48
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ELIOT E. CLARK
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APR^ 15
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Figs. 1 to 8 Camera lucida drawings of blood-vessels in a section of the dorsal fin expansion of the tail of a rana sylvatica larva, on April 15, 16, 17, 18, 20, May 12, 20 and 31. Length of larva April 15, 10.5 mm., May 31, 29 mm. Arrows indicate direction of circulation. Relative rate of circulation indicated as FAST, MOD., moderate, SLOW, NO C, no circulation. Corresponding vessels are numbered. Vessels present in one drawing which have been retracted in the next are cross-hatched. The positions formerly occupied by vessels which have retracted are indicated by dotted lines. In figure 8, the vessels which were present in figure 1 are stippled, enl. (1: 50).
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CxROWTH OF BLOOD-VESSELS IN FROG LARVAE
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49
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on the left, with a continuous hunon, but without circulation, and a fourth which ends blindly. The arteriole ends with a bend to the right, from which a long thread extends to another non-circulating vessel. Following the two branches to the right, it is seen that the first pursues a winding course, while the second passes fairly directly to the main venule or venous capillary, near the right. Between the two branches are three communicattng vessels, the last of which forms a non-circulating loop. Below the venule there is a rather elaborate plexus of vessels
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APR 17
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APR 18
 +
 
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50 ELIOT R. CLARK
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 +
containing, for the most part, lumina, but without circulation. Extending peripherally from them are several blind-ending projections. As regards the rate of circulation, through branch 6 it is relatively 'fast,' while through branch 10 it is 'slow.' To summarize the condition of the vessels for the small area selected, there is a simple irregular plexus of capillaries with wide and rather irregular lumen, some of them with and some without circulation. For convenience, the principal afferent and effefent capillaries have been called arteriole, or arterial capillary, and venule, or venous capillary, though they are of capillary size and appearance.
 +
 
 +
In figure 2 (a day later) a 'slow' circulation has started in a numlDer of vessels which had been non-circulating on the previous day, and several new sprouts and connections between sprouts have formed. In figure 3 this has continued, and has been accompanied by an increase in the rate of circulation in some of the vessels. On the other hand, there are some vessels in which the circulation has diminished, or ceased altogether. In figures 4 and 5 the same processes have continued — a slight formation of new vessels, with modification of the rates of circulation in many of the vessels, an increase in some and a decrease in others, in addition a new change has become marked, namely, the disappearance of certain capillaries, in which the circulation had ceased, or in which the circulation had never started.
 +
 
 +
Owing to the fact that the prolonged use of chloretone had caused a slowing of growth processes, the larva was allowed to develop in fresh water, with observations at less frequent intervals, in order to see the fate of the vessels being watched, after a considerable amount of growth had taken place. A record was made on April 22, which showed very little change from the record of April 20. The succeeding records were made May 12, May 20 and May 31. "While these later records are not close enough together to show all the growth changes, they show very well the new capillary areas which have developed, their relation to the vessels already present, and the changes which the earlier formed vessels have undergone, in consequence. Thus it will be
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GROWTH OF BLOOD-VESSELS IN FROG LARVAE
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51
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seen that in figure (j and 7 the no n- vascular zone toward the margin of the fin has become much reduced in .extent by the formation of new vessels, until only a very narrow non-vascular zone is left. It will also be noted that there has been a general expansion of the tail, so that the meshes between the vessels are not
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A ^ R 2,
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52 ELIOT R. CLARK
 +
 
 +
iceably larger, and the vessels longer. The last record shows this enlargement of the tail most markedly. ' The tail has increased not only in length and height, but also in thickness, and with the enlargement there has been a very great development of new capillaries, in the widened spaces of the blood-vessel meshwork. In the half of the fin next the muscle, where the growth in thickness has been most pronounced, many of the new capillaries are in new planes, more superficial than the earlier formed vessels. As regards the fate of the vessels present in the earlier stages, it is seen that there has been a marked differentiation. In figure 1 the vessels present are nearly all of a uniform diameter. In each successive record there is a progressive differentiation, in which certain capillaries increase in size, others remain of the same, or slightly diminished caliber, while others disappear. In the last stage this differentiation is seen at its maximum; definite arterioles and venules have formed, which supply and drain considerable capillary areas. In this elaborated system there are present many of the same vessels and parts of vessels which w^ere present in the first stage recorded. Some have been incorporated as parts of the larger vessels, others are still capillaries, while others have disappeared.
 +
 
 +
Considered as a whole, then, this series shows strikingly that arterioles and venules develop, at least in this region in tadpoles, not by a steady outgrowth of a single vessel, which grows straight ahead into a new region, giving off branches where they are needed, and fulfilling its predetermined destiny to grow in a particular place, but rather by the sending out of numerous capillaries, in various directions, which anastomose, adding new loops of circulating capillaries to those already present. Of these new loops some are so placed that a circulation is never established through them, and they disappear; others are incorporated as parts of arteriole or venule or remain as capillaries. The effect of the addition of new capillaries on the system already present depends upon the relation' which the older parts bear to the new; thus a vessel which is at one stage the chief vessel of the region may entirely disappear, while another vessel, w^hich is small, and has a slow circulation at one stage, may later be a
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GROWTH or BLOOD-VESSELS IN FROG LARVAE
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53
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part of the main arteriole or venule of the region. It is impossible to predict at one stage, which way a capillary will go — whether it will increase in size, remain the same, or atrophy and disappear; it all depends upon the relation which it bears to the other vessels in existence at the time, and to those which are developed later. The endothelium is equipotential, then, and its differentiation into arteries, veins and capillaries is determined by factors outside the endothelial wall or in the lumen.
 +
 
 +
Further evidence for this view is found in the following experiments on chick embryos. They were performed to test another point, but the results are sufficiently interesting in their bearing on the problem of blood-vessel growth to deserve brief mention.
 +
 
 +
The anterior cardinal view of one side, from a point anterior to the otic vesicle to and including a part of the duct of Cuvier, was dissected out from chicks of two and one-half to three days incubation. The method employed is as follows : Berlin Blue is injected into the vein through a very fine glass cannula. As
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MAY 20-2!
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THE AMERICAN JOURNAL OF ANATOMY, VOL. 23, NO. 1
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54 ELIOT R. CLARK
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soon as the Berlin Blue mixes with the blood it forms a precipitate which plugs the vessel and sticks to the endothehal wall, outlining the position of the vein. Using this as a guide, the vessel is dissected out, with considerable of the surrounding tissue, to make certain that all is removed. The egg is then sealed and the chick allowed to develop further.
 +
 
 +
This experiment was performed successfully six times and in every case, there was found to be a large vein in the place of the one removed. In one case, the vein on the operated side was larger than the one on the unoperated side. The chicks were examined four to eight days after the operation.
 +
 
 +
The conclusion seems justified that the secondary development of a large vein in the neck, in the place of the one removed, indicates that the mechanical conditions of the circulation favor the growth of a large vein in this region. Surely the new vein can hardly be considered as due to inheritance.
 +
 
 +
What are the laws which govern the growth of the endothelium, making the differentiation of such an elaborate system possible? What, in other words are the modes of reaction of bloodvascular endothelium?
 +
 
 +
THE FORMATION OF NEW CAPILLARIES
 +
 
 +
The first property to be noted is the capacity of the endothelium to send out sprouts. This process has been frequently observed in the transparent tails of living frog larvae, and verified by other studies, and is the generally accepted mode of spreading of the vascular system, after its primary differentiation. The sprout consists of an elevation of the endothehum which is sent out, usually starting at right angles to the vessel wall, and with a lumen continuous with the lumen of the parent vessel. The end of the sprout consists of a solid process of varying length, which may be in the form of a single thread, or of a thread with one or more branches. This process usually extends in a straight line from the parent vessel, for a varying distance, and may then curve. Sooner or later it reaches a similar sprout, or approaches a fully formed capillary, when it shows itself possessed of a prop
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GROWTH OF BLOOD-VESSELS IN FROG LARVAE
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55
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erty most important for the development of a system of anastomosing vessels, namely, that blood-vascular endothelium has an affinity for blood-vascular endothelium, of such a nature that if two processes of blood-vascular endothelium draw near one another in their growth, a union will be formed between them ('cytotropism,' Roux). Equally important is the fact, readily observable in the tail of the frog larva, that blood-vessel endothelium avoids, in its growth, the cells of other tissues among which it grows, such as mesenchyme, and lymphatic endothelium. As a rule, the lumen eventually extends through the entire extent of this new sprout, it widens, and after a varying amount of time
 +
 
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56 ELIOT R. CLARK
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the circulation of blood cells commences, and a new circulating capillary has been added to the system. This whole process may, however, not be completed, for some sprouts grow out a short distance and are retracted, while some in which the lumen has been formed, never have a circulation, but retrogress — becoming solid, and disappearing. Throughout this process the endothelium remains complete, the lumen being separated from the tissue fluid outside by a complete investment of endothelium.
 +
 
 +
The facts concerning the morphological changes which take place in the formation of sprouts are clear enough; the question then arises as to why sprouts are sent out, to what sort of stimulus the endothelium responds when it sends out a sprout. The answer to this question is not entirely clear, yet certain facts together with certain general considerations justify the proposal of an hypothesis.
 +
 
 +
A study of the positions at which sprouts are formed and of the general direction taken in their growth shows that they are preceded in their formation by the growth of the other tissues and that they extend into regions where the amount of tissue not yet vascularized is greatest in amount. In the tad-pole's tail, at early stages, vessels develop first along the muscle — the thickest part of the tail. Later they grow out into the fin expansions, which attain a considerable size before vessels reach them. Growth of new capillaries continues in a general direction toward the dorsal and ventral margins of the fin, until eventually the plexus reaches nearly to the margin. During the growth of this first set of vessels, the fin remains thin, and the capillaries — save for the thickest part next the muscle — are all in a single plane. Later, the fin becomes much thicker and there occurs a corresponding new growth of capillaries, from the older parts of the plexus, which pass toward the epidermis, and form plexuses in two new planes.
 +
 
 +
In both cases it is clear that the growth of new blood-capillaries has been secondary to the growth of the outside tissue. It has been suggested by Thoma as an hypothesis that the stimulus responsible for sprout formation lies in an increase in bloodpressure. If this wxre so, one would expect to find them growing
 +
 
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GROWTH OF BLOOD-VESSELS IN FROG LARVAE 57
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 +
out from the arterial rather than the venous end of the capillary, since, obviously, the pressure is higher in the arterial end. This, however, is not the case — at least, in the tad-pole's tail new sprouts grow out as frequentl}^ from the venous as from the arterial ends. Loeb ('93) suggests that the explanation of the new growth of blood-capillaries must l^e sought in the stimulus exerted by specific chemical substances outside the capillary. A similar suggestion is made by Evans ('09 B, note, p. 296), who says in discussing vascular and non- vascular areas in embryos : ^Ve have to do here, perhaps, with a matter of cell chemistry or tropisins, for endo.thelium apparently avoids certain areas in the embryo — the non-vascular areas." In discussing this paper (see also '12, p. 584), Thoma ('11) argues that the findings of Evans fit in wdth his hypothesis, explaining nonvascular areas in the embryo as areas in which the pressure is high, due to the compactness of the tissues in such areas. As a result of this, according to Thoma, the difference in pressure betw^een the blood inside the capillary and the fluid outside is less in such areas than in looser tissues where he supposes the pressure to be lower. There appears, however, to be a valid objection to the suggestion of Loeb and of Evans, which is found in the variations in the richness of the capillary plexus in the different organs and tissues of the adult. This is summarized as follows in Kolliker's Gewebelehre ('02, p. 670) :
 +
 
 +
Bestimmend ftir die Anordnung der Kapillaren ist die physiologische Leistung, und ergiebt sich als allgemeines Gesetz, dass, je grosser die Thatigkeit eines Organes, beziehe sie sich nun auf Bewegung oder Empfindung, auf Ausscheidung oder Aufsaugung, vor allem in den Lungen, der Schilddriise, der Leber, den Nieren, dann in den Hiiiiten und den Schleimhaiiten, viel weiter in den Organen, die nur behufs ihrer Ernahrung imd zu keinen anderen Zwecken Blut erhalten, wie*in den Muskeln, Nerven, Sinnesorganen, serosen Hauten, Sehnen imd Knochen.
 +
 
 +
Thus we find that richness of capillary plexus may occur where the chief factor concerned is the passage of substances through the w^all of the blood-capillary from the lumen outward, as in the kidney ; again where the absorption- of substances is apparently the chief factor, as in the intestine; and again, where removal
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58
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ELIOT R. CLARK
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and absorption are approximately equal, as in the liver. It seems almost inconceivable that the great richness of capillaries in each of these cases can be due to the presence of an unusual amount of tropic substances.
 +
 
 +
The only common factor that can be discovered would seem to be the total amount of passage of substances through the endothelial wall, whether the direction be to or from the lumen of the capillary. That it is the quantity of substances passing through, and not some specific chemical body is strongly indicated by the fact that, in different organs, the substances which pass through the capillary wall are, in many cases, of a widely different nature.
 +
 
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 +
 
 +
 
 +
 
 +
Fig. 9 Diagram to represent the modification in amount of tissue supplied by a capillary as the result of the addition of a new capillary. The dotted area indicates the area supplied by the portion of capillary DBF; the lined area that supplied by the new capillary DEF.
 +
 
 +
 
 +
 
 +
This, then, would seem to me to be the most likely hypothesis as to the nature of the stimulus which is chiefly responsible for the formation of new sprouts — that it is the total quantity of passage of substances through the endothelial wall. This conclusion is in agreement with that of Roux ('95) which has previously been fully quoted. According to this hypothesis, when the amount of fluid passing through any part of the endothelial wall exceeds a certain point, the endothelium reacts by sending out a sprout, which eventually becomes a new capillary, thereby increasing the endothelial surface and diminishing the relative amount of interchange through any part of the wall. Figure 9 has been constructed as a diagram to show how such a law, re
 +
 
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GROWTH OF BLOOD-VESSELS IN FROG LARVAE 59
 +
 
 +
duced to its simplest terms, would operate. The square (fig. 9 A) represents, diagrammatically, the amount of tissue supplied by the capillary ABC. The large area, supplied by the portion DBF, shown by stippling, causes the amount of interchange through the wall near D and F to become excessive, and new sprouts are sent out which form a new capillary, DEF (fig. 9 B). The relatively diminished area supplied by this new capillary is shown by cross hatching, while the greatly diminished areS, left for DBF is shown by stippling.
 +
 
 +
Let us see how this hypothesis accords with the observations of the present study. When capillaries first enter the fin expansion there is an extensive, growing non-vascular area, for the fin attains a considerable development before the blood-capillaries reach it. The formation of new sprouts at this stage is extremely rapid, a rapidity which may easily be accounted for by the excessive amount of interchange involved in the relatively enormous non-vascular area. The new formation takes place in one plane — the sprouts being sent out toward the dorsal and ventral margins of the fin, in the direction to and from which there is clearly the greatest amount of interchange, for the fin^ at this stage, is quite thin. New sprout formation continues, in this plane, until the plexus of capillaries reaches nearly to the fin borders. As the borders are approached, sprout formation diminishes in rapidity, and this may be explained by the relatively smaller amount of tissue beyond the furthermost capillaries. Later, the tissue through the fin increases in all dimensions — in thickness, as well as in length and height, the capillaries increase in length, and a secondary formation of new sprouts takes place in the interstices of the old plexus. Many of the capillaries of this secondary set are in new planes, nearer the surface, especially in the thicker portions of the fin, near the muscle. It is significant that, in this secondary formation, new capillaries may grow out at places in the wall where vessels have been present but have been retracted, at an earlier stage. This secondary formation is best explained by the increase in exchange of substances due to the increase in amount of tissue.
 +
 
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60 ELIOT R. CLARK
 +
 
 +
It would seem difficult to explain the formation of new sprouts — especially the exact location at which they are sent out from the older capillaries — on the basis of the action of specific chemical substances. Such substances, if present, should act equally on all parts of the endothelium, resulting in streams of sprouts, sent out by each endothelial cell affected. We find, however, that excessive sprout formation occurs only in early stages, when the atnount of tissue entirely non-vascular is very great. Later, when the tissue has received its primary supply, and when new sprouts are clearly associated with the general enlargement of the organs, the formation of new sprouts occurs in a much more orderly fashion, a single new sprout here, another there, a condition which seems much better explained by the hypothesis that the new formation is due to increase in interchange of substances beyond a certain point, than by supposing the presence of specific chemical substances.
 +
 
 +
On more general grounds, also, the proposed hypothesis seems the most plausible. It has been brought out especially by Roux that the growth, maintenance and atrophy of tissues is to a considerable extent regulated by the extent of their performance of certain functions in the body as a whole. Increased or diminished function results in increased or diminished growth. The endothelium of blood capillaries functions as a membrane through which substances pass to and fro between the lumen and the fluid outside. It would be in harmony with Roux' general conclusions, if it were found that the new growth, maintenance and atrophy of capillaries is regulated by the intensity of this passage of substances through their endothelium.
 +
 
 +
To be sure, it is impossible to go with certainty beyond the conclusions of Mall that, with the new formation of tissue new blood-vessels may grow into it"— that it is "the growth of the tissue which leads the way," and that into this new-formed tissue the capillaries grow." Nevertheless the proposed hypothesis as to the precise formative stimulus seems to the author to be more in accordance with the facts than the other hypotheses which have been suggested.
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GROWTH OF BLOOD-VESSELS IN FROG LARVAE 61
 +
 
 +
INCREASE IN SIZE OF CAPILLARIES TO FORM ARTERIOLES AND
 +
 
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VENULES
 +
 
 +
It is obvious, in looking over the series of changes which take place in the capillaries, that, while some remain capillaries or are retracted, others gradually increase in size to form arterioles and venules. A study of the position of those which increase in size, shows that the increase takes place in the capillaries or parts of capillaries which are so placed that they form vessels for the suppty or drainage of larger and larger capillary areas, so that their endothelium is subjected to the action of the passage of an increasing amount of blood. It would seem, then, that the conclusion is justified that increase in the size of the lumen of a capillary is regulated by the amount of blood flow. This is somewhat similar to the conclusion of Thoma, expressed in his first histomechanical law, that the size of the lumen depends upon the rate of the blood flow, at a minute distance from the wall. There is, however, this difference, that according to Thoma the rate of flow is the determining factor, while the present studies indicate that it is the total amount rather than the rate.
 +
 
 +
The capillaries in early stages — that is, during their early extension into the fin — are markedly wider than a few days later. At the same time the rate of flow in all vessels, at the earlier stages, is decidedly less than later — so that, coincidently with the increase in rate, there is, at this stage, a general diminution in the size of the lumen of all vessels. Moreover, in later stages, new capillaries are, for a time, relatively wide, with a slow circulation, and become narrower as the rate of circulation through them increases. Again, many instances may be seen, in any growing tad-pole's tail, of vessels remaining the same size or even diminishing in size until the lumen is obliterated, through which the rate of blood flow is relatively rapid, but, because of frequent complete stoppage of the flow, with the total amount very small, while, side by side with them, new capillaries, with a decidedly slower circulation, though they may diminish slightly in size, are not obliterated.
 +
 
 +
Another fact which must be referred to here is the well-known one that veins, in general, have larger diameters than arteries,
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62 ELIOT R. CLARK
 +
 
 +
and yet the rate of circulation in veins is less than in arteries, while the amount of blood flowing through the two sets of vessels is the same.
 +
 
 +
It is clear, then, that the size of the lumen is not solely dependent upon either the rate or the amount of blood-flow. The chief difference in the condition existing in arteries as compared with veins, aside from the rate of blood flow, is the difference in blood-pressure. It would, therefore, seem 'that, with the same amount of blood to be propelled, the size of the lumen varies inversely as the pressure, providing the resistance is such that, with the greater pressure, there is a higher rate of blood-flow.
 +
 
 +
The diminution in caliber of blood-capillaries, after the early stage, is probably to be explained in this way. At the early stage the strength of the heart-beat is relatively small, as is shown by the slow rate of the circulation. Later the heart-beat evidently becomes stronger, for the rate of circulation increases markedly in all vessels. With this increase in rate there is diminution in caliber of all vessels.
 +
 
 +
If, however, we consider the changes which take place in a number of vessels which are subjected to approximately the same pressure conditions and rate of circulation, we find that the lumen varies with the amount of blood flowing through. Given a sufficient amount of blood to fill all vessels, and a sufficient strength of heart-beat to keep the capillary circulation up to the necessary standard, and it is found that the size of the vessel varies with the amount of blood flowing through. Thus, of two capillaries near one another, the one so placed that it forms a pathway for the supply or drainage of an enlarged capillary area has an increased circulation and increases in caliber, while the one not so situated remains the same size or becomes smaller.
 +
 
 +
The objection might be raised that the movement of the blood is not a formative factor — that the vessel merely fits the stream, and that its size is solely the result of the mechanical distention. That this is not valid is shown by the fact that vessels in which the circulation ceases altogether, or in which the circulation never starts, grow smaller and smaller until their lumen is entirely
 +
 
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GROWTH OF BLOOD-VESSELS IN FROG LARVAE 63
 +
 
 +
obliterated. (This will be taken up more fully later.) Thoma has made similar observations on ligated vessels — finding that, in spite of the mechanical distention due to blood-pressure, the lumen of ligated vessels becomes reduced to zero between the point where the last branch is given off and the point of ligature. We are, then, in agreement with Thoma, on this point, that the endothelium responds to the action of a moving fluid. To us, however, it appears that Thoma' s claim that it is the rate of flow is not justified. Rather, it appears that it is the amount of blood flow through a vessel which determines the size of its lumen. To be sure the factor of rate of flow, as well as the closely related factor of blood pressure, cannot be disregarded. To a certain extent, however, their action appears to be the opposite of that claimed by Thoma, for with increased rate there may be a diminution in size.
 +
 
 +
THE REGRESSION OF VESSELS
 +
 
 +
It has already been mentioned that in the growth of blood vessels many capillaries and parts of capillaries disappear. This process was referred to briefly in a former paper (2), '09 and will now be examined more in detail. On the series of records shown, there are many instances of the disappearance of vessels, in some cases of vessels which had never attained a circulation, in others of vessels which had had an active circulation.
 +
 
 +
First let us see what are the morphological changes which take place in the 'disappearance' of a vessel. The process has been watched carefully many times, and two sets of records are reproduced to show the details (figs. 10 and 11). The first change to be noted is a narrowing of the lumen. Next there appears an interruption of the lumen by the formation of a solid portion. The solid part may start near the middle of the capillary or nearer one end, and it gradually increases in extent toward the two ends. As it increases, the solid portion becomes narrower and narrower, until a varying amount of the former capillary is represented by only a fine thread. This thread becomes thinner until it is barely visible, and eventually disappears, leaving the two ends forming blind-ending projections from the
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64
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ELIOT R. CLARK
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vessels to which the former capillary was connected. These remains of the capillary shorten by the retraction of the endothelium into the vessels with which they are connected, until they form onl}^ a slight swelling on the surface. This eventually disappears, and there is nothing left to mark the site of the former capillary. In figure 11 may be seen the movement back into the connecting vessel of a nucleus and a small pigment granule, from the capillary which is undergoing retrogression.
 +
 
 +
In this process of retrogression of blood-vessels as it is seen in the living animal, there is nothing that even remotely suggests the
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Maijl^ / /' Mai^l5 |/^ Max^II j 1^
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Maui/l
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Mdi|Z.
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11
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Fig. 10 Several stages in the narrowing and retracting of a vessel in the tad-pole's tail (rana palustris). NO C, no circulation.
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 +
transformation of an endothelial cell into a mesenchjnme cell, or of any other type of cell. The impression is gained that the entire protoplasm is withdrawn into the parts of the system which persist, and that none of it is lost. Certainly some of it is withdrawn, as for example, in the case of the nucleus mentioned above. If any part fails to be withdrawn, it must be that it is dissolved, for the last that can be seen is a thread so minute that it is barely visible with high power lenses.
 +
 
 +
In the tail of the frog larva parts of blood-vessels rarely become completely isolated from the rest of the system, as has been de
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GROWTH OF BLOOD-VESSELS IN FROG LARVAE 65
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>^-^}=
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vV_JL
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Fig. 11. Stages in the retraction of a capillar}- in the tail fin of a rana pipiens larva.
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66 ELIOT R. CLARK
 +
 
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scribed for vessels elsewhere. In order to observe the fate of such a capillary I completely isolated a small section of a capillary by cutting through its connections. Much to my surprise, this isolated capillary was found, two day.s later, to have formed an anastomosis with one of the circulating capillaries (figs. 12). Thus a capillary which has become isolated, does not lose its blood-vascular-endothelial properties, and may be reincorporated in the vascular system. It is perhaps worth noting that this vessel was in a region, near the margin of the fin, where active new formation of vessels was taking place.
 +
 
 +
What, then, are the factors which lead to the regression of capillaries? In keeping a record of the developing vessels, a record was also kept of the presence or absence of circulation in each capillary, and of the approximate rate of the circulation. On looking over the previous records of the circulatory conditions of any vessel which has undergone retrogression it is found that this process is preceded by a period in which the circulation has ceased. This period is usually preceded in turn, by a period in which the circulation has diminished in quantity. This latter, of course, applies only to the vessels in which a circulation has been established, for, as has been said, some vessels are withdrawn before any circulation has been established in them. Thus many definite records have been obtained in which the retrogression of a capillary has been associated with the stoppage or absence of circulation. The conclusion, therefore, seems justified that a vessel which is connected with the rest of the circulating system of vessels, retrogresses and disappears if the circulation within it ceases for a sufficient length of time.
 +
 
 +
The finding that the diminution in size of lumen which precedes the retraction of capillaries involves the property, on the part of the endothelium, of reacting to the amount of blood flowing through the vessel, agrees, in part, with Thoma's first histomechanical law. According to this law, however, it is the rate ('Geschwindigkeit') of blood flow which is the determining factor ('93, p. 37 ff). In these studies it appears that, in capillaries, at least, it is not the rate but the amount of blood flow which is
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GROWTH OF BLOOD-VESSELS IN FROG LARVAE
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67
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12 a
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12 b
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Fig. 12 Drawing to illustrate fate of an isolated portion of blood-vessel. The portion A was cut through at the two points labeled X in drawing a. Two days later (b) this isolated portion had formed a connection with the rest of the blood-vascular system at B.
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68 ELIOT R. CLARK
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important. For while in some capillaries there is diminution in rate before retraction, others become smaller and have the lumen reduced to zero through which the rate never has become slower. The only common factor between these two is the total amount of blood flowing through. In the second type this is shown by the fact that though the flow is rapid, it is scanty and intermittent. The question naturally arises why the circulation becomes slower and even ceases altogether in certain capillaries, in a region where an active new growth of capillaries is taking place. In the case of many of the capillaries, the answer is pretty clear. With the continuous formation of new vessels, the pressure conditions in the vessels already present are changed, and a capillary which formerly may have been the only vessel between vein and artery, may later form merely a cross connection between two parallel vessels, in which the pressure is equal. With an equal pressure in the two ends of the capillary, the flow of blood through the capillary ceases. The majority of the cases are evidently of this character, as may be seen by looking over the records (cf. branches 12 and 16, figs. 1 to 5). There are, however, other vessels to which this does not seem to apply, particularly branches of the larger arterioles (cf. branch 3, figs. 1 to 6). A considerable percentage of the branches of each vessel which differentiates into an arteriole disappear. In the case of these capillaries the retraction is preceded by a period during which there is no flow through the vessel, but the period of absence of circulation is not preceded by a slowing of the circulation. Instead of a slowing there is a diminution in the amount of blood passing through, while the rate remains rapid. Periods of total absence of circulation alternate with periods in which a few cells pass through at a rapid rate. In this condition the capillary branch affected leaves the arteriole at a right angle, and is relatively small as compared with the arteriole. Frequently the entrance to the branch becomes plugged by an erythrocyte or a leucocyte, which causes a stoppage of the circulation. The retrogression of such vessels takes place usually much later than in the case of the other retrogressing vessels, such a vessel often remaining for several days, with a gradual increase in the
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(iltOWTH OF BLOOD-VESSELS IN FROG LARVAE 69
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length of the periods of no circulation before retrogression takes ])lacc. I have no entirely satisfactory explanation to offer to account for the stoppage of cinuilation in these branches. It is possible that the increasing thickness and elasticity of the arteriole causes a constriction a])()ut the opening of the branch, until it becomes so small that a blood cell cannot pass through. Another possil^ility which has suggested itself is that the narrowing is due to a suction on the branch caused by the rapid passage of fluitl past an opening into a branch going off at a right angle. A\'hate\er the explanation, however, the important fact remains that the retrogression of these branches is associated with a diminution in the total amount of blood which passes through them.
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There remain vessels in which the blood-flow diminishes and the lumen decreases, with resultant retraction, for which none of the factors thus far suggested seems to offer a satisfactory explanation. It is .quite a striking fact that, in most new capillaries the blood flow is at first slow — usually slower than in older capillaries — that the circulation through the newer capillaries increases while that through some of the older ones diminishes. It would seem that here, as in the case of the formation of new sprouts, a regulating factor must be looked for in the rate of interchange of substances through the wall. A glance at the diagram (fig. 9) will illustrate the significance of this suggestion. While the portion of the capillary DBF, in figure 9 A, is so placed that it forms the medium of interchange for the large stippled area; in figure 9 B, the new capillary DEF, which has developed peripherally, has taken over the greater portion of this area, leaving only the small stippled area for capillary DBF. It seems logical to suppose that, if the area . supplied by such a capillary is sufficiently reduced so that the amount of interchange through the wall falls below a certain point, the capillary wdll diminish in caliber and eventually retract.
 +
 
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The capillary is concerned chiefly with this interchange of substance, and it is difficult to escape the conclusion that the growth processes of endothelium are regulated by it. Were they not, it seems impossible to conceive of how an organ becomes
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THE AMERICAN JODRXAL OF ANATOMY. VOL. 23, NO. 1
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70 ELIOT R. CLARK
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sufficiently vascularized and how supernumerary capillaries are disposed of — in fact, how any equilibrium is established between the extent of metabolism of the various organs and the richness of their blood capillary supply.
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The precise nature of the stimulus may well be conceived as a physical one — the frictioii produced by the passing through the endothelium of fluid substances, just as the other regulating factor — the blood-flow over the interior of the endothelium — is a physical character. With a • certain (undetermined) amount of interchange, the endothelial cell remains unchanged; with increased interchange beyond a certain (undetermined) point, the endothelial cell sends out a sprout; with diminished interchange below a certain (undetermined) point the endothelial cells constrict the lumen and are eventually withdrawn into the active capillaries, leaving no trace of the capillary which had fallen into disuse. It is probable, if this hypothesis is correct, that the retraction of capillaries through which circulation has ceased as the result of equalized pressures at the extremities or of plugging of the vessel, is due to the operation of this factor of endothelial response to diminished interchange.
 +
 
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These two factors, then, amount of interchange through and amount of flow over the inner surface of the endothelial wall of capillaries appear to be the chief ones concerned in the regulation of the new growth of capillaries, their maintenance, increase in diameter to form arterioles and venules, or decrease in diameter with e^'entual solidification and retraction. Of these two factors, the amount of interchange is primary and the amount of flow secondary, since increased or diminished blood-flow depends in the main upon changes incident to the formation of new capillaries.
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Endothelium subjected to either of these factors will survive, grow, or retract according to the intensity of the stimulus. If the blood-flow is increased, there is increase in the diameter of the vessel. If the rate of interchange is increased there results sprout formation. Diminution of blood-flow causes diminution in diameter, and diminution of interchange, narrowing, solidification and retraction of endothelium.
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(JROWTH OF BLOOD-VESSELS IN FROG LARVAE
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71
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.>'P
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Fig. 13 From series shown in figures .1 to 8, showing vessels of stage of May 31 (dotted lines) superimposed on vessels of stage of April 15 (solid linesj. The corresponding vessels are lettered. Enlargement the same for both stages.
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Fig. 14 Same vessels as shown in figure 13. The vessels from stage of May 31 (dotted lines) have been reduced, by means of the pantograph, until they have the same dorso-ventral length as vessels from stage of April 15 (solid lines).
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72 ELIOT R. CLARK
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THE INCREASE IN THE LENGTH OF VESSELS
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With the general growth expansion of the tissue, the question arises as to what effect this has on the blood vessels present in the midst of the organ. In figures 13 and 14 there have been superimposed capillaries and vessels which were present at the earliest and at the latest records. In one case (fig. 13), they are both drawn at the same degree of enlargement. In the other (fig. 14) the drawing of the older stage has been reduced, by means of the pantograph, enough so that the two sets of vessels have the same dorso-ventral dimensions. Measurements show that the increase has been greater in the antero-posterior than in the dorsoventral direction. The increase in the dorso-ventral measurement of the fin expansion has been in the proportion of 1 to 2.22; while in the antero-posterior measurement (the total length of he tadpole) the increase has been in the ratio of 1 to 2.73. Thus the ratio of the dorso-ventral to the antero-posterior increase is about 7: 10. Almost the same proportion is found to exist between the measurements of corresponding parts of the capillary plexus at the same two stages. Thus the ratios obtained are, for the dorso-ventral increase: 1. to 2.2, for the antero-posterior increase: 1. to 2.9. It is possible that the agreement between the two sets of antero-posterior measurements would be even closer, had the measurement been made of the increase in length of the tail, instead of the increase in the length of the entire larva, including the head. It is obvious that the growth of the tissue has caused a proportionate growth in the length of the blood capillaries, and the size of the capillary mesh-work.
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There would seem to be but one possibility as to the factors responsible for this increase in length of vessels, namely, the one proposed by Thoma ('11), as his second histomechanical law, that increase in length of vessels results from a tension exerted in a longitudinal direction on the vessel wall, by the surrounding tissue. In the tail of the frog larva, the space between the blood capillaries is occupied mainly by branched mesenchyme cells, from which fine fibrillae in great abundance are given off in all direction, surrounding and supporting the blood-capillaries, lym
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GROWTH OF T3LOOD-VESSELS IN FROG LARVAE 73
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phatics and nerves, and extending between the right and left layers of epiderniis. As the tail grows there is an increase in the number of these cells, and in the size and complexity of their l)rocesses. It is clear that such an increase in the tissue filling up the space formed by a blood-capillary mesh- work, the fine processes of the tissue being in contact with the blood-vessels, would lead to a pushing and pulling on the capillary wall, and it seems (luite safe to infer, as Thoma has done, that the growth in length of vessels is due to the response of the vessel wall to this mechanical tension.
 +
 
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CHANGE IN THE ANGLE OF BRANCHING
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It was somewhat surprising to find how closely the direction and pattern of the capillaries which persisted resembled, in the latest stage, their pattern in the earliest stage. Thus, bends, present in the capillary at its first formation, although they may diminish, or disappear entirely, may be retained throughout. To a considerable extent, also, the angles between branches remained nearly the same as when first formed. It is apparent that the capillaries, once formed, are held fairly rigidly by the surrounding network of fine connective tissue fibrillae. There is, howe\'er, a marked change in the angle of branching, in the case of the arterioles. Here one sees the working out, in a general way, of the laws of branching discovered by Roux, for the smaller the branch, relative to the main stem, the nearer its angle of branching approaches a right angle, and the larger the branch, the more acute — relatively — its angle of branching. Figure 15 illustrates the change. On March 18, the two branches — B and C — into which A divides are approximately equal, and the angle of branching of each is nearly 90°. This stage is an early one — the capillaries are newly formed, and are characteristically wide, and the angles are those which happened to form as a result of the direction taken by the new sprouts. March 20, branch B is slightly larger than C, and there is a tendency for its angle of branching to become slightly more acute. March 24, April 13, and April 26, however, show a progressive increase in the size of branch C over branch B, and a corresponding dimi
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74
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ELIOT R. CLARK
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nution of its angle of branching, as compared with that of branch B, until, April 26, it forms, as it leaves the main stem A, almost a direct continuation of A, while the angle formed by B is much nearer a right angle. It will be noted that, in the later stages, there is a reduction in the size of all the vessels. With this reduction, however, there is marked increase in rate of flow. Other examples may be seen by comparing, in the successive stages of the main series shown, the branches from the chief arteriole.
 +
 
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Marl^'
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Mar.ZO
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Mar, li
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Apr. \3
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A}}r.Z6
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Fig. 15 Several stages of blood-vessels in the tail of rana sylvatica larva, to show changes in angle of branching. In x, stage of April 26 is shown in dotted lines, superimposed on stage of March 18.
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In these cases, the angle of branching is found to vary according to the relative size of the branch — the larger the branch the smaller the deviation from the line continuing the axis of main stem, while the smaller the branch, the larger the angle — until, when the disproportion is great, the angle reaches 90°.
 +
 
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These results corroborate the results of Roux' studies, which were based on measurements of arterial branches in adult animals. Roux stated his results in the form of a law, namely, that the size of the branch divided by the size of the main stem gives a series of figures which vary about as the cotangent of the angle of branching. He considered that the size of the angle represents
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GROWTH OF BLOOD-VESSELS IN FROG LARVAE 75
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the response of the tissue to hydrodynamical factors, and that the angle formed is always the one by which the minimum of friction is permitted.
 +
 
 +
The same problem is discussed by Thoma, ('11, p. 26) who agrees in the main, with Roux' findings, but considers that the blood-vessel, in assuming this shape, is merely responding to the rate of blood-flow, according to his first histomechanical law.
 +
 
 +
It would seem that Thoma's explanation (if amount of flow is substituted for rate) is sufficient, for, if fluid flowing through any tube tends to leave at a greater and greater angle the smaller the opening, then, if the blood vessel did not correspond with the direction naturally taken by the fluid stream, part of its wall would be subjected to the action of a greater flow of blood, than other parts, and would enlarge, while other parts would retract from the opposite cause. By this, the branch would be, remodelled until its angle of branching represented that in which the blood flowed in even amounts over all parts of the wall.
 +
 
 +
My observations on the angle of branching add nothing new to the results obtained by Roux, except that they represent studies on the same vessels at different stages, and give a picture of the actual changes in shape going on, hand in hand with the changes in the relative sizes of the branches, together with an approximate record of the relative amount of blood flow.
 +
 
 +
STUDY OF VESSELS IN TAD-POLES WITHOUT HEARTS
 +
 
 +
Since it has been found that a considerable development takes place in embryos deprived of a circulation by eliminating the heart-beat, it would seem, at first sight, that an opportunity offered itself here to test certain of the hypotheses dealt with in this paper — to find out whether any, and, if any, how much development of arteries, veins and capillaries takes place — particularly in the portion of the tad-pole's tail studied.
 +
 
 +
Brief reference has been made to such studies, and they will now be referred to more fully.
 +
 
 +
Roux ('95, p. 83) refers to a picture given by Dareste ('77, PI. VII, fig. 6) of a chick embryo in which the embryo proper failed to develop. In commenting, Roux says:
 +
 
 +
 
 +
 
 +
76 ELIOT K. CLARK
 +
 
 +
Nach dicser Abbildung Dareste's und einigcn von mir zufallig aufgefiindenen, weit ausgebildeten Fallen treten in dieseni Capillarnetz schon einige den normalen grosseren Gefassen entsprechende Richtigungen deutlich hervor, wie ich sehe, die entsprechende Erweiterung derselben und die Verdickung ihrer Wandung f ehlt ; der Sinus terminalis ist jedoeh ausgebildet und beweist so allein schon die vererbte localisierte Anlage eines typischen Gef asses.
 +
 
 +
Embryos without circulation were produced by J. Loeb ('93) who found that, by growing embryos of fundulus, a salt water minnow, in the proper concentration of potassium chloride, the beat of the heart could be entirely eliminated, or so diminished that all circulation of the blood was absent. The development of the embryo, however, continued for several days — about half the normal hatching time. In such embryos Loeb found an extensive development of blood-vessels which, except for irregu"larities in caliber, resembled in distribution the vessels in normal embryos. He concluded that:
 +
 
 +
Die mechanischen Ursachen fiir das Wachstum der Gefasswande sind deshalb nicht im Gefasslumen zu sucheii, sondern in alien oder einzelnen Zellen der Gefasswande und die Abgabe von Aesten ist bestimmt durch innere Ursachen in den Zellen der Gefasswande oder durch Reizursachen, die von der Umgebung ausgehend, diese Zellen treffen, ahnlich wie im Falle der Stolonenbildung von Hydroidpolypen.
 +
 
 +
Patterson ('09, pp. 87-88) studied the area vasculosa of chick embryos in which the development of the embryo proper had been prevented by operation upon the unincubated blastoderm. He describes the finding of vessels which radiate toward the remains of the embryo, and which he interprets as omphalomesenteric arteries.
 +
 
 +
Knower ('07) studied the development of frog larvae from which the heart anlage had been removed before pulsation had started. In a brief summary he states that — the aorta, the large veins and the segmental vessels are laid down." "Both arteries and veins are very abnormal and have a few well-defined branches. All vessels become much distended and follow very irregular courses." He found no vessels in the fin expansions of the tail.
 +
 
 +
 
 +
 
 +
GROWTH OF BLOOD-VESSELS IN FROG LARVAE i i
 +
 
 +
Stockard found in fundulus embryos, in which the circulation of blood was inhibited from the start by the use of alcohol, that the vessels of the yolk sac and many of the vessels of the embryo, including the two aortae, are formed, that some of them become much distended, and that they may persist without circulation for many days. While he gives no detailed or careful study of the exact amount of development of the vascular system, or the amount of retraction of vessels, he states ('15, B, p. 586) with reference to the aorta:
 +
 
 +
Tlie aorta in old embryos that never had their blood to circulate and in which the heart is actually a solid stream of tissue, grows and attains a well-developed lumen and a wall .lined with endothelium and surrounded by concentric fibers of connective tissue, as is shown in figure 4 a in the previous paper, drawn from such a specimen. This vessel is very slow to degenerate, in fact, it shows no sign of degeneration and actually persists as long as the embryo is able to exist without a circulation, for 30 days or more." .... The function of the vessel as a blood conductor, therefore, seems in these embryos of Fundulus, to have little if anything to do with its early development and not much effect on its ability to survive These facts are most significant in a consideration of the influence of function on growth and development, auto-differentiation. Here it is seen that the structure both grows and develops in entire absence of function.
 +
 
 +
The descriptions of the extent of vascular development which takes place without a blood circulation, as given by these investigators, w^hile meagre and incomplete, agree in the finding of an extensive vascular system, in which at least some of the main vessels appear to have developed sufficiently to give the impression of being fairly similar to the vessels in normal embryos. Thoma ('93, p. 28) recognized, in chick embryos, that there is not only an extensive development of capillaries in the extraembryonic area, but part of the aorta is well developed before the heart beat commences.
 +
 
 +
He offers t\vo possible explanations of the early development of the aorta. One is that there is an inheritance of an anatomical structure which is in agreement with the structure resulting from the action of mechanical forces. He says (p. 28) :
 +
 
 +
Man kann somit nur feststellen, dass die vererbte Form sich in Uebereinstimmung befindet mit jenem allgemeinen von mir aufgestell
 +
 
 +
 
 +
78 ELIOT R. CLARK
 +
 
 +
ten Gesetze, welches das Wachstum der Gefasswand von den Stromungsverhaltnissen des Blutes abhangig erscheinen lasst.
 +
 
 +
At another place, however, (p. 32) he suggests that the development of large vessels in the site of the two primitive aortae may be due to favorable mechanical or nutritional conditions.
 +
 
 +
That other main vessels develop, in chick embryos, before circulation commences, has been shown by Miss Sabin ('17) and referred to earlier in this paper.
 +
 
 +
In order to study the character of the blood-vessels in the tail fin of frog embryos deprived of a circulation, the type of plexus formed, and the mode of growth, I have employed Knower's method on frog larvae, and studied the vessels in the fin expansion of the tail. The heart was removed after it was sufficiently developed to be clearly visible, under the binocular microscope, but before it had started to beat, by making an opening through the skin, into the pericardial cavity grasping the heart with a pair of forceps, and dissecting it loose with a needle. In some cases a small pulsating fragment was left but there was no bloodcirculation. Embryos operated on in this way rarely live more than seven days, if the weather is warm, though they survive for ten to twelve days in cool weather. As Knower had described them, they become greatly swollen, due to the accumulation of fluid in the body cavities. They are very active, swim about the dish restlessly, and respond quickly to stimulation.
 +
 
 +
Unlike Knower, I found a considerable vascular development in the tail fins. Since the tail is opaque in early stages, due to the yolk and pigment, it was not possible to obtain very striking records of the growing blood-vessels. In several cases, however, the tail became sufficiently clear to permit records to be made. In one case records were made covering three days of growth of the vessels in the dorsal fin, and a considerable amount of growth was observed. The blood-vessels in the dorsal fin, in these larvae without hearts, form a primitive close-meshed plexus, of delicate vessels. In some cases the vessels are distended with blood cells, in others they are distended with a clear fluid, while in others they are very narrow. The blood cells are pushed about in the vessels by the movements of the embryos, and are
 +
 
 +
 
 +
 
 +
GROWTH OF BLOOD-VESSELS IN FROG LARVAE
 +
 
 +
 
 +
 
 +
79
 +
 
 +
 
 +
 
 +
 
 +
Fig. 16 Vessels from a portion of the tail of Rana Pipiens larva from which the heart was removed on April 27, before pulsations had started. Drawings made May 6, 7 and 9 of same region of dorsal fin. A, B, C, D— indicate the same blood-vessels. Lymphatics are dotted.
 +
 
 +
 
 +
 
 +
80 ELIOT R. CLARK
 +
 
 +
also moved about to some extent by the action of gravity. Thus, vessels which have been empty, have a little later been found to be packed with cells, and vessels filled wdth cells, may later be quite empty, or filled only with a clear fluid. Figure 16 shows some of the vessels in the dorsal fin of such a larva, on three different days.
 +
 
 +
On comparing the three records, it is obvious that growth has taken place by the formation of sprouts, which are at first narrow threads, but which later acquire a lumen. Anastomoses form between neighboring sprouts, and no additions are made by outside cells. In fact the vascular plexus extends in essentially the same manner as in the normal tail. There is, however, a larger number than in the normal, of fine solid processes. In this particular specimen the vessels are very narrow, and contain no blood cells.
 +
 
 +
It is to be noted that the vessels, once formed, show no tendency to differentiate further — into arterioles or venules. All diminish somewhat in caliber. In other specimens all the vessels are considerably distended. While three days is not a sufficient time, even in the embryo with a circulation, for much differentiation, still, in stages as early as this, at least a beginning differentiation is noticeable, as may be seen in the series shown in figures 1 to 3.
 +
 
 +
It is of interest to note the growth of Ijrmphatics in the embryos deprived of blood circulation. Knower's observation that the anterior lymph hearts in such embryos are larger and beat more strongly than in normal embryos, was confirmed. In the tail fin, lymphatics grow out often beyond the bloodvessels, although in the normal embryos at this stage and in this species (Rana pipiens) the blood-vessels in this region grow out well in advance of the lymphatics. The lymphatics are somewhat wider than in normal embryos. The mode of growth of lymphatics is the same as in normal embryos, by the extension outward of sprouts, and there is no tendency for the lymphatic and blood- vascular endothelium to form anastomoses with one another.
 +
 
 +
The enlarged caliber of the lymphatics is of interest, especially in connection with the enlarged lymph hearts, and with the ob
 +
 
 +
 
 +
GROWTH OF BLOOD-VKSSKLS IN FROG LARVAE 81
 +
 
 +
servation made by Knower, and confirmed by myself, that there is movement of lymph, as shown by the passage through the l>'ni])h h(>art of an occasional blood-cell, for it shows that passage of lymph into lymphatics is not dependent upon the maintenance of a certain amount of l^lood pressure.
 +
 
 +
The studies on embryos without circulation show that without the action of the mechanical factors concerned with circulation an extensive development of blood-vessels takes place ; that some vessels — at least the aorta in chick and fundulus embryos — differentiate bej^ond the capillary stage. My finding that, in such embryos, growth may occur by the usual process of sprouting, indicates that this property of endothelium is not dependent for its earh^ manifestation upon the action of the specific mechanical or chemical factors which seem to regulate it in later stages. 80 far as they bear on the main problem of this investigation, these results are important as indicating the extent and character of development of the vascular system which may take place without the mechanical factors concerned with the circulation of blood. It has been clearly brought out, especially by Roux, that there are two chief stages in the development of each organ or tissue. The first stage is the stage of 'auto-differentiation,' in which, by virtue of what our ignorance forces us to call heredity, or as Noel Paton expresses it, 'hereditary inertia,' each tissue differentiates and develops to a certain point. The second stage is the stage of 'functionelle anpassung,' functional adaptation, in which the further grow^th is regulated mainly by factors concerned in a quantitative way with the especial function of the organ or tissue. It is, therefore, consonant with our knowledge of many other organs and tissues to find that bloodvessel endothelium differentiates and grows for a certain period, and even that a vessel such as the aorta develops, as the results of 'heredity,' and without the action of mechanical forces. Such a finding affords no objection to the thesis that, in their later growth, blood-vessels are subject to the regulative action of the moving blood stream, the blood-pressure, the mechanical tension exerted on the wall by outside tissues, and the amount of passage of substances through the vessel wall. In fact, were it
 +
 
 +
 
 +
 
 +
82 ELIOT R. CLARK
 +
 
 +
not true of blood-vessel, as it is of other tissues, that two such phases exist, the vascular system would furnish a marked exception — at least, so far as our present knowledge of other tissues and organs goes. The question as to how far the vascular system might develop without heart-beat has not yet been satisfactorily worked out and presented.
 +
 
 +
SUMMARY AND DISCUSSION
 +
 
 +
The results of these studies on living blood-vessels are:
 +
 
 +
a. An extensive vascular development takes place, in the early embryonic stages, which is independent of the mechanical factors concerned with the circulation of the blood and the interchange of substances through the endothelial wall. During this stage, which to some extent precedes the inauguration of cardiac pulsation, it has been found that the aorta develops beyond the capillary stage (Thoma ('93) ) and that a number of other main arteries and veins are formed (Miss Sabin, '17) while several observers, by producing embryos with the heart beat eliminated, have found, apparently, that a number of the other main vessels develop. My own studies on frog embryos without hearts, show that extension of the blood-vascular system during this primary stage takes place by sprouting, and by the formation of anastomoses between sprouts.
 +
 
 +
Thus the vascular system, like other systems about which we have knowledge, differentiates and is carried a considerable distance on its developmental course — manifesting the property of extension by sprout formation, and forming some of the larger arteries and veins — as a result of 'hereditary inertia,' or 'self development.'
 +
 
 +
This stage, however, comes to an end relatively early; and the vascular system, for its further development into the complicated and nicely balanced system of the adult animal comes to be dependent upon the mechanical factors concerned with the pull and push of outside tissues, with blood-pressure and bloodcirculation, and with the interchange of substances through the wall. The picture presented in the tails of a-cardiac tad-poles
 +
 
 +
 
 +
 
 +
GliOWTH OF BLOOD-VESSELS IN FROG LARVAE 83
 +
 
 +
by the irregular indifferent plexus with no tendency toward transformation into arterioles, venules and capillaries, showing little more than the ability to send out and withdraw sprouts, is in marked contrast to the picture presented by the vessels in the same region, in tad-poles with a healthy circulation.
 +
 
 +
b. In normally developing tad-poles, the establishment of circulation brings into play, on the endothelium, the distending action of the blood-pressure, the mechanical friction of the moving blood stream, the mechanical (and possibly chemical) action produced by the passage of substances through the endothelial wall, in the interchange between the blood and the tissue fluid, and the pull and push of the enlarging and shifting outside tissues and organs. These studies, which have been principally confined to this stage, indicate, in general agreement with Thoma, Roux, Mall and Evans, that the new factors come to play a predominant part in the regulation of the growth of the vascular system, a part so important that the vascular endothelium may be said to depend for its growth on its response to the action of these forces.
 +
 
 +
The morphological changes are as follows:
 +
 
 +
The blood-vascular system extends by the well-known method of sprout formation. Blood-vascular endothelium has an affinity for other blood-vascular endothelium, cytotropism, (Roux), and avoids cells of other types of tissue, so that connections form between one sprout and another, or between a sprout and a fully formed capillary, while no connections form betw^een a sprout and foreign cells. Thus the vascular system growls as a continuous network.
 +
 
 +
A sprout once formed may have one of several fates; it may enlarge to form part of an arteriole or venule, which may become an artery or vein, or it may remain a capillary, or it may retrogress, losing its lumen, separating in the middle, and retracting into the capillaries with which it is connected at its two ends.
 +
 
 +
Changes take place in the angle of branching, and vessels increase in length.
 +
 
 +
The mechanical conditions which regulate these morphological changes are the following :
 +
 
 +
 
 +
 
 +
84 ELIOT R. CLARK
 +
 
 +
The new formation, enlargement, maintenance and atrophy of capillaries is dependent upon two factors (1) the amount of blood flow and (2) the amount of interchange of substances through the wall.
 +
 
 +
1. A capillary or part of a capillary so located that an increased amount of blood passes through it, increases in diameter until it may form part of an arteriole or venule; one so located that there is no especial increase or decrease in the amount of blood passing through remains a capillary; while to decrease or absence of blood-flow the capillary reacts by a diminution in lumen to final solidification and complete retraction. This, in a general way, agrees with Thoma's first histomechanical law, according to which the diameter of the vessel responds to the action of the moving blood stream. A difference, however, lies in this, that, while Thoma assigns chief importance to the rate of the blood stream, I find, in capillaries, that it is rather the amount of blood flow.
 +
 
 +
The changes in the amount of flow through different capillaries are brought about in various ways. The addition of new capillaries beyond, may cause an increase in a capillary so placed as to help supply or drain the new area. Again, the opening up of a new capillary may place an older capillary in such a position that it forms merely a cross connection between two parallel vessels, in which the pressure is equal, bringing about a slowing or stoppage of circulation in the older capillary. In certain cases of stoppage of circulation the cause is more puzzling — that is, in case of branches of capillaries which enlarge to form arterioles. It is suggested that the stoppage, here, is due to the constriction about the beginning of the branch, resulting from the increased thickness and elasticity of the arteriole. Some support is lent to this explanation by the fact that blood cells are often seen to plug the entrance to such branches often for long periods.
 +
 
 +
It is possible, however, that the narrowing of such vessels, as well as the slowing of circulation in the case of other capillaries, is due to the second factor mentioned.
 +
 
 +
2. The amount of interchange through the wall. This is pro
 +
 
 +
 
 +
(iROWTH OF BLOOD-VESSELS IN FROG LARVAE 85
 +
 
 +
]:)()scd as an hypothesis, because it seems to fit the facts better than any other. According to this hypothesis, the endothehum of blood-capillaries responds to the passage through it of various substances, in the interchange which takes place between the blood and the outside tissues. To an increase, beyond a certain maximum, the endothelium is thought to react by sending out a sprout; to a diminution, beyond a certain minimum, in a capillary which is not so placed as to form part of an arteriole or venule, the capillary is thought to react by narrowing its lumen; while for the maintenance of a capillary, a certain intensity of interchange is thought to be necessary.
 +
 
 +
The formulation of this hypothesis, which agrees, in general with Roux' conception, is merely carrying the explanation for the new formation of capillaries a step further than Mall, who recognized, as did Thoma, that the ultimate cause for new growth of capillaries lies in the growth and metabolism of outside tissues. It seems to fit more facts than the suggestion of Loeb and Evans that the cause lies in the action of specific substances outside the capillaries, or Thoma's hypothesis that it results from increase in capillary blood-pressure. It needs, however, further proof, before it can be accepted as a law of growth."
 +
 
 +
The changes in the angle of branching, which were observed, represent a response to the relative amount of blood-flow through the branch, as compared with the main stem. If a branch remains or becomes relatively large, as compared with the stem vessel, the angle between the two approaches 0°, if relatively small, 90°. This is, in general, in agreement with the studies of W. Roux who has made elaborate mathematical estimations of the angle of branching, and finds that the relation is so precise that it can be expressed within limits, as a mathematical formula.
 +
 
 +
The growth in the length of vessels goes hand in hand with the increase in outside tissue, and is clearly, as Thoma has expressed in his second histomechanical law, brought about by the reaction of the vessel to the mechanical pull, exerted in a longitudinal direction on the vessel.
 +
 
 +
Thoma's third law, according to which the thickness of the
 +
 
 +
THE AMERICAN JOURNAL OF ANATOMY, VOL. 23, NO. 1
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 +
 
 +
 
 +
86 ELIOT R. CLARK
 +
 
 +
4
 +
 
 +
vessel-wall is regulated by blood-pressure, has not been tested in these studies.
 +
 
 +
In general, my findings are that, while blood- vascular endothelium differentiates, develops the power to grow by sprouting, and forms a primitive system of arteries, veins and capillaries as the result of hereditary factors, it very early becomes dependent, for its complete and orderly w^orking out into the elaborate and beautifully proportioned adult vascular system, upon the regulative action of outside factors, to which it reacts in definite ways and upon which it comes to be completely
 +
 
 +
dependent.
 +
 
 +
BIBLIOGRAPHY ^
 +
 
 +
Arnold, J. 1871 Experimentelle Untersuchungen iiber der Blutkapillaren.
 +
 
 +
Virchow's Arch., vol. 53, p. 70. BoBRiTZKY 1885 Entwickelung der Capillargefasse. Centralbl. f. d. medicin.
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Wissensch., vol. 23. Bremer, J. L. 1912 The development of the aorta and aortic arches in rabbits.
 +
 
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Am. Jour. Anat., vol. 13, p. 111.
 +
 
 +
1914 The earliest blood-vessels in man. Am. Jour. Anat., vol. 16, p. 447.
 +
 
 +
Clark, E. R. 1909 Observations on living growing lymphatics in the tail of the frog hirva. Anat. Rec, vol. 3, p. 183.
 +
 
 +
1912 Further observations on living growing lymphatics: their relation to the mesenchyme cells. Am. Jour. Anat., vol. 13, p. 351.
 +
 
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1915 Studies of the growth of blood-vessels, by observation of living tadpoles and by experiments on chick embryos. Anat. Rec, vol. 9, p. 67.
 +
 
 +
Dareste, Cam. 1877 Recherches sur la production artificielle des Monstrositc's, Paris.
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Elze, C. 1912 Entwickeln sich die Blutgefjissetamme aus 'netzformigen Anlagen' unter dem Einflusse der mechanischen Factoren des Blutstromes? Anat. Anzeig., Erganz. Heft, zum, vol. 44, p. 102.
 +
 
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Evans, H. M. 1909 A On the development of the aortae, cardinal and umbilical veins and the other blood-vessels of vertebrate embryos from capillaries. Anat. Rec, vol. 3, p. 498.
 +
 
 +
1909 B On the earliest blood-vessels in the anterior limb buds of birds and their relation to the primary sulclavian artery. Am. Jour. Anat., vol. 9, p. 281.
 +
 
 +
1912 The development of the vascular system in Keibel and Mall, Human Embryology, vol. 2, p. 570.
 +
 
 +
GoLUBEW, A. 1869 Beitrage zur Kenntniss des Baues und der Entwickelungsgeschichte der Capillargefasse des Frosches. Arch. f. mikr. Anatom., vol. 5, p. 49.
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His, W. 1869 Untersuchungen iiber die erste Anlage der Wirbelthiere. Leipzig.
 +
 
 +
 
 +
 
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GROWTH OP^ BLOOD-VESSELS IN FROG LARVAE 87
 +
 
 +
Knoweu, H. McE. 1907 Effects of early removal of the heart and arrest of the circulation on the development of frog embrj^os. Anat. Rec., vol. 1, p. 16L
 +
 
 +
KoLLiKKK 1886 Zei'schrift F. wissensch. ZooL, vol. 43. 1902 Gewebelohre, Bd. 3, pt. 2, 6 ed., p. 670.
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 +
LoKB, J. 1893 . Ueber die Entwickelung von Fischembryonen ohne Kreislauf. Pfliiger's Archiv, vol. 54, p. 525.
 +
 
 +
Mall, F. P. 1906 A study of the structural unit of the liver. Am. Jour. Anat., vol. 5, p. 227.
 +
 
 +
Mauchand, F. 1901 Der Process dcr Wuiullieilung, mit Einschluss der Transplantation. Stuttgart.
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1913 Uber die Ifcrkunft der Leucocyten und ihre Schicksal bei der Entziindung. Verhandl. d. deutsch. path. Gesellsch., pp. 11-14.
 +
 
 +
Meyer, Jos. 1853 Ueber die Neubildung von Blutgefjissen in plastischen Exudaten serosen Membranen und in Hautwunden. Annalen des Charite-Krankenhaus zu Berlin, pp. 41-140.
 +
 
 +
Miller and McWhorter 1914 Experiments on the development of blood-vessels, 'etc. Anat. Rec, vol. 8, p. 203.
 +
 
 +
MiNOT, C. S. . 1912 The development of the blood, in Keibel and Mall, 'Human Embryology,' p. 498.
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MoLLiER, S. 1909 Die Blutbildung in der embryonalen Leber des Menschen und der Saugethiere. Archiv. f. mikr. Anat., vol. 74, p. 474.
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MiJLLER, E. 1903-1904 Beitrage zur Morphologic des Gefasssystems I. Die Armarterin des Menschen. Anat. Hefte, 1903. II. Die Armarterin der Saugetiere. Anat. Hefte, 1904.
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 +
NoTHNAGEL 1888 Die Entstehung des Collateralkreislaufes. Zeitsch. f. klin. Med., vol. 15 (ref. to in Oppel '10).
 +
 
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Oppel, a. 1910 Ueber die gestaltliche Anpassung der Blutgefasse unter Beriicksichtigung der funktionellen Transplantation, (mit einer Originalbeigabe von W. Roux). Vortriige und Aufsitze iiber Entwickelungsmechanik der Organise en. No. 10.
 +
 
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Patterson, J. T. 1909 An experimental study on the development of the area vasculosa of the chick blastoderm. Biol. Bull., vol. 16, pp. 87-88.
 +
 
 +
Rabl, H. 1907 Die erste Anlage der Arterien der vorderen Extremitiiten bei • den Vogeln. Arch. f. mikr. Anat., Bd. 69.
 +
 
 +
Reagen, F. p. 1917 Experimental studies on the origin of vascular endothelium and of erythrocytes. Am. Jour. Anat., vol. 21, p. 39.
 +
 
 +
RouGET, Ch. 1873 Mcmoire sur les Capillaires Sanguins et Lymphatiques. Archives de Physiol., vol. 5.
 +
 
 +
Roux, W. 1879 Die Bedeutung der Ablenkung des Arterien systems bei der Astabgabe. Jenaische Zeitschr. fiir Naturwiss., vol. 13, p. 321-337. (repub. in Roux, 1895, No. 2, A, pp. 77-100).
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1895 Gesammelte Abhandlungen der Entwickelungs mechanik der Organismus. Leipzig, verlag \V. Engelmann.
 +
 
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1910 Theorie der Gestaltung der Blutgefasse einschliesslich des Kollateralkreislaufs. ■ (included in Oppel's articel, q.v.)
 +
 
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RucKERT and Mollier 1906 Die Entstehung der Gefasse und des Blutes bei Wirbelthieren. Handbuch der Vergl. und exp. Entwicklungslehre d. Wirbelth., O. Hertwig, vol. 1, p. 1019.
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88 ELIOT R. CLARK
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Sabin, F. R. 1917 Origin and development of the primitive vessels of the chick and of the pig. Contributions to Embryology, No. 18, publication 226 of the Carnegie Institution of Washington.
 +
 
 +
ScHULTE, H. VON W. 1914 Early stages of vasculogenesis in the cat, etc Memoirs of the Wistar Institute, No. 3.
 +
 
 +
Smith, H. W. 1909 On the development of the superficial veins of the bodywall in the pig. Am. Jour. Anat., vol. 9, p. 439.
 +
 
 +
Stockard, C. R. 1915 A The origin of blood and vascular endothelium, etc. Am. Jour. Anat., vol. 18, p. 227.
 +
 
 +
1915 B A study of wandering mesenchymal cells on the living yolk sac, etc. Am. Jour. Anat., vol. 18, p. 525.
 +
 
 +
Thoma, R. 1893 Untersuchungen iiber die Histogenese und Histomechanik des Gefasssystems Stuttgart. .
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1911 tJber die Histomechanik des Gefasssystems und die Pathogenese der Angiosklerose. Virchow's Archiv, vol. 204, p. 1.
 +
 
 +
ZiEGLER, E. 1905 Lehrbuch der allgemeinen Pathologic, vol. 1, 11th edit., p. 303.
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 +
 
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 +
author's adsthact of this papeh issued by the binliographic service, september 28
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 +
==THE FATE OF THE ULTIMOBRANCHIAL BODIES IN THE PIG (SUS SCROFA)==
 +
 
 +
J. A. BADERTSCHER
 +
 
 +
Deparlmenl of Anatomy, Indiana University School of Medicine, Bloomington,
 +
 
 +
Indiana
 +
 
 +
FOUR PLATES
 +
 
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I. Introduction ^ ' 89
 +
 
 +
II. Historical 90
 +
 
 +
III. Material and methods 92
 +
 
 +
IV. Description of stages 93
 +
 
 +
V. Summary and discussion Ill
 +
 
 +
VI. Conclusions 119
 +
 
 +
VII. Bibliography 121
 +
 
 +
I. INTRODUCTION
 +
 
 +
The fate of the ultimobranchial bodies is one of the many unsettled questions associated with the development of the thyroid gland. While most of the results of the considerable amount of investigation that has been done in recent years on these structures in various mammalian species have led to the interpretation that they do not contribute to the structural elements of the thyroid gland, there is still a diversity of views as to their actual fate. The variety of views expressed in the literature in regard to their fate in mammals is apparently due to several factors among which may be mentioned: (1) the possibility of a variable developmental behavior of these structures in different mammalian types, (2) inadequate series of successively older embryonic stages (especially embryos of larger mammals) , and (3) faulty technique (principally poor fixation of the thyroid gland in the older embryos of the larger mammals, especially man).
 +
 
 +
1 Some of the younger stages used in this study were prepared during the summer of 1914 while a guest in the Department of Histology and Embryology in Cornell University. I wish to express my appreciation to Prof. B. F. Kingsbury for the facilities so generously extended to me during that time.
 +
 
 +
89
 +
 
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90 J. A. BADERTSCHER
 +
 
 +
A study of the ultimobranchial bodies in a wide range of successively older developmental stages of pig embryos of which the thyroid gland was well fixed, has resulted in bringing to light some interesting and important developmental features of these structures in this mammalian type.^
 +
 
 +
II. HISTORICAL
 +
 
 +
A brief historical outline representing in a general way the different views in regard to the fate of the ultimobranchial bodies is here given. The works of Verdun ('98) and Grosser ('12) render an extensive bibliography in this article unnecessary.
 +
 
 +
Born ('83) claims for the thyroid in the pig a triple origin. In a 21 mm. embryo he finds that the nuclei and cytoplasm of the cells composing the lateral thyroids stain more intensely than the cells of the median thyroid, but in a 37 mm. embryo in which the lateral thyroids have become imbedded in and fused with the median thyroid there is no histological difference between these structures and the median thyroid.
 +
 
 +
According to Moody ('12) in pig embryos 100 mm. in length no difference is to be observed between the central and lateral parts of the gland in vascularity, colloid formation or connective tissue development." He believes that the ultimobranchial bodies contribute to the structural elements of the thyroid gland.
 +
 
 +
Simon ('96) claims that in mammals (guineapig, rabbit, cat, calf, sheep, and pig) the lateral thyroids do not actually fuse with the elements of the median thyroid, although they become entirely inbedded in the latter. The lateral thyroids in early developmental stages show signs of growth and further development. During this period, which he designates the periode d'activite, the lateral thyroids are broken up into cell cords and cell
 +
 
 +
^ No consideration was given to the origin of the structure variously termed 'ultimobranchial body,' 'postbranchial body,' 'suprabranchial body,' 'telqbranchial body,' and 'lateral thyroids.' The morphological value of these terms have been discussed by Greil ('05), Rabl ('09), Kingsbury' ('14), and others. Throughout the descriptive part of this work the term 'ultimobranchial body' will l^e used.
 +
 
 +
 
 +
 
 +
FATE OF THE ULTIMOBRANCHIAL BODIES 91
 +
 
 +
masses. This is brought about in an entirely passive way, by the ingrowth of vascular tissue and of elements from the median thyroid. These cell cords and cell masses stain more intensely than do the elements of the median thyroid. Traces of the lumen persist mainly in the more central part of the lateral thyroids. These structures in later developmental stages undergo degenerative changes. This period, which he designates the periode de survivance, is characterized by a disappearance of the cell cords and the degeneration of the more centrally located epithelial cells, forming cysts lined with cuboidal or columnar epithelium which may or may not be ciliated. He also claims that the formation of cysts in the lateral thyroids of pig embryos is not a constant occurrence. Cysts in these structures in the pig were found only in five out of eleven embryos which he examined. In a 210 mm. embryo, the largest examined, no traces of the lateral thyroids were found. He is of the opinion that these structures in the pig disappear entirely.
 +
 
 +
Rabl ('08) finds that in the older mole embryos the lateral thyroids are reduced to insignificant structures, being represented by cell cords and cysts.
 +
 
 +
Verdun ('98) finds that in birds (chicken and duck) the postbranchial bodies remain independent structures of a glandular character but do not produce colloid. He regards these structures as special glands for birds. In the thyroid of mammals (rabbit, cat, dog, mole) the postbranchial bodies are represented by cysts and cell cords. The cysts vary greatly in size in the different species studied. Neither during the embryonic nor the postnatal life of these mammals was he able to demonstrate the transformation of the epithelial cords of the postbranchial bodies into thyroid follicles. He beheves that the cysts and cell cords represent atrophied vestiges of the special gland in birds.
 +
 
 +
According to Tourneaux and Verdun ('97) the lateral thyroids in human embryos can for some time be recognized as a rather densely staining mass on the posterior surface of the lateral lobes of the thyroid gland. They undergo the same structural changes as the median thyroid, but more slowly. From the
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92 J. A. BADERTSCHER
 +
 
 +
median thyroid anlage, however, is derived the larger part of the structural elements of the thyroid gland.
 +
 
 +
According to Christiani, in rodents (rat) the lateral thyroid gives rise to an epithehal body.^
 +
 
 +
Maurer ('99) finds that in the Echidna the postbranchial bodies do not fuse with the median thyroid anlage. In the adult condition the thyroid lies posterior to the postbranchial bodies. The latter are represented by two alveolar structures which developed colloid (judged by staining reaction). The first traces of colloid formed in the postbranchial bodies appears, however, in later developmental stages than does that of the median thyroid.
 +
 
 +
Prenant ('94) finds that in sheep embryos the lateral thyroid develops into a central canal with an irregular lumen from the walls of which cell cords and cell masses (recognized by their dense structure) extend into the substance of the median thyroid. An intimate fusion takes place between the lateral and medial elements. In later developmental stages the tissue, which in earlier stages can be recognized as derived from the lateral thyroids, disappears. He was unable to determine whether or not the lateral thyroids contribute to the structural elements of the gland.
 +
 
 +
In this brief historical sketch the following views as to the fate of the ultimobranchian bodies were brought out : (1) They contribute to the structural elements of the thyroid gland; (2) They develop into cysts; (3) They develop into a gland of a different structure from that of the thyroid gland; (4) They develop into epithehal bodies, and (5) They disappear entirely.
 +
 
 +
III. MATERIAL AND METHODS
 +
 
 +
The material used for this investigation was collected in great abundance at a packing house. The upper jaw, cranium, and thorax were removed from embryos ranging from 15 to 25 mm. in length. The part containing the thyroid was thus made comparatively small and fixed well. From embryos 26 to 75 mm. in
 +
 
 +
» Cited from Zuckerlandl ('03).
 +
 
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FATE OF THE ULTIMOBRANCHIAL BODIES 93
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length only the neck, from which the sides and the cervical vertebrae were removed, was reserved. From embryos 100 to 270 mm. in length (full term) only the thyroid with some of the surrounding structures — trachea, esophagus, a portion of the thymus, etc., was removed. The length in millimeters of the different developmental stages of which the thyroid was prepared for a study of the ultimobranchial bodies is as follows: 15, 16, 17, 17, 18, 18, 19, 19.5, 20, 20, 21, 21, 21.5, 22, 22, 23, 23, 23, 24, 24.5, 25, 25, 26, 27, 27, 28, 29.5, 30, 33, 35, 37.5, 38, 40, 48, 53, 60, 65, 65, 75, 100, 100, 111, 125, 125, 145, 150, 160, 175, 225, 245, 270, and 270. These figures represent the length of the embryos while in a fresh condition.
 +
 
 +
The fixing fluids employed were Zenker's fluid, Zenker-formol, and Picro-aceto-formol. The materia) was imbedded in paraffi.n. The earlier embryos were cut transversely in sections 5 microns thick, while those of later stages in sections 8 to 10 microns thick. Various stains were used. For embryos 15 to 65 mm. in length, iron hematoxylin gave the best results. The thyroid gland of later developmental stages was stained with Chloral hematoxylin and eosin, and eosin-methylene blue.
 +
 
 +
IV. DESCRIPTION OF STAGES
 +
 
 +
The earliest stage chosen for description is one just before the ultimobranchial bodies have fused with the thyroid gland. 'These structures will be described in two embryos of the same size only when there is a marked contrast in their size, structure, or position in the thyroid in the two embryos.
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 +
Embryo of 18 mm. (fig. 1). The ultimobranchial bodies have lost their connection with the fourth (?) pharyngeal pouch and extend cephalad beyond the anteror margin of the thyroid gland. Their anterior portion is in form a slender tube, ovaF in cross section, and with wall two to three layers of cells (nuclei) thick. Caudalward the walls of these structures gradually becomes thicker. In the portion in relation to the thyroid gland the lumen in places is obliterated and the remnants persist as mere slits. Anteriorly the surface of these tubules is quite smooth, while caudal] y irregularities occur on their surface. Their cau
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94 J. A. BADERTSCHER
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dal halves lie at varjdng distances dorsal to the lateral margin of the thyroid gland which has the general shape of a crescent with its horns directed anteriorly and dorsally. In a few sections they are separated from the thyroid only by a very thin layer of connective tissue iU). They extend almost to the caudal margin of the thyroid gland. Their caudal ends lie more closely together than their anterior ends.
 +
 
 +
The ultimobranchial bodies at this stage are composed of a syncytium. No cell walls are present. Vacuoles are found throughout their entire extent, although their distribution is not uniform. In places they can be found throughout an entire cross section of an ultimobranchial body while in other places they are confined to its more central portion. The vacuoles vary in size, the largest being almost as large as some of the nuclei.
 +
 
 +
The nuclei vary somewhat in size and in shape. Some are oval, some round, while others are irregular in outline. They contain from one to three nucleoli and a rather generous amount of chromatin which is in the form of granules and threads. The more centrally located nuclei have no regular arrangement while those near the periphery are in places quite regularly arranged. They are more closely packed together in the nonvacuolar than in the vacuolar portions of the ultimobranchial bodies. A consideration of this feature is of particular importance in stages in which the ultimobranchial bodies have fused with the thyroid gland. Mitoses of the nuclei can readily be found, especially in the larger more caudal part of the bodies, thus indicating a growth tendency of these structures. Neither blood vessels nor connective tissue are present in the ultimobranchial bodies at this stage.
 +
 
 +
The thyroid {T) is composed of nonvacuolar cell masses and cell cords^ the latter of which are for the most part transversely arranged. No cell walls can be demonstrated, hence the cell
 +
 
 +
■* Norris ('16) finds that in early developmental stages of human embiyos the cell cords seen in cross sections of the thyroid gland represent in reality sections of fenestrated epithelial plates. As I have not made a careful study of the formation of the follicles in the thyroid gland I shall use the term 'cell cords' which is the microscopic picture presented in cross sections of the gland.
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FATE OF THE ULTIMOBRANCHIAL BODIES . 95
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cords and cell masses have a syncytial structure. The nuclei vary in shape and somewhat in size but their form, average size, and structure in this stage is the same as in the ultimobranchial bodies. The nuclei of the thyroid are more closely packed together than in the vacuolar portions of the ultimobranchial bodies, but when a nonvacuolar portion of the latter is brought into the same microscopic field wdth a portion of the thyroid gland, no difference in structure can be seen between them even under high magnification (1500 diameters). Some of the spaces between the cords of cells are lined with endothelium and contain blood.
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 +
Embryo of 19.5 mm. (figs. 2 a and 2 b). The ultimobranchial bodies extend slightly farther cephalad than the thyroid gland. Only slight traces of their lumen still persist. They lie along the dorsal surface of the thyroid gland but are located nearer the mesial plane than those of the preceding stage. In some places there is actual fusion between these structures and the thyroid gland (fig. 2 a, right side), while in other places a thin layer of connective tissue intervenes (fig. 2 a, left side). The ultimobranchial body on the left side extends almost to the caudal margin of the thyroid gland (fig. 2 b), w^hile on the right side it terminates twelve sections (5 microns in thickness) earlier. The shape and orientation of the thyroid gland is similar to that in the preceding stage.
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 +
In this stage, as in the preceding one, both vacuolar and nonvacuolar areas are found in places along the periphery of the ultimobranchial bodies. In some places where actual fusion has taken place wdth the thyroid gland it is impossible to tell w'here the two striictures meet. Fusion with the thyroid gland has taken place along the ventro-lateral surface of the ultimobranchial bodies. The dorso-medial surface of these structures is in places studded with epithelial buds (fig. 2 a) .
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In the ultimobranchial bodies of this developmental stage are found a few nuclei in which the nucleoplasm stains quite deeply in comparison with that in the large majority of nuclei present. In some of these nuclei the chromatin is more abundant than in the more numerous and more lightly stained ones but in both
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96 . J. A. BADERTSCHER
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types of cells it is distributed in the form of threads and granules. With comparatively low magnification they appear as dark specks among the other nuclei (fig. 2 a). Since these have apparently been regarded by some investigators as degenerating nuclei, they deserve special attention in successively older developmental stages.
 +
 
 +
Embryo of 20 mm. (figs. 3 a, 3 b, and 3 c). The uLtimobranchial bodies are small anteriorly, and extend slightly farther cephalad than the thyroid gland. The one on the left side is separated for a short distance from the extreme anterior part of the thyroid (fig, 3 a). Caudal ward these structures rapidly become larger and form the greater portion of the horns of the crescent shaped tripartite complex. The one on the left side is slightly larger, and eleven sections (5 microns in thickness) longer than the one on the right side and extends as far caudally as does the thyroid gland. The extreme caudal portion of these structures is not fused with the thyroid gland. Remnants of the lumen are present in two places in the anterior third of the left one.
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 +
A feature quite noticeable in the ultimobranchial bodies of this developmental stage is the presence of unusually small nuclei which are found in small groups and promiscuously scattered among those of usual size. From their similarity in structure to the larger nuclei they seem to be normal. Deeply stained nuclei, which are somewhat more numerous throughout these structures than in the preceding stage, are also present in these groups.
 +
 
 +
It is impossible to determine definitely the exact place of fusion between the ultimobranchial bodies and the thyroid gland. Judging, however, from the uniformity of the distribution of the deeply stained nuclei, from the absence of cell cords along the greater portion of their dorso-mesial free border, from the manner in which they terminate, as stated above, from the absence of blood vessels, and from the distribution of the small nuclei and vacuoles, it seems that the cell masses labeled ultimobranchial bodies in the figures 3 a, 3 b, and 3 c represent exclusively the ultimobranchial bodies.
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FATE OF THE ULTIMOBRANCHIAL BODIES 97
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Epithelial buds, as represented in figure 3 b (Ep.B.), are present in various places along the free border of these structures. These buds are fused to the more or less vacuolar mass of cells. Mitosis can be found without much searching in both the ultimobranchial bodies and the thyroid gland.
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Embryo of 21 nmi. (fig. 4). Both ultimobranchial bodies are as long as the thyroid gland. They are fused to the latter along their entire extent excepting the extreme caudal end of the left one which is separated from the gland by a thin layer of connective tissue. The one on the right side has a comparatively regular outline and makes up nearly all of the lateral portion of the tripartite complex ([/). The ultimobranchial body on the left side is more deeply embedded in the thyroid gland than the right one which makes it difficult to follow its extent in transverse sections. In places blunt and both vacuolar and nonvacuolar epithelial buds are attached to these structures.
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Groups of small nuclei in the ultimobranchial bodies are present but they are not as mumerous as in the preceding stage. The darkly stained nuclei are no more numerous than in the previous stage. A few darkly stained nuclei were found in the cell cords of the thyroid gland. These have a structure similar to the darkly stained nuclei of the ultimobranchial bodies but are not nearly as numerous and can be found only after prolonged searching. Mitoses can readily be found in all the different components of the tripartite complex.
 +
 
 +
Embryo of 21.5 mm. (fig. 5). The ultimobranchial bodies lie along the entire extent of the dorso-medial margin of the thyroid gland and compose the largest portion of the tripartite complex. The anterior extremity of both ultimobranchial bodies and the posterior extremity of the left one are not fused with the thyroid gland. Their largest diameter (U) is about midway between their extremities from which they gradually taper to blunt points. Their greater portion is vacuolar but nonvacuolar areas are present in their deeper parts as well as along their periphery. Large blunt epithelial buds, some of which are vacuolar, are present in various places along their free border. The darkly
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98 J. A. BADERTSCHER
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stained nuclei and groups of small nuclei are more numerous than in the preceding stage.
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Embryo of 22 mm. (figs. 6 a and 6 b). The tripartite complex presents extremely varied pictures. Its anterior fourth is composed entirely of typical thyroid cell-cords while its caudal portion is composed chiefly of the ultimobranchial bodies (fig. 6 b) . The caudal portion of each ultmiobranchial bodj^ is composed of a cell mass of irregular outline in which remnants of the lumen, lined with columnar epithelium, still persist (fig. 6 b, L). Anteriorly, they are largely broken up into coarse cell cords which process marks the beginning of important developmental features in these structures (fig. 6 a, U). Nonvacuolar areas can be found throughout their entire extent. The deeply stained nuclei in the coarse cell cords, in the more central unbroken masses, and in the epithelial buds are more numerous than in any of the preceding stages. They can be quite readily found in the cell cords of the thyroid gland (figs. 6 a and 6 b), but are not nearly as numerous as in the ultimobranchial bodies. No degenerating nuclei, such as pyknotic or fragmented nuclei, were found. Small nuclei, in groups and diffusely scattered, are more numerous in the ultimobranchial bodies of this stage than in the previous one. In another 22 mm. embryo the size, shape, and extent of the ultimobranchial body along the thyroid gland is quite similar to that of the 21.5 mm. embryo described above.
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Embryo of 23 mm. (figs. 8 a, 8 b, and 8 c). The ultimobranchial bodies are small anteriorly and extend slightly farther cephalad than the thyroid gland. For a short distance anteriorly the ultimobranchial bodies and the thyroid are not fused. From their point of fusion with the thyroid they rapidly become larger so that the caudal portion of the tripartite complex is largely composed of the ultimobranchial bodies (figs. 8 a, 8 b, and 8 c, U). The epithelial buds attached to the ultimobranchial bodies are in general not as large as those in the 22 mm. embryo. The darkly stained nuclei and groups of small nuclei are also less numerous than in the preceding stage. Only an occasional darkly stained nucleus can be found in the cell cords of the thy
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FATE OF THE ULTIMOBRANCHIAL BODIES 99
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roid gland. A few blood vessels of a capillary character are found in the larger portion of the ultimobranchial bodies.
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Embryos of 24 to 30 mm. During this developmental period quite marked changes occur in the ultimobranchial bodies, the most pronounced of which is a breaking up of their major portion into cell cords which, when first formed, are usually larger than those of the thyroid gland. Two factors are apparent during the formation of cell cords, namely, a continued growth and division of the epithelial buds, and their invasion by mesenchymal and vascuolar connective tissue. The extent to which this process occurs during this developmental period varies. In some embryos these structures are almost entirely broken up into cell cords while in others a centrally located, more or less vacuolar and irregularly outlined core, variable in size, persists for some time longer. This process is illustrated in figure 9 (U), which represents a section through almost the middle portion of the tripartite complex in a 27 mm. embryo. In most stages of this developmental period (24 to 30 mm.), and even in some later stages, the caudal portion of the ultimobranchial bodies is for a time less broken up into cell cords than their more anterior part. Also, these structures never become entirely vacuolar. Some of the coarse cell cord^ are composed of a nonvacuolar syncytium. Nonvacular areas are also present in the more centrally located syncytial mass and in the larger and less unbroken caudal portion of these structures. Groups of small nuclei which appear normal in structure are preseilt in both vacuolar and non vacuolar parts. In places, instead of being arranged in groups, the small nuclei are quite uniformly scattered among the larger nuclei. Mitoses in both the thyroid gland and ultimobranchial bodies can readily be found.
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It is during this developmental period and also in somewhat earlier and later stages that the darkly stained nuclei are most numerous. In only two developmental stages, namely, in a 23 mm, embryo (not the one described above), and in a 24 mm. embryo (fig. 7, D.N.), were degenerated (pyknotic and fragmented) nuclei found in sufficient numbers to .suggest a general degeneration of these structures. The degenerated nuclei in
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100 J. A. BADERTSCHER
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these stages were not generally distributed throughout the ultimobranchial bodies but were found in localized areas. A few darkly stained nuclei can be found in the cell cords of the thyroid gland during this developmental period. In the thyroid, however, they are never very numerous and in some they are found only after prolonged searching.
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Embryo of 29.5 mm. (figs. 10 a, 10 b, and 10 c). The tripartite complex in this embryo is of interest in that a large portion of it is asymmetrical in shape, due to the unequal length of the ultimobranchial bodies. Nearly all of the anterior fourth of the complex is symmetrical and is composed of cell cords of the thyroid gland only (fig. 10 a). The greater portion of the middle two-fourths of the tripartite complex is characterized by the presence of massive cell cords of the left ultimobranchial body and the entire absence of the right ultimobranchial body (fig. 10 b). Along the posterior fourth of the thyroid gland both ultimobranchial bodies are present. The left one terminates rather abruptly thirty sections (150 microns) anterior to the caudal end of the thyroid while the right one tapers to a point and extends as far caudally as the thyroid gland. The extreme caudal portion of each ultimobranchial body is less broken up into cell cords than is represented in figure 10 c. Small disconnected vacuolar areas are present in the more caudal portion of both branchial bodies. The large cell cords are almost entirely free from vacuoles but are characterized by a comparatively large number of small nuclei. The deeply stained nuclei, which are comparatively few in number, are most confirfed to the ultimobranchial bodies. Only a few are present in the cell cords of the thyroid gland. Only a very few degenerated nuclei were found.
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In embryos from about 30 mm. in length to stages in which colloid is first present in the folUcles of the thyroid gland (75 mm.), the ultimobranchial bodies present a varied appearance. They are largely broken up into cell cords and in the progress of development the cell cords of the thyroid gland and usually those of the ultimobranchial bodies have become closely packed together so that a sharp demarcation between the
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FATE OF THE ULTIMOBRANCHIAL BODIES 101
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median and lateral elements of the tripartite complex is not always evident. A description of a few stages will suffice to bring out the general character of the ultimobranchial bodies during this developmental period. Since the thyroid gland in previous stages is free from vacuoles, it is, I believe, safe to assume that the vacuolar areas found in the succeeding stages represent portions of the ultimobranchial bodies.
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Embryo of 33 mm. The ultimobranchial bodies are limited to the posterior half of the tripartite complex. They are located on each side of the median plane, deeply buried beneath the dorsal surface of the thyroid gland and are represented by disconnected vacuolar areas the majority of which are not sharply circumscribed but gradually give place to the compactly arranged cell cords of the thyroid gland with which they are fused. The thyroid terminates posteriorly in two short blunt processes. In these processes small vacuolar areas are promiscuously scattered among the cell cords. A few" small vacuolar areas which are round in cross section and sharply demarcated by connective tissue from the surrounding cell cords were also found. Only a few darkly stained nuclei are present. No degenerated nuclei were found.
 +
 
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Embryo of 35 mm. The only traces of the ultimobranchial bodies are small disconnected vacuolar areas on each side of the median plane of the thyroid gland. The gland terminates posteriorly in two short blunt processes of nearly equal length, both of which are partly vacuolar. Only a few deeply stained nuclei are present.
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Embryo of 37.5 mm. The anterior portion of the tripartite complex is very large and strongly crescent in outline. Caudalward it gradually loses its crescent outline and ends in a single blunt cone-shaped process. The ultimobranchial bodies lie in the posterior four-fifths of the thyroid gland. Their anterior ends lie imbedded beneath the dorsal surface of the thjrroid lateral to its median plane. Caudal ward they rapidly increase in size and shift in position so that in places they extend to the free surface on the lateral margin of the thyroid gland. Their posterior ends are fused and compose by far the largest part of the
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THE AMERICAN JOURNAL OF ANATOMY, VOL. 23, NO. 1
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102 J. A. BADERTSCHER
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caudal one-fifth of the tripartite complex. The greater portion of the ultimobranchial bodies are in the form of vacuolar syncytial cores which give off coarse cell cords. Some of the coarse cell cords are vacuolar and many are fused to the cell cords of the thyroid gland. The central core is more or less' invaded with mesenchymal and vascular connective tissue. Deeply stained nuclei are quite numerous in the. ultimobranchial bodies and a few are found in the cell cords of the thyroid gland. No degenerated nuclei were found. The variableness in the size of the nuclei in the ultimobranchial bodies is more marked than in the nuclei of the thyroid gland, the former having a proportionally larger number of small nuclei. The extent to which the transformation of the ultimobranchial bodies has taken place in this stage is about equal to that in the 29.5 mm. embryo.
 +
 
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Embryo of 38 mm. The ultimobranchial bodies are limited to the posterior two-thirds of the tripartite complex. Their anterior ends are small and entirely imbedded in the thyroid gland near its dorsal surface. Caudalward they rapidly increase in size. The tripartite complex ends in two blunt cone-shaped processes the greater portion of which are composed of the ultimobranchial bodies. The ultimobranchial bodies are composed of irregularly outlined syncytial cores which gradually merge into the compactly arranged cell cords, of the thyroid gland. Only a few capillaries are found in them. Mitoses in the vacuolar areas as well as in the cell cords of the thyroid are quite numerous. Only a few deeply stained nuclei are present. No degenerated nuclei were found.
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Embryo of 40 mm. The ultimobranchial bodies lie in the posterior half of the thyroid gland. Their anterior parts are represented by small disconnected vacuolar areas which lie deeply buried in the thyroid gland lateral to its median plane. Caudalward these areas become large and branched so that the caudal fifth of the thyroid gland is largely invaded by a vacuolar syncytial mass which is not sharply demarcated from the closely packed cell cords of the thyroid gland. The tripartite complex ends in two blunt and slightly vacuolar processes of unequal length. A small number of deeply stained nuclei are present
 +
 
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FATE OF THE ULTIMOBRANCHIAL BODIES 103
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throughout the entire complex. Only a few degenerated nuclei were found in the vacuolar areas. There are realtively more small nuclei present in the vacuolar areas than in the non vacuolar portions and mitoses are more numerous in the latter than in the former areas.
 +
 
 +
Embryo of 48 mm. (figs. 11a and lib). Both ultimobranchial bodies extend anteriorly as far as the thyroid gland. The one on the right side is isolated from the anterior fourth while the one on the left side is isolated from the anterior third of the thyroid gland (fig. 11a, U). Excepting a vacuolar area present in the anterior end of the left one and traces of the lumen caudal to the vacuolar area, the isolated portions of the ultimobranchial bodies have a structure identical to that of the thyroid gland along which they lie. The right ultimobranchial body near its fusion with the thyroid is quite large (fig. 11a, U). The part fused to the thyroid gland is difficult to follow, yet traces of it may be seen in the form of small vacuolar areas that are promiscuously scattered beneath the dorso-lateral margin of the tripartite complex. Some of these areas are sharply outlined while others gradually merge into the compactly arranged cell cords.
 +
 
 +
The left ultimobranchial body caudal to its most anterior point of fusion with the thyroid gland is characterized in some places by very irregular vacuolar areas, while in other places by large, closely packed cell cords. In some places also it is only partially fused to the thyroid gland while in other places it is entirely separated from it by connective tissue (fig. 11 b, U.) Traces of the lumen still persist (L). The tripartite complex ends in two large conical processes which have, excepting a small vacuolar area found in each, a typically thyroid structure. Only a few darkly stained nuclei were found. Mitoses throughout the entire complex can be found without much searching.
 +
 
 +
Embryo of 53 mm. (fig. 12). Traces of the ultimobranchial bodies are present in the caudal half of the tripartite complex. The anterior end of each is represented by a small irregularly outlined vacuolar area which lies lateral to the median plane just below the dorsal surface of the gland. For some distance
 +
 
 +
 
 +
 
 +
104 J. A. BADERTSCHER
 +
 
 +
caudalward these areas become larger, in places very irregular in outline, in places broken up with typical thyroid structures, and are located more deeply in the lateral halves of the thyroid gland. The thyroid ends in a single blunt process that has a typically thyroid structure. In a few places the ultimobranchial bodies are unusually vacuolar. In these places the nuclei do not stain deeply (U). Similar lightly stained areas were observed by Kingsbury ('14) in the thyroid gland of human embryos. Also, a few groups or nests of small, closely packed nuclei were found. In some of these groups the nuclei had a normal structure, while in others they were only slightly stained. A few degenerated nuclei were found in the vacuolar areas and in their immediate neighorhood. Deeply stained nuclei are present in small numbers in both vacuolar and nonvacuolar parts.
 +
 
 +
Embryo of 60 mm. (fig. 13). The ultimobranchial bodies are limited to the posterior third of the thyroid gland. The right one is fused to the dorso-lateral margin of the gland and along its greater extent is composed of loosely arranged cell cords (U). Near the caudal end of the thyroid it merges into a vacuolar area which is interspersed with typical thyroid structures. The nuclei of the cell cords are on an average smaller than those found in the thyroid gland but, excepting a few darkly stained nuclei, they have a normal structure. The left ultimobranchial body lies just lateral to the median plane and is more deeply imbedded in the thyroid than the right one. It is largely composed of loosely arranged cell cords. Small vacuolar areas are present throughout its entire extent. The tripartite complex ends in a single process which is partly vacuolar. Mitoses can readily be found in the loosely arranged cell cords.
 +
 
 +
Embryos of 65 mm. The ultimobranchial bodies in two embryos of this developmental stage are described in order to contrast the structure of these bodies in two embryos of the same age. In one embryo these structures are represented by small disconnected vacuolar areas which are promiscuously scattered in the caudal half of the tripartite complex. In the other embryo the ultimobranchial bodies are located in the posterior third of the thyroid gland, and each one is characterized by a large and
 +
 
 +
 
 +
 
 +
FATE OF THE ULTIMOBRANCHIAL BODIES 105
 +
 
 +
very irregularly outlined and continuous vacuolar mass to which coarse and loosely arranged cell cords, some of which are vacuolar, are attached. In the extreme caudal portion of the thyroid gland these structures fuse with each other and make up a large portion of its blunt termination. In each embryo a few deeply stained nuclei and a few degenerated nuclei were found.
 +
 
 +
Embryo of 75 mm. This is the youngest developmental stage in which colloid is found in the thyroid gland (picro-aceto-formol and hematoxylin and eosin). The follicles containing colloid are not numerous but are quite uniformly distributed throughout the anterior portion of the gland. The ultimobranchial bodies are limited to the posterior two-thirds of the gland lateral to the median plane and bordering the dorsal surface of the gland. They are represented by a continuous area of cell cords w^hich contains no colloid. Within these areas are found small, irregularly outlined, and disconnected syncytial masses which contain an unusually large number of small nuclei. These nuclei have the same structure as those found in the cell cords of the thyroid gland. Many of the cell cords which do not contain colloid are fused to these syncytial masses. Vacuoles are almost entirely lacking. A few^ degenerated nuclei are present, found only after prolonged searching. The tripartite complex ends in two blunt processes which have a typical thyroid structure.
 +
 
 +
Embryos of 100 mm. The tripartite complex of two embryos deserves notice. In both the colloid is more abundant than in the previous stage.
 +
 
 +
Embryo No. 1 (fig. 14). The ultimobranchial bodies are limited to the middle tw^o-fourths of the thyroid gland. The right one lies partially exposed on the dorsal surface of the thyroid lateral to its medial plane. In some places it is composed of coarse and loosely arranged cell cords (U), while in other parts the cell cords merge into a large syncytial mass. In places the connection between it and the thyroid is more intimate than is shown in figure 14. In the syncytial masses the nuclei are on an average a little smaller than those in the cell cords of the thyroid, but in both their structure is the same. No vacuoles are
 +
 
 +
 
 +
 
 +
106 J. A. BADERTSCHER
 +
 
 +
present. A few deeply stained nuclei are present. Some are also found in the cell cords of the thyroid gland. A few degenerated nuclei were found. The ultimobranchial body on the left side has a similar structure to the one on the right side but lies deeply buried below the dorsal surface of the gland. The portion of the tripartite complex which can be distinctly recognized as a derivative of the ultimobranchial bodies, and the cell cords in their immediate neighborhood contain no colloid although the cell cords have a typical thyroid structure of somewhat earlier stages.
 +
 
 +
Embryo No. 2. The ultimobranchial bodies cannot be identified structurally. However, it is to be noted that on the right side, lateral to the median plane and along the dorsal border in the middle third of the gland is an area which contains no colloid. This area is composed of closely packed cell cords which have the same structure as the cell cords of the thyroid just before the appearance of colloid, such as in a 60 mm. embryo. On the left is an area similar in structure to the one on the right side only its cephalo-caudal extent is considerably less. These areas which are free from colloid correspond favorably in position to that of the ultimobranchial bodies in some of the previous stages. The thyroid terminates in two rather blunt processes the extreme caudal portions of which contain no colloid.
 +
 
 +
Embryo of 111 mm. The ultimobranchial body on the right side of the gland is represented by two small and widely separated syncytial masses which extend through a series of ten and six sections respectively (10 microns in thickness), and on the left side it is represented by a syncytial mass extending through a series of eleven sections. These syncytial masses are quite vacuolar and the nuclei are comparatively small and clear and many are irregular in outline. Only a few darkly stained and degenerated nuclei are present.
 +
 
 +
It is of importance to note that on the right side lateral to the median plane in the middle two-fourths of the gland and in line with the syncytial masses described above is an irregularly outlined area of closely packed cell cords. This area is quite large in cross section and is free from colloid. A similar area of about the same length is present in the left side of the thyroid but it
 +
 
 +
 
 +
 
 +
FATE OF THE ULTIMOBRANCHIAL BODIES 107
 +
 
 +
extends farther caudally and not so far anteriorly as the right one. These areas which are free from colloid correspond favorably in position to that of the ultimobranchial bodies in some earlier developmental stages.
 +
 
 +
Embryos of 125 mm. The ultimobranchial bodies in two embryos of this developmental stage were examined.
 +
 
 +
Embryo No. 1 (fig. 15). The ultimobranchial body on the right side lies in the middle two-fourths of the thyroid gland. Its anterior end is deeply imbedded beneath the dorsal surface of the thyroid and is composed of a very irregularly outlined and vacuolar syncytial mass in which the nuclei have about the same size as those in the follicular epithelium of the thyroid. A few pale nuclei are present. Cell cords, some of which are coarse and vacuolar, lead from the vacuolar area and are fused with the surrounding thja-oid structures. Slightly farther caudal it reaches the free surface on the ventro-lateral side of the gland and is composed of a loose network of cell cords some of which are vacuolar. From this place it gradually occupies a more dorsal position in the thyroid gland and is composed of closely packed cell cords, having a structure similar to that of the thyroid gland just before the appearance of colloid. Its more caudal portion reaches the free surface of the thyroid gland on its dorsal aspect (U) and contains a large cyst (C).
 +
 
 +
The ultimobranchial body on the left side is represented by a. series of six small disconnected, and irregularly outlined syncytial masses which lie just lateral to the mesial plane of the gland. These areas are more or less vacuolar and do not contain any colloid. The thyroid ends in a single process throughout which the colloid is cjuite uniformly distributed.
 +
 
 +
Embryo No. 2 (figs. 16 a and 1Gb). The tripartite complex of this embryo is of interest in that the ultimobranchial bodies are only partially imbedded in the thyroid gland. The ultimobranchial body on the right side lies along the lateral margin of about the middle two-fourths of the thyroid gland to which it is fused. It is fusiform in shape, with its greatest diameter about midway between its ends (fig. 16 a, U). The free portion along its entire extent is composed of syncytial masses and coarse and tortuous cell cords in both of which are found cystoid follicles which are
 +
 
 +
 
 +
 
 +
108 J. A. BADERTSCHER
 +
 
 +
lined with cuboidal and columnar epithelium. Some of the nuclei in the syncytial masses and in the coarse cell cords stain more deeply than others, and in general they are more variable in size than those in the follicular epithelium of the thyroid gland. The nuclei in the epithelial lining of the cystoid folicles lie closely together and stain uniformly. While the cystoid follicles are free from colloid in this developmental stage, small follicles containing colloid are thinly scattered throughout its free portion (fig. 16 a, Co). Along the line of fusion of the free portion of the ultimobranchial body to the thyroid gland there is in the latter an area composed of cell cords in which colloid is just beginning to form (fig. 16 a). The cell cords of this area have a structure similar to those in earlier stages in which colloid formation has just begun. In the free portion of this structure vacuoles are almost entirely lacking and only a few degenerated nuclei were found.
 +
 
 +
The length of the ultimobranchial body on the left side is equal to the length of the right one. It also lies along the lateral margin of the thyroid gland but is more deepl}^ imbedded in it (fig. 16 b, U). In cross section it is smaller than the right one but, excepting the absence of cystoid follicles, it has a similar structure. By referring to the figure it will be seen that it merges gradually into the thyroid gland. The follicles containing colloid gradually become smaller toward the more central portion of the ultimobranchial body in which only an occasional small follicle can be found.
 +
 
 +
Embryo of 145 mm. (fig. 17). The ultimobranchial bodies on both sides are limited to the middle two-fourths of the thyroid gland just lateral to its median plane. The right one along nearly its entire extent is partly exposed to the free surface along the dorsal border of the gland. The portion most deeply imbedded in the thyroid gland is represented by an area of cell cords in which the follicles containing colloid are quite numerous but all very small (U). _ In places along its free margin are found cystoid follicles which also contain colloid {C.F.).^
 +
 
 +
' The substance in the cystoid follicles is called 'colloid' on the ground that it has a staining reaction identical to that of the colloid in the follicles of the thyroid gland.
 +
 
 +
 
 +
 
 +
FATE OF THE ULTIMOBRANCHIAL BODIES 109
 +
 
 +
The ultimobranchial body on the left side is exposed to the free surface only in a few places. The larger portion lies immediately beneath the dorsal border of the thyroid gland. In one of its exposed parts are found cystoid follicles which contain colloid. The imbedded parts have the same structure as the imbedded portion of the right one. No darkly stained nuclei were found in either of the ultimobranchial bodies.
 +
 
 +
Embryo of 150 mm. The ultimobranchial bodies are located in the middle and a part of the posterior third of the thyroid gland near its lateral borders. They are represented by areas of typical thyroid structures in which the follicles containing colloid are small and not very numerous. In places they reach the free surface of the gland along its lateral border. No cystoid follicles or deeply stained and degenerated nuclei are present.
 +
 
 +
Embryo of 160 mm. (fig. 18). The only structures present in the thyroid gland indicative of the presence of the ultimobranchial bodies are areas (C/), on each side lateral to the median plane along the dorsal surface of the gland. In these areas the follicles containing colloid are quite small in comparison to the large majority present in the thyroid gland, but appreciably larger than those found in corresponding areas in the thyroid of 145 and 150 mm. embryos. These areas extend from about the caudal end of the anterior third well into the posterior fourth of the thyroid gland which terminates in a rather blunt single process. In the caudal end are a very few large follicles containing colloid but it was impossible to determine whether or not they developed in connection with the ultimob ranchial bodies. No darkly stained or degenerated nuclei were found.
 +
 
 +
Embryo of 175 mm. The follicles in the thyroid gland are on an average considerably larger than those found in the preceding stage. They vary greatly in size but are uniformly distributed throughout the gland. No structures are present which can be interpreted as derivatives of the ultimobranchial bodies.
 +
 
 +
Embryo of 225 mm. (fig. 19). The only apparent traces of the ultimobranchial bodies are areas of considerable extent in which the follicles are comparatively small ([/). These areas are located in the middle third on each side of the median plane and
 +
 
 +
 
 +
 
 +
110 J. A. BADERTSCHER
 +
 
 +
along the dorsal surface of the thyroid gland, and compare in position to the ultimobranchial bodies in some other comparatively late developmental stages. The areas of small follicles on the right side is a little shorter than that on the left side.
 +
 
 +
Embryo of 245 mm. (fig. 20). On the right side along the lateral margin of the posterior two-thirds of the thyroid gland is an area containing many cystoid follicles which contain colloid and which are lined with cuboidal epithelium. This area, small anteriorly, gradually becomes larger and reaches its greatest cross-section area near the posterior fourth of the thyroid gland. From this position it decreases in size and near its termination it is almost separated from the thyroid gland. This area occupies a position similar to that of the ultimobranchial bodies in some earlier stages and apparently represents a partially imbedded ultimobranchial body similar to the right one in No. 2 of the 125 mm. embryo (fig. 16a).
 +
 
 +
On the left side lateral to the median plane and below the dorsal surface of the thyroid gland is an area in which the average size of the follicles is appreciably smaller than the large majority of follicles in other portions of the thyroid gland. This area lies in the posterior half of the thyroid gland but does not extend as far caudally as the area of large follicles on the right side. It also corresponds favorably in position to that most generally occupied by the ultimobranchial bodies in earlier stages.
 +
 
 +
Embryos of 270 mm. (full term). The thyroid glands of two full term embryos were examined.
 +
 
 +
Embryo No. 1. The follicles containing colloid are variable in size but uniformly distributed throughout the gland. The only portion of the gland which can be interpreted as a derivative of an ultimobranchial body is an area of only small follicles on the right side lateral to the median line in the posterior half of the gland. This area extends through a series of only sixty sections (10 microns in thickness) and lies hear the dorsal surface of the gland.
 +
 
 +
Embryo No. 2 (fig. 21). The thyroid gland extends through a series of 827 sections (10 micrxjns in thickness). The left ulti
 +
 
 +
 
 +
FATE OF THE ULTIMOBRANCHIAL BODIES 111
 +
 
 +
mobranchial body is not completely transformed into typical thyroid structures. It lies in the posterior half of the gland and can be traced through a series of 234 sections (2.3 mm.). It is characterized by a small area of tortuous and nonvacuolar syncytial cords free from colloid which is eccentrically located in an area of small follicles (U). The nuclei in the syncytial cords correspond in size and structure to those in the follicular epithelium. A few nuclei in mitotic division are present. No deeply stained or degenerated nuclei are present.
 +
 
 +
The right ultimobranchial body extends through a series of 243 sections and is found in the middle third of the thyroid gland. It is characterized by an area of small follicles. In both ultimobranchial bodies from their more central portion toward their periphery the follicles gradually become larger. There is no sharp line of demarcation between these structures and the thyroid gland.
 +
 
 +
V. SUMMARY AND DISCUSSION
 +
 
 +
By comparing the rate of growth of the ultimobranchial bodies and the thyroid gland, it is seen that a more uniform size ratio is maintained in early than in later developmental stages. During this 'periode d'activite' (Simon) of the ultimobranchial bodies, which extends from an 18 mm. or earlier developmental stage to about a 33 mm. stage, the cephalo-caudal extent of the ultimobranchial bodies is nearly or entirely equal to that of the thyroid gland. In later stages (33 mm. to full term) in which the ultimobranchial bodies can be recognized structurally, their cephalocaudal extent is generally much less . than that of the thyroid gland, which indicates that in later developmental stages the rate of growth of the thyroid exceeds that of the ultimobranchial bodies. In embryos from about 50 mm. in length to full term the ultimobranchial bodies are usually located in the posterior half of the thyroid gland. In a few stages they occur in the middle third or the middle two-fourths of the gland. Simon ('96) claims that during this period of retarded growth of the ultimobranchial bodies, which he calls the 'periode de survivance,' they undergo degenerative changes which is manifested prin
 +
 
 +
 
 +
112 J. A. BADERTSCHEU
 +
 
 +
cipally by cystic formations (guinea-pig, rabbit, cat, calf, sheep) or their complete disappearance (pig) .
 +
 
 +
The ultimobranchial bodies first fuse with the thyroid gland along their ventro-lateral border. In early stages (19 mm. to about 27 mm.) they make up a considerable portion of the horns of the crescent-shaped tripartite complex. The extent of their fusion to the thyroid gland during their period of retarded growth (from about 33 mm. to full term) is variable. In the majority of late stages they are entirely imbedded in the thyroid gland while in some they are only partially imbedded. The latter condition is particularly the case in the following embryos; 48 mm. (figs. 11 a and 11 b); 60 mm. (fig. 13) ; 100 mm. (fig. 14); 125 mm. (fig, 16 a and 16 b); and 145 mm. (fig. 17). In the later stages they usually lie more or less deeply imbedded below the dorsal surface of the thyroid gland lateral to its medial plane, but occur less frequently along the lateral or dorso-lateral margin of the gland.
 +
 
 +
The formation of vacuoles in the ultimobranchial bodies begins before their fusion with the thyroid gland has occurred and continues after fusion. However, in the various stages examined no ultimobranchial body was found that is vacuolar throughout. In human embryos Kingsbury ('14) finds that vacuolation, 'reticulation,' continues until the entire structure is altered in this way. The extent to which vacuolation takes place varies in embryos of the same and different developmental stages. For example in No. 1 of the 125 mm. embryos the more central portion of these structures are quite vacuolar while in No. 2 of the 125 mm. embryos no vacuoles are present. Also no vacuoles are present in the left ultimobranchial body in No. 2 of the 27G mm. embryos. In early stages non-vacuolar portions are present along the periphery as well as in the deeper portions of these structures. In later stages in which the ultimobranchial bodies are largely broken up into cell cords the vacuoles are most numerous in their more central unbroken portion although vacuolated syncytial cords were found. In a few stages of which the embryo 53 mm. long is an example, the only part of the ultimobranchial body that can be recognized structurally as such are
 +
 
 +
 
 +
 
 +
FATE OF THE ULTIMOBRANCHIAL BODIES 113
 +
 
 +
small vacuolar syncytial masses entirely surrounded by typical thyroid structures (fig. 12).
 +
 
 +
Up to about a 24 mm. stage a marked contrast exists in the structure of the ultimobranchial body and the thyroid gland, in that the former are largely unbroken syncytial masses, \\'hile the latter is broken up into cell cords (as seen in cross section) . Although epithelial buds produce irregularities on the surface of the ultimobranchial bodies even in a 21 mm. stage and indications of cell cord formation were found in one 22 mm. embryo, the process of extensive cell-cord formation in these structures is particularly active in stages ranging from 24 to 27 mm. in length. The larger caudal end becomes broken up somewhat later than the smaller anterior end. Usually, also, the more central portion breaks up into cell cords later than the periphery. The syncytial cords when first formed are usually larger or coarser than those of the thyroid gland. Many are vacuolar for some distance away from the central more or less vacuolar core to which they may be attached. The time of breaking up of the central core into cell cords is very variable. The extent to which the ultimobranchial bodies become invaded with vascular tissue corresponds closely to the extent of cell cord formation. The first blood vessels, which are of a capillary nature, are found in these structures in a 23 mm. embryo.
 +
 
 +
According to Simon ('96) the cell cores of the ultimobranchial bodies are formed in an entirely passive way, namely, by the ingrowth of vascular tissue and of structural elements of the median thyroid. That the former is a potent factor in this process is, I believe, beyond doubt. It appears to me, however, that he lays too much stress on the formation of cell cords by the ingrowth of thyroid structures which will be considered later. Another active factor in the process of cell cord formation is a continued growth and branching of the epithelial buds found on their surface in early stages. The buds by continued growth and branching take the form of coarse cell-cords which can in many instances be recognized structurally from the smaller cell cords of the thyroid gland by the larger proportion of small nuclei which they contain and by vacuoles which, when present, are
 +
 
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114 J. A. BADERTSCHER
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 +
found in their more proximal ends near their attachment to the more central unbroken portion of these bodies. Also, in stages in which the darkly stained nuclei are numerous many can usually be found in the coarse cell cords. The presence of nuclei in mitotic "division in these cords is further evidence that they really grow.
 +
 
 +
The cell cords of the ultimobranchial bodies when first formed are generally more loosely arranged than those of the thyroid gland (figs. 9, 10 c, and 13). The time at which they become more compactly arranged and resemble in appearance the thyroid gland previous to the appearance of colloid in the latter, varies greatly. For example, in embryos of 48 and 53 mm. in length, excepting the small vacuolar portions, they have a structure, similar to the thyroid gland, while in both 125 mm. embryos cell cords in portions of these structures have still a quite loose arrangement.
 +
 
 +
The deeply stained nuclei are most numerous in the ultimobranchial bodies in stages from 20 mm. to about 30 mm. in length. In the first half of this brief developmental period (20 to 30 mm.) the ultimobranchial bodies attain their largest size as unbroken or solid structures while in the latter half of this period the process of cell cord formation is very active. The deeply stained nuclei diminish in number in stages beyond 30 mm. in length and finally disappear altogether. Their decrease in number is, however, not uniform in successively older stages. For example, in a 35 mm. embryo in which the only structural traces left of the ultimobranchial bodies are small disconnected vacuolar areas, the darkly stained nuclei are comparatively few in number, while in a 37.5 mm. embryo in which these structures are still large and easily traceable, the darkly stained nuclei are quite numerous. In late developmental stages in which the ultimobranchial bodes can be structurally recognized as such the darkly stained nuclei have largely or entirely disappeared. For example in No. 2 of the 125 mm. embryos there are some present although not in large numbers, while in the ultimobranchial bodies in No. 2 of the full term embryos no darkly stained nuclei are present.
 +
 
 +
 
 +
 
 +
FATE OF THE ULTIMOBRANCHIAL BODIES 115
 +
 
 +
The deeply stained nuclei have been regarded by Simon ('96) as degenerating nuclei. In only two developmental stages (23 and 24 mm. embryos, fig. 7) were degenerated nuclei found in sufficient number to suggest a general degeneration of these structures. In some of the later developmental stages degenerated nuclei were also found but always in small numbers. It appears that the degenerated nuclei are derived from the darkly stained nuclei although I was unable to trace their source through intermediate forms directly to them. Some of the nuclei in connective tissue cells, in developing muscle fibers, in epithelial cells lining the esophagus, and also in some stages in the cell cords of the thyroid gland, stain deeply. This gives them a structural appearance quite similar to those found in the ultimobranchial bodies. The presence of these nuclei in various developmental structures suggested the probability that the dark nuclei in the ultimobranchial bodies are in a certain physiological state. This, however, is mere conjecture. If they represented a general degeneration of the ultimobranchial bodies one would naturally expect to find large numbers of degenerated nuclei in later developmental stages, but a contrary condition is the case. They gradually decrease in number while the ultimobranchial bodies continue in their growth. This fact seems to me to be strong evidence in favor of the persistence of these structures.
 +
 
 +
A feature quite noticeable in the ultimobranchial bodies in most of the earlier developmental stages and in some of the quite late stages is the small and variable size of some of the ultimobranchial nuclei. Grosser ('10) and Kingsbury ('14) also observed small ultimobranchial nuclei in human embryos. The small nuclei are very variable in number in stages of about the same age. Some of these nuclei also stain deeply in stages in which deeply stained nuclei are present, and in a few instances groups of pale small nuclei were found. However, the large majority of the small ultimobranchial nuclei have a normal structure, in all stages in which they occur. In late stages no small nuclei are present. Although the significance of the darkly stained and the small ultimobranchial nuclei are unknown to me I am
 +
 
 +
 
 +
 
 +
116 J. A. BADERTSCHER
 +
 
 +
convinced that they do not represent a general degeneration ot the iiltiniobranchial bodies.
 +
 
 +
Follicles containing colloid appear first in the thyroid gland in a 75 mm. embryo. In the ultimobranchial bodies the follicles containing colloid are first quite numerous, though small (excepting the cystoid follicles), in a 145 mm. embryo (fig. 19). A few small follicles containing colloid were found in these structures in the 125 mm. embryos. The retarded development of colloid in the ultimobranchial bodies in the pig corresponds with a similar retardation in its development in these structures in the Echidna in which, according to Maurer ('99), they remain independent structures. The time at which the transformation of the ultimobranchial bodies into typical thyroid structures is completed, that is, when they can no longer be distinguished from the derivatives of the median thyroid anlage, is variable. For example in a 175 mm. embryo their transformation is complete while in No. 2 of the full term embryos the left one is composed of an area of small follicles in which is located a small area of cell cords free from colloid (fig. 21). A comparison of the structure of the right ultimobranchial body, which is composed of an area of small follicles, and the left one in No. 2 of the full term embryos also shows that one ultimobranchial body may undergo a more rapid transformation into typical thyroid structures than the other in the same embryo.
 +
 
 +
Cell cords are formed from the periphery of the ultimobranchial bodies usually sooner than from their more central portion, as stated above. It is also in the cell cords of the peripheral portion of the ultimobranchial bodies that colloid is first formed, so that the older peripheral follicles of these structures in many stages are larger than the more centrally located ones. Figures 18, 19, and 21 show that the follicles containing colloid gradually decrease in size toward the more central portions of these structures. Since colloid appears first in the thyroid gland many of the follicles are quite large before colloid is first formed in the ultimobranchial bodies. It would thus seem that if the ingrowth of structural elements of the thyroid gland into the ultimobranchial bodies is a factor in breaking up the latter into cell cords,
 +
 
 +
 
 +
 
 +
FATE OF THE ULTIMOBRANCHIAL BODIES 117
 +
 
 +
as claimed by Simon, there would be some quite large thyroid follicles found in the deeper portion of the ultimobranchial bodies among the smaller ultimobranchial follicles which begin to develop comparatively late. However, excepting the cystoid follicles in the ultimobranchial bodies in some of the later stages, this condition is not found. The follicles containing colloid gradually increase in size from the more central portion to the periphery of these structures. It therefore seems that the contention of k^imon is incorrect.
 +
 
 +
It also appears that in a few stages by far the larger portion of the ultimobranchial bodies undergo a transformation into typical thyroid structures even before colloid is formed in the thyroid gland. For example in embryos of 35 and 53 mm. in length the only structural features of the tripartite complex that can be interpreted as derivatives of the ultimobranchial bodies are small vacuolar areas (fig. 12) in contrast with the loosely arranged cell cords of these structures as found in embryos 37.5 and 60 mm. in length. Since in early stages it is impossible to distinguish the minute structure of the nonvacuolar portions of an ultimobranchial body from that of the thyroid gland when both are seen in the same microscopic field under high magnification, I believe that the vacuolar areas in embryos of 35 and 53 mm. in length represent only the more central cores of ultimobranchial bodies of which their more peripheral portion has undergone an early transformation into typical thyroid structures. This interpretation is supported by the conditions presented in a 48 mm. embryo in which the anterior portion of each ultimobranchial body is isolated from the thyroid gland. Excepting a small vacuolar area and traces of a lumen found in the isolated portion of the left one, the isolated portion of each of these bodies has a structure similar to the thyroid gland along which it lies.
 +
 
 +
I am of the opinion that the so variable developmental behavior of the ultimobranchial bodies in pig embryos throws light on a disputed point in connection with the development of these structures in human embryos. Grosser ('10) writes of a 'dichtere Zellgruppierung' in the thyroid gland of a human embryo
 +
 
 +
THE AMERICAN JOURXAL OF ANATOMY, VOL. 23, NO. 1
 +
 
 +
 
 +
 
 +
118 J. A. BADERTSCHER
 +
 
 +
50 mm. long. He however does not believe that this dense cell area is derived from an ultimobranchial body but that it is ■'niir der Ausdruck intensivem Wachstums der ganzen anlage, wiihrend die Differenzierung der neugebildeten Strange mehr oberflachlich stattfindet; die Zellen sind durchwegs typische Thyreoideazellen." Kingsbury ('14) finds that a human embryo 25 mm. long is the last stage in which the ultimobranchial body is clearly outlined. Their position in succeeding stages up to 41 mm. is occupied by a poorly circumscribed area of denser tissue." He is of the opinion that this "inner condensation" marks the place of disappearance of the ultimobranchial body although it may also well be as Grosser has stated, a center of growth." He further states that in 41 mm. and later developmental stages the "condensation is no longer recognizable." Although he was unable to satisfy himself as to the actual fate of the ultimobranchial bodies, he is of the opinion that they disappear.
 +
 
 +
From a study of the material used in this investigation I feel confident that the structure described by Grosser represents an ultimobranchial body. The process of cell cord formation at the periphery of the 'dichtere Zellgruppierung,' as described by him, corresponds favorably to the process of their formation in the ultimobranchial l3odies in pig embryos. Both the 'inner condensation' (Kingsbury) and the 'dichtere Zellgruppierung' (Grosser) apparently represent the central core which in the ultimobranchial bodies of pig embryos is found in a very wide range of developmental stages, even in a full term embryo (fig. 21). It seems that if the 'dichtere Zellgruppierung' represented a proliferati\'e center for the thyroid gland one would expect to find a rather large number of mitotic figures in them as an expression of rapid tissue growth. This, however, is not the case. No more nuclei in division are found in these areas than in any other portion of the thyroid gland.
 +
 
 +
The stages in which a comparatively early transformation of the greater portion of the ultimobranchial bodies takes place are comparatively few in ninnl)er. Also there are comparati\'ely few stages before full term in which there are no areas of small follicles
 +
 
 +
 
 +
 
 +
FATE OF THE ULTIMOBRANCHIAL BODIES 119
 +
 
 +
(ultimobranchial bodies) present. Judging, therefore, from the so variable developmental behavior of the ultimobranchial bodies it seems that the 175 mm. stage referred to above is one in which the ultimobranchial bodies underwent an early transformation into typical thyroid structures.
 +
 
 +
The portion of the structural elements of the thyroid gland at birth derived from the ultimobranchial bodies is small in comparison to the part derived from the median thyroid anlage. Owing to the variable de\'elopmental behavior of the former structures the comparative proportion contributed by them and the median thyroid anlage undoubtedly varies in different embryos. Figures 22 a, 22 b, and 22 c are diagrammatic representations of the portions derived from the median thyroid anlage and the ultimobranchial bodies in No. 2 of the 270 mm. (full term) embryos.
 +
 
 +
In the posterior portion of the right ultimobranchial body in No. 1 of the 125 mm. embryos is a cyst which extends through a series of sixty-seven sections (10 microns in thickness). It is lined with cuboidal epithelium the cytoplasm of which stained only very faintly. In one place in its lumen an isolated group of cells is found. The nature of its formation is unknown to me. According to Simon ('96) the formation of cysts in these structures is a regular occurrence during their 'periode de survivance' in all animals examined by him, excepting in the pig in which they occurred in five out of eleven specimens. Since cyst formation occurred in only one specimen out of those I studied, it seems to be an exceptional developmental feature in the pig.
 +
 
 +
VI. CONCLUSIONS
 +
 
 +
1. The ultimobranchial bodies in the pig participate in the formation of thyroid follicles. However, the portion of the gland in full term embryos that is derived from these structures is small in comparison with the part derived from the median thyroid anlage.
 +
 
 +
2. The cephalo-caudal extent of the ultimobranchial bodies is equal to or nearly equal to that of the thyroid gland in embryos up to about 33 mm. in length. From this stage on to full term
 +
 
 +
 
 +
 
 +
120 J. A. BADERTSCHER
 +
 
 +
the latter grows more rapidly in size than the former so that in stages from about 50 mm. in length to full term the ultimobranchial bodies usually lie in the posterior half of the thyroid gland but may be found in the middle third or in the middle two-fourths of the gland.
 +
 
 +
3. The developmental stages in which the ultimobranchial bodies transform into typical thyroid structures (that is, when they can no longer be recognized structurally from the median thyroid anlage) , vary greatly. The transformation of the greater part of these structures may take place as early as in a 35 mm. stage, before colloid is present in the thyroid gland, but in the majority of stages examined it takes place in later stages. Even in full term embryos an entire ultimobranchial body may not be completely transformed.
 +
 
 +
4. The ultimobranchial bodies in a thyroid gland may vary in size, in shape, in the degree of their transformation, and in their location in the lateral halves of the thyroid gland. This variability is particularly pronounced in some of the later developmental stages.
 +
 
 +
5. Colloid first appears in the follicles of the thyroid gland in embryos of 75 mm. in length. A few small follicles containing colloid appeared first in the ultimobranchial bodies of a 125 mm. embryo. In a 145 mm. embryo the follicles containing colloid in these structures are quite numerous although on an a\'erage small in comparison with those in the thyroid gland.
 +
 
 +
6. Large cystoid follicles containing colloid may develop in the ultimobranchial bodies.
 +
 
 +
7. The ultimobranchial bodies usually become entirely imbedded in the thyroid gland. In a few developmental stages they were found to be only partially imbedded.
 +
 
 +
8. The formation of cysts in the ultimobranchial bodies of pig embryos is of rare occurrence.
 +
 
 +
 
 +
 
 +
FATE OF THE ULTIMOBRANCHIAL BODIES 121
 +
 
 +
VIII. BIBLIOGRAPHY
 +
 
 +
Born, G. 1883 Uebcr die Derivate dcr embryonalen Schlundbogen und Schlund spalten bei Sjiugctieren. Arch. f. mikr. Anat., Bd. 22. FiscHELis, P. 1885 Beitriige zur Kenntniss der Entwickelungsgeschichte der
 +
 
 +
Gl. Thyreoidea und Gl. Thymus. Arch. f. mikr. Anat., Bd. 25. Fox, H. 1908 The pharyngeal pouches and their derivatives in the mammalia,
 +
 
 +
Am. Jour. Anat., vol. 8. Getzowa, S. 1907 Ueber die glandula parathyreoidea, intrathyreoidale Zell haufen derselben und Reste des postbranchialen Korpers, Arch. f.
 +
 
 +
path. Anat., Bd. 188. Greil, a. 1905 Ueber die Aniage der Lungen, sowie der ultimobranchialen,
 +
 
 +
(postbranchialen, suprapericardialen) Korper bei anuren Amphibien,
 +
 
 +
Anat. Hefte, Bd. 29. Grosser, O. 1910 Zur Kenutnis des ultimobranchialen Korpers beim Mem chen, Anat. Anz., Bd. 37.
 +
 
 +
1912 The development of the pharynx and of the organs of respiration.
 +
 
 +
Manual of Human Embryology, edited by F. Keibel and F. P. Mall,
 +
 
 +
vol. 2. Herrmann, G. and Verdun 1899 Persistance des corps post-branchiaux chez
 +
 
 +
I'homme. Remarques sur I'anatomie comparce des corps post-branchiaux. Comptes Rend. Soc. Biol. Paris.
 +
 
 +
1900 Note sur les corps post-branchiaux des Cameliens. Les corps
 +
 
 +
post-branchiaux et la thyroide; vestiges kystiques. Comptes. Rend.
 +
 
 +
Soc. Biol. Paris. Kast.schenko, N. 1887 Das Schicksal der embryonalen Schlundspalten bei
 +
 
 +
Saugetieren. Arch. f. mikr. Anat., Bd. 30. Kingsbury, B. F. 1914 On the so-called ultimobranchial body of the mammalian embryo: Man. Anat. Anz., Bd. 47. KoHN, A. 1897 Studien liber die Schilddriise, II. Arch. f. Mikr. Anat., Bd. 38. Maurer, F. 1899 a Die Schilddriise, thymus und andere Schlundspaltenderi vate bei der Eidechse. Morph. Jahrb., Bd. 27.
 +
 
 +
1899 b Die Schlundspalten-Derivate von Echidna. Anat. Anz.
 +
 
 +
Ergjinzungsheft, Bd. 16. Moody, R. M. 1912 Some features of the histogenesis of the thyroid gland in
 +
 
 +
the pig. Reprints of Papers from the Dept. of Anat. of the Univ. of
 +
 
 +
Cal., vol. 4. NoRRis, E. H. 1910 The morphogenesis of the follicles in the human thyroid
 +
 
 +
gland. Am. Jour. Anat., vol. 20. Prexant, a. 1894 Contribution a I'etude organique et histologique du thymus,
 +
 
 +
de la glande thja-oide et de la glande carotidienne. La Cellule, T. 10. Rabl, H. 1913 Die Entwicklung der Derivate des Kiemendarms beim Meer schweinchen. Arch. f. mikr. Anat., Bd. 82. Schapfer, J., AND Rabl, H. 1908 and 1909 Das thyreothymische System des
 +
 
 +
Maulwurfs und der Spitsmaus. I. Morphologic und Histologic by J.
 +
 
 +
Schaffer. II. Die Entwicklung des thyreothymischen System beim
 +
 
 +
Maulwurf by H. Rabl. Sitzber. kais. Akad. Wiss. Wien, vols. 117
 +
 
 +
and 118.
 +
 
 +
 
 +
 
 +
122 J. A. BADERTSCHER
 +
 
 +
Simon, Ch. 1896 'I'hyroide later;ilc et glandule thyroidienne chez les mam miferes, These de Xancy. Symington, J. 1897 t ber Tliyreoidea, Glandylae parathyreoidcae und Thymus beim dreizehigen Faulthier (Ai, Bradypus tridactylus). Arch. f.
 +
 
 +
Anat. u. Physiol. Supplement-Band zur Anat. Abt. TouRNEUX, F., and Verdun, P. 1897 Sur les premiers developpements de la
 +
 
 +
thyroide, du thymus et des glandules thyroidiennes Chez L'Homme.
 +
 
 +
Journ. de Anat. et de la Phys., T. 23. Verdun, P. 1898 Contribution a I'etude des derives Branchiaux chez les ver tcbres superieurs, These, Toulouse. ZucKERKANDL, E. 1903 Die EntM icklung dcr Schilddruse und der thymus bei
 +
 
 +
der Ratte. Anat. Hefte, Bd. 21.
 +
 
 +
 
 +
 
 +
PLATES
 +
 
 +
 
 +
 
 +
123
 +
 
 +
 
 +
 
 +
PLATE 1
 +
 
 +
EXPLANATION OF FIGURES
 +
 
 +
1 From a photogi'aph of a transverse section of the ultimobranchial bodies and the thyroid gland about midway between the anterior and posterior ends of the latter. Before the fusion of the ultimobranchial bodies with the thyroid gland. From an embryo 18 mm. long. X 60.
 +
 
 +
2 a and 2 b From photographs of transverse sections of the ultimobranchial bodies and the thyroid gland taken respectively near the anterior and posterior ends of the latter. P'usion between the ultimobranchial bodies and the thyroid gland has in some places taken place. From an embryo 19.5 mm. long. X 60.
 +
 
 +
3 a, 3 b, and 3 c From photographs of transverse sections of the ultimobranchial bodies and the thyroid gland taken respectively near the anterior, middle, and posterior portions of the latter. The numerous deeply stained nuclei in the ultimobranchial bodies are represented by small black dots. From an embryo 20 mm. long. X 60.
 +
 
 +
4 From a photograph of a transverse section about midway between the two ends of the tripartite complex. The horns of the crescent are largely composed of the ultimobranchial bodies the left one of which is quite irregular along its dorso-mesial surface due to epithelial buds. From an embryo 21 mm. long, X 60.
 +
 
 +
5 From a photograph of a transverse section about mid.vay between the two ends of the tripartite complex showing the large size of the ultimobranchial bodies. From an embryo 21.5 mm. long. X 60.
 +
 
 +
6 a and 6 b From photographs of transverse sections through near the middle and caudal portions respectively of the tripartite complex, showing nvunerous deeply stained nuclei (small black dots in the figures) in the ultimobranchial bodies and a few in the cell cords of the thyroid gland. The figures also show that the caudal portion of the ultimobranchial bodies are less broken up into cell cords than their more anterior portion. From an embryo 22 mm. long. X 60.
 +
 
 +
7 From a photograph of a portion of an ultimobranchial body showing degenerated and deeply stained nuclei. From an embryo 23 mm. long. X 650.
 +
 
 +
D.N., degenerated nuclei T., thyroid gland
 +
 
 +
D.S.N. , deeply stained nuclei Tr., trachea
 +
 
 +
Ep. B., epithelial buds U., ultimobranchial body L., lumen
 +
 
 +
 
 +
 
 +
124
 +
 
 +
 
 +
 
 +
FATE OF THE ULTI.MOHKAXCHIAI. BODIES
 +
 
 +
J. A. BADERTSCHEU
 +
 
 +
 
 +
 
 +
PLATE 1
 +
 
 +
 
 +
 
 +
Wm^'
 +
 
 +
 
 +
 
 +
 
 +
'^^^^M
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
^
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
6 a
 +
 
 +
 
 +
 
 +
 
 +
125
 +
 
 +
 
 +
 
 +
PLATE 2
 +
 
 +
EXPLANATION OP FIGURES
 +
 
 +
8 a, 8 b, and 8 c From photographs of transverse sections through near the anterior, middle, and posterior portions respectively of the tripartite complex, showing the gradual enlargement of the ultimobranchial bodies from their anterior to their posterior ends. The posterior end of the tripartite complex is largely composed of the ultimobranchial bodies. From an embryo 23 mm. long. X 60.
 +
 
 +
9 From a photograph of a transverse section about midway between the two ends of the tripartite complex showing the ultimobranchial bodies largely broken up into coarse cell cords. From an embryo 27 mm. long. X 56.
 +
 
 +
10 a, 10 b, and 10 c From photographs of transverse sections through the anterior, middle and posterior portions respectively of the tripartite complex. The ultimobranchial body on the left side extends along the posterior threefourths of the thyroid gland while the right one along only its posterior fourth. The unecjual size of the two ultimobranchial bodies, which are largely broken up into coarse cell cords, produce the asymmetry of the tripartite complex. From an embryo 29.5 mm. long. X 56.
 +
 
 +
11 a and 11 b From photographs of sections through the anterior and nearly the middle portions respectively of the tripartite complex showing both ultimobranchial bodies separated from the anterior portion of the thyroid gland (fig. 11 a) and the left one with traces of the lumen also separated from the thyroid (fig. 11 b). From an embryo 48 mm. long. X 60.
 +
 
 +
Ep.B., epithelial buds Tr., trachea
 +
 
 +
L., lumen U., ultimobranchial body
 +
 
 +
T., thyroid gland
 +
 
 +
 
 +
 
 +
126
 +
 
 +
 
 +
 
 +
FATE OF THE ULTIMOBRANCHIAL BODIES
 +
 
 +
J. A. BADEHTSCHEU
 +
 
 +
 
 +
 
 +
PLATE 2
 +
 
 +
 
 +
 
 +
V.
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
^ ,!i
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
A^m:^
 +
 
 +
 
 +
 
 +
'.. U.^':
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
10 b.
 +
 
 +
 
 +
 
 +
 
 +
'^^i?^'-^"-;-;^
 +
 
 +
 
 +
 
 +
< ^?-V"*-4^S^^2y!SSj-^'^''^'"~'^' '•
 +
 
 +
 
 +
 
 +
 
 +
127
 +
 
 +
 
 +
 
 +
PLATE 3
 +
 
 +
EXPLANATION OF FIGURES
 +
 
 +
12 From a photograph of a small portion of a section through an ultimobranchial ])ody (the light area in the figure) which merges gradually into typical thyroid structures. In the light area the majority of the ultimobranchial nuclei do not stain deeply. From an embryo 53 mm. long. X 337.
 +
 
 +
13 From a photograph of a portion of a transverse section of the tripartite complex showing the right ultimobranchial body which is largely composed of coarse and loosely arranged cell cords. From an embryo 60 mm. long. X 60.
 +
 
 +
14 From a photograph of a portion of a section of the tripartite complex showing the right ultimobranchial body which, in this particular place, is composed of loosely arranged cell cords in which no colloid is present. From an embryo 100 mm. long. X 56.
 +
 
 +
15 From a photograph of a portion of a transverse section through the posterior portion of the tripartite complex showing the compactly arranged cell cords of the right ultimobranchial body in which is located a cyst. The area inside the dotted circle is free from colloid. From an embryo 125 mm. long. X 45.
 +
 
 +
16 a and 16 b From photographs of portions of a transverse section of ths tripartite complex showing respectively the right and left ultimobranchial bodies. The right one is only partially imbedded in the thyroid gland and contains many cystoid follicles (C.i^.)'which do not contain colloid and a few small follicles which contain colloid (Co). The black dots in the portion of the figure labeled 'thyroid' represent colloid. The left ultimobranchial body is more deeply imbedded in the thj-roid gland. From an embryo 125 mm. long. X 38.
 +
 
 +
17 From a photograph of a portion of a transverse section of the trip:.rtite complex showing the right ultimobranchial body in which are found both small and cystoid follicles that contain colloid. From an embryo 145 mm. long. X 38.
 +
 
 +
18 From a photograph of a portion of a section of the tripartite complex showing the left ultimobranchial body which is represented by an area of small follicles. The black dots in the figure represent colloid. From an embryo 160 nun. long. X 38.
 +
 
 +
C, cyst T., thyroid gland
 +
 
 +
C.T., cystoid follicles U., ultimobranchial body
 +
 
 +
Co., colloid
 +
 
 +
 
 +
 
 +
128
 +
 
 +
 
 +
 
 +
FATE or THE ULTIMOBRANCHIAL BODIES
 +
 
 +
J. A. IlAnEHTSCHF.R
 +
 
 +
 
 +
 
 +
PLATE 3
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
f»«At'V;.«tJti-«<V <</I •^/JLilfc . • -1 " Wets.
 +
 
 +
 
 +
 
 +
 
 +
 
 +
^'^i*-v;v-^" '■■■■■■ -^V
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
'-16a
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
CF :
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
,'i*^ v.. •
 +
 
 +
 
 +
 
 +
?'f ^"»'V •v:-'"'^K. -j «
 +
 
 +
 
 +
 
 +
■17
 +
 
 +
 
 +
 
 +
i?.-*.
 +
 
 +
 
 +
 
 +
V' 'V.<...v..- ^ ■ ■;.'■'■
 +
 
 +
 
 +
 
 +
J
 +
 
 +
 
 +
 
 +
■••■'•..■ ,■>-.■•? '■■>■'■ ■^ v^»f
 +
 
 +
 
 +
 
 +
 
 +
129
 +
 
 +
 
 +
 
 +
PLATE 4
 +
 
 +
EXPLANATION OF FIGURES
 +
 
 +
19 From a photograph of a portion of a section through the left ultimobranchial body and a portion of the thyroid gh\nd surrounding it. The ultimobranchial body is characterized by follicles which contain colloid and which are on an average appreciably smaller than the follicles of the thyroid gland. From an embryo 225 mm. long. X 38.
 +
 
 +
20 From a photograph of a portion of a section through the right ultimobranchial body and a portion of the thyroid gland surrounding it. The ultimobranchial body contains many cystoid follicles which contain colloid. The colloid dropped out from some of the follicles during the process of staining. From an embryo 245 mm. long. X 38.
 +
 
 +
21 From a photograph of a portion of a section through the left ultimobranchial body and a portion of the thyroid gland. The ultimobranchial body is characterized by an area of small follicles in which is located a small area free from colloid. The light dots represent follicles from which the colloid has fallen. This figure represents the ultimobranchial body at C in figure 22 a. From No. 2 of the embryos 270 mm. long (full term). X 38.
 +
 
 +
22 a, 22 b, and 22 c These figures show the relative size of the ultimobranchial bodies and the thyroid gland in No. 2 of the embryos 270 mm. long (full term). The extent of the ultimobranchial bodies is outlined by a dotted line. Inside the left ultimoljranchial body is a small area (X), also outlined by a dotted line, which is free from colloid (figs. 22 a and 22 c). The portion of the left ultimobranchial body outside the area X and all of the right one is characterized by follicles which are on an average appreciably smaller than those of the thyroid gland. Figures 22 b and 22 c represent cross sections through the tripartite complex at b and c respectively of the structures represented in figure 22 a. X 7.5.
 +
 
 +
T., thyroid U., ultimobranchial body
 +
 
 +
 
 +
 
 +
130
 +
 
 +
 
 +
 
 +
FATE OF THE ULTIMOBRANCHIAL BODIES
 +
 
 +
J. A. BADERTSCHER
 +
 
 +
 
 +
 
 +
PLATE 4
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
!-•;:'■■ *' ■/'
 +
 
 +
 
 +
 
 +
.*. . .^'» '■,o^
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
22 b.
 +
 
 +
 
 +
 
 +
 
 +
22 c.
 +
 
 +
 
 +
 
 +
131
 +
 
 +
 
 +
 
 +
ATTHOR S ABSTRACT OF THIS PAPER ISSUED BY THE BIBMOGRAPHIC SERVICE, DECEMBER 1
 +
 
 +
 
 +
 
 +
CHONDRIOSOMES IN THE TESTICLE-CELLS OF
 +
 
 +
FUNDULUS
 +
 
 +
J. DUESBERG
 +
 
 +
Carnegie Institution, Department of Embryology, Baltimore, Maryland
 +
 
 +
TWENTY-ONE FIGURES (tWO PLATES)
 +
 
 +
Our knowledge of chondriosomes in the spermatogenesis of fishes is limited, as far as I know, to an incomplete account on Myx3^noids by A. and K. E. Schreiner ('05, '08). In the ripe spermatozoon, however, the same bodies have been studied, especially by Retzius, in quite a large number of species.
 +
 
 +
According to A. and K. E. Schreiner, the chondriosomes are represented in the spermatogonia as well as in the spermatocytes of Myxine glutinosa by very small granules, tightly crowded together in the neighborhood of the 'Sphare.' No change in shape is observed during mitosis ; furthermore, the behavior of the mass of mitochondria seems to be entirely passive and consequently its segregation between the daughter-cells is often unequal. Concerning the process of spermiogenesis, these authors mereh^ state that the mitochondria build a sheath around the axile filament. It must be added that the preservation of the chondriosomes in the material used by A. and K. E. Schreiner can hardly be considered as satisfactory. ♦
 +
 
 +
Retzius has studied the ripe spermatozoon of Amphioxus ('05 b), of several selachians ('09 c; '10 b), of one ganoid (Amia calva, '05 b) and of a number of teleosts ('05 b; '10 b). As data concerning the process of spermiogenesis in selachians are lacking, in reference to the chondriosomes at least, it is hardly possible to decide what part of the spermatozoon is formed by these bodies. In the other classes however, their identification is easier and the concordant observations of Retzius on Amphioxus, Amia and teleosts can be summarized as follows : the chondriosomes of the ripe spermatozoon are located at the posterior part of the head
 +
 
 +
133
 +
 
 +
THE AMERICAX JOURNAL OF ANATOMY, VOL. 2.3, NO. 1
 +
 
 +
 
 +
 
 +
134 J. DUESBEKG
 +
 
 +
and surround usually for a short distance the proximal part of the tail. The shape of this sheath varies with the different species. In Amphioxus the chondriosomes are represented by a rather voluminous body in which, by careful study, one can make out three to five granules. In Amia, such a body appears indistinctly granular and fits the posterior part of the head as the cup fits the acorn. In teleosts similar dispositions are found, for the details of which I refer to Retzius' papers. I wish to emphasize that in a number of species the granules are very distinct and even constant in number. In Lophius piscatorius for instance, Retzius ('10 b) invariably found four of them, disposed in regular order around the origin of the tail.
 +
 
 +
It may well be recalled that a similar disposition of the chondriosomes has been observed in invertebrates. For instance, according to the observations of Meves ('00, '03), each spermatid of the apyrene generation of Paludina vivipara contains four chondriosomes. They assume the form of spheres and occupy the posterior part of the head, where they surround the axile filament. Bonnevie ('07) gives a similar description for Membranipora pilosa. In these cases however, this stage is a transitory one, for the shape of the chondriosomes changes during the further evolution of the spermatid, while in other invertebrates the same arrangement is, according to Retzius, retained in the ripe spermatozoon, namely, i-n a number of celenterates ('04 a and b; '05 a; '09 a), in many echinoderms ('04 a and b; '05 a; '10 a), ^ in worms ('04 a and b; '05 a; '06 b, c and d; '09 b) and in moMuscs ('04 a and b; '05 b; '06 a; '10 b). In many species belonging to the two last-named classes the numerical constancy of the chondriosomal spheres and the regularity of their arrangement around the axile filament are conspicuous features of the ripe spermatozoon. Especially remarkable is the disposition in
 +
 
 +
1 Meves ('12) contests the accuracy of Retzius' description for Parechinus miliaris. He finds that the so-called 'Mittelstiick' is not granular, as stated by Retzius, but homogeneous, and that it has the shape of a ring, through which runs the axile filament. I take this opportunity to remind how inadequate is the expression 'Mittelstiick' or 'middlepiece,' as, between the 'Mittelstiick' of the spermatozoon of an echinoderm, of a selachian or a urodele amphibian and of a mammal, there is no homology whatever.
 +
 
 +
 
 +
 
 +
TESTICLE-CELLS OF FUNDULUS 135
 +
 
 +
polychetes: the granules in many species are consistently four in number, their form being exactly spherical, their volume equal and their disposition around the proximal part of the tail perfectly regular.
 +
 
 +
The origin of these bodies is briefly referred to by Retzius ('04 a and b) who states (for molluscs) that the large spheres are formed by the confluence of smaller granules. Previously, Pictet ('91) and Field ('95) both had described the formation of the so-called 'mittelstiick' in the spermatozoon of echinoderms through fusion of highly refractive granules which, however, they erroneously derived from remnants of the spindle.
 +
 
 +
Quite recently M. R. Lewis ('17) has stained the chondriosomes (the so-called 'middlepiece') of the spermatozoon of Cerebratulus lacteus and of Echinorachnius parma in vivo, by using a solution of Janus-green in sea-w^ater. The object of the present investigation is the testicle of Fundulus (heteroclitus and majalis), the main purpose being to study the behavior of the chondriosomes during spermatogenesis. The material was collected in Woods Hole, Mass., in June, 1916, and fixed in Regaud's or in Benda's fluid, the latter either with or without acetic acid. The sections, 5 n thick, were stained in the first case with iron-haematoxyhn or acid f uchsin-methylgreen ; after Benda's fixation, I resorted to iron-haematoxylin acid, fuchsin-methylgreen or Benda's stain, the lattef giving, as previously stated for embryos ('17), a very small percentage of good preparations. A number of sections were stained with safranin, hi order to study the chromatin.
 +
 
 +
Once more I found that the preservation of the tissue is much better after Benda's fixatian than after Regaud's. This last reagent has a pronounced tendency to make the seminal cells
 +
 
 +
2 In the same paper, M. R. Lewis (p. 33) quotes my opinion, as expressed in my review ('12j, on the vital staining of chondriosomes and, from this quotation, one might be induced to conclude that, for me, neutral red and methylenblue can be used or have been used to stain the chondriosomes in vivo. To prevent any misunderstanding, I wish to recall that this has never been my opinion, as appears clearly in the quoted place of my article (p. 608), as well as in several others (for example, page 823, in the discussion of Arnold's plasmosomes).
 +
 
 +
 
 +
 
 +
136 J. DUESBERG
 +
 
 +
swell. The extent to which the ground substance is affected is well illustrated by the difference in size exhibited by the cells represented in figures 10 and 11, both in exactly the same stage of evolution, the first from material fixed in Benda's fluid, the second from material treated with Regaud's. Thus, cells which normally float freely in the cystic cavity are made to conglomerate and stick together. The chondriosomes are also sw^ollen, and the chromosomes are transformed into an undecipherable clump. In contrast to this, the last-named bodies are well preserved in Benda's material, an appearance which confirms that swelling in Regaud's rather than shrinking in Benda's fluid is responsible for the differences between the two sets of preparations.
 +
 
 +
The testicle of Fundulus is at the time of spawning a rather voluminous organ formed by a considerable number of tubular cysts in which spermatogenesis proceeds from the periphery towards the hilus.^ The excretory system of the gland consists of a number of ducts lined with cubic or cylindric epithelium. In the distal part of these ducts the cells (fig. 1) contain, besides secretion-granules, a large number of chondriosomes. These are mostly long chondrioconts running along the nucleus in a direction perpendicular to the basis of the cell and intertwining at both poles of the nucleus. This disposition reminds one somewhat of the structure of the cells of the tubuli contorti (Heidenhain's rods) or of the salivary ducts (Pfliiger's rods). The inner part of the cell is often free of chondriosomes and irregularly delimited, an appearance which may be due to the action of the fixing fluid.
 +
 
 +
In the cells lining the proximal part of the excretory ducts, the chondriosomes are all replaced by granules of pigment. This recalls an observation made by Prenant ('11) on the skin and cornea of the frog. Prenant found that the cells of both layers in the skin contain mitochondria and pigment-granules. In the upper layer the granules of pigment are located near the surface, the mitochondria in the lower part of the cell, w^hile in the deeper layer mitochondria and pigment-granules are mixed
 +
 
 +
' Degenerating cells are, as in other testicles and especially in invertebrates, b}' no means infrequent in Fundulus.
 +
 
 +
 
 +
 
 +
TESTICLE-CELLS OF FUNDULUS 137
 +
 
 +
together. In the cornea no pigment is present. If one studies the point of transition between cornea and skin, one can see how the mitochondria gradually take the place of the pigment-granules. This observation is interpreted by Prenant, apparently not without reason, as indicating the transformation of chondriosomes into pigment and in the same sense could be interpreted the conditions just described in" the excretory ducts of the fishtesticle.
 +
 
 +
The seminiferous cysts are reunited by thin sheets of connective tissues containing blood-vessels and cells. Some of these are conspicuous by their large size and by the presence of a great number of bacilli-shaped chondrioconts (fig. 2) ; others contain also granules which I am inclined to consider as secretion-products. In places where the connective tissue is somewhat more abundant, for instance in such stellar spaces as appear between the cross sections of the cysts, they usually build groups of two or more elements. The nearest interpretation of these cells is that they correspond to the interstitial cells of the ma nmalian testicle. Supposing I were right, this would be the first mention of them in fishes, or, as far as I know, the literature does not contain any mention gf interstitial tissue in this class of vertebrates: in fact Friedmann ('98) and Ganfini ('02) state positively that they could not find it.
 +
 
 +
The distal part of the cysts is occupied by cells which are obviously the stem of the whole seminal lineage and as such should be designated as spermatogonia. Since, as we shall see, several generations of spermatogonia can be distinguished, I would call these 'primary spermatogonia.' Their size is relatively large (fig. 3, two cells on the top row and two cells at the right). Each nucleus contains usually only one large, sharply delimited, spherical block of chromatin-. The eventual occurrence of multiple nucleoli is often accompanied by the presence of indentations (the process is just indicated in figure 3, in the cell of the top row, to the right), which are suggestive of direct division. Mitosis however was repeatedly observed (figs. 4 and 5). It would not be surprising if these indentations were indicative of a process described as occurring in the spermatogonia of
 +
 
 +
 
 +
 
 +
138 J- DUESBERG
 +
 
 +
Salamandra after the period of sexual activity (namely by Meves '91), as once that period over, the testicular conditions are very similar in both the amphibian and the fish.
 +
 
 +
The chondriosomes of the primary spermatogonia deserve special mention. In the resting cell they are numerous, coarse and irregular granules or rods. Most of them are located very close to the nucleus and cover its surface. This disposition might be interpreted in favor of Goldschmidt's chromidial theory. Such a claim however would be unfounded: Goldschmidt and his pupils basing themselves upon defective observations, expected to demonstrate that the chondriosomes of the germ-cells were formed during the growth-period and they have failed utterly.^ The continuity of the chondriosomes on the other hand has been demonstrated in a number of animals and is strongly supported for fishes by my observations on the fishembryo ('17).^ It is however far from my mind to deny the
 +
 
 +
^ For a complete historical and critical account of the chromidial theory, see the third chapter of my review ('12). Shaffer, who seems inclined to believe (p. 414) in a nuclear origin of the chondriosomes, gives as an argument that "in nearly all the growth-stages of the first spermatocytes, there is present a denser and more deeply staining perinuclear zone," formed by the chondriosomes. I should take exception to this statement, for it is characteristic, even if not quite general, that the male auxocytes have their chondriosomes accumulated at one pole of the nucleus, around the idiozome.
 +
 
 +
In a paper on the testicle of opossum, Jordan ('11) claims that he has demonstrated the discontinuity of the chondriosomes in the seminal cells. I have
 +
been investigating lately the same object and my observations are in direct contradiction with Jordan's claim: chondriosomes exist in abundance in all the stages of the evolution of the seminal cells.
 +
 
 +
Shaffer ('17) enters against the theory of the continuity of the chondriosomes in the following way; " (p. 423) the progressive increase in the amount of mitochondria (during the evolution of the seminal cells) seems to indicate that they are differentiation-products. Hence, if there is any genetic continuity between the mitochondria of successive cell-generations, it is only of a limited sort. The conception that the mitochondria present in the somatic cells are the direct descendants of those of the germ-cells, from which thej' have arisen, certainly has very little evidence in its favor." I must state that I entirely fail to see an argument against the continuity of the chondriosomes in the fact that their amount may increase. Concerning the continuity of the chondriosomes in the somatic cells with those of the germ-cells, -Shaffer overlooks apparently the numerous observations which have shown this continuity, from the egg at least to the embryonic cells. I limit myself to remind of my own observations on the bee,
 +
 
 +
 
 +
 
 +
TESTICLE-CELLS OF FUNDULUS 139
 +
 
 +
existence of nucleocytoplasmic exchanges, as the nucleus is certainly not a sort of impermeable rubber-vesicle enclosed in the cell. But it would be rash to base on the mere existence of such appearances as described above any definite conclusion. The arguments for the cytoplasmic nature of the chondriosomes I do not want to repeat here and refer the reader to former papers, limiting myself to state that no indications of a nuclear origin can be found in the staining reactions.^
 +
 
 +
During the mitotic division of the primary spermatogonia the shape of the chondriosomes changes somewhat: they round up and become more regular (figs. 4 and 5). Their location in the cell is also modified: at the stage of metaphase they surround the spindle (fig. 4) and later are found between the daughternuclei (fig. 5).'^
 +
 
 +
Next to these cells are others differing but slightly from them. They are somewhat smaller in size and their chondriosomes are not quite so coarse. These cells are assembled in rosettes of
 +
 
 +
the rabbit and quite lately on Ciona, where the chondriosomes form the material of the yellow crescent, the continuity of which has been demonstrated by Conklin.
 +
 
 +
^ The original colors of the preparations could not be reproduced in the plates; as is well known, they are, in acid fuchsin-methylgreen preparations, red for the chondriosomes and green for the chromatin; in Benda's preparations, dark purple for the chondriosomes and pale brown for the chromatin.
 +
 
 +
' Concerning the fate of the chondriosomes during the mitotic division of the spern.atogonia of Passalus, Shaffer expresses himself as follows (p. 410): "the spermatogonial cysts which are in mitotic activity, stand out very clearly in contrast with the resting cysts. This is because of their lighter staining capacity; whether this in turn is due to the partial disappearance of the mitochondria could not be ascertained." Shaffer quotes Buchner as having found that in Gryllotalpa vulgaris, the chondriosomes disappear during or just before celldivision and gives three possible explanations "for the partial loss of mitochondrial structure dating mitotic activity." Interesting though they may be, these explanations appear to me for the present useless, as, after my own experience, chondriosomes do not disappear during mitosis, no more in Gryllotalpa, as I have shown ('10), than in any other case I know of.
 +
 
 +
Payne ('17) quotes both Buchner and me and sees no reason why we should differ so much in our observations: "In this case, one or the other has certainly made a mistake." Between a negative result, however, and a positive one, there is, in my opinion, no room for hesitation. It must be added that since, Buchner has considerably modified his attitude towards the chromidial theory, as appears from a text-book he recently published.
 +
 
 +
 
 +
 
 +
140 J. DUESBERG
 +
 
 +
three or more (fig. 3 on the left below) . So unvarying are these features that I feel justified in considering these cells as a distinct generation of spermatogonia and term them 'secondary spermatogonia.' The primary and secondary spermatogonia are in close contact with each other, the cystic cavity being at these stages only virtual, in contrast with all later stages, when some room, in well fixed material, is left between the cells.
 +
 
 +
The spermatogonia belonging to a third generation are, if any, not much smaller than the secondary spermatogonia. In the nucleus several blocks of chromatin are present. The chondriosomes are granules, most of them regular, some larger and coarser. Instead of surrounding the nucleus, as in the preceding generations, they are all located at one of its poles (fig. 6). During mitosis a breaking-up into smaller granules appears to take place. Their behavior is the same as described above and is illustrated for the stage of metaphase by figure 7. In fact, the size of the spindle is in proportion to the size of the cell so large that the chondriosomes have to take whatever place they can in the cell-body, which is practically filled by the karyokinetic figure.
 +
 
 +
In the first spermatocytes (fig. 8) the polar location of the chondriosomes persists throughout the whole growth-period until the prophase of the first division and coincides as always with the polar field, while in the nucleus the usual structural changes take place. The chondriosomes are now granules all equal in size and regularly spherical and most of them are very closel}^ heaped together. It must be noted that during this so-called growthperiod the spermatocytes of Fundulus actually grow very little and that there is no evidence, as in other spermatocytes, of an increase in the mass of chondriosomes.
 +
 
 +
At the prophase of the first division the mitochondria become scattered all around the nucleus and, when the spindle is formed, they are as previously pushed towards the periphery of the cellbody and very close to it ; for here again the spindle is very large in proportion to the cell. I may mention in passing that the centrioles appear very conspicuously at the poles of the spindle (fig. 9). During the anaphase all the mitochondria are found
 +
 
 +
 
 +
 
 +
TESTICLE-CELLS OF FUNDULUS 141
 +
 
 +
between the daughter-nuclei (figs. 10 and 11). The same process is repeated during the second division (fig. 12). Though the cells are very small, it is easy enough to distinguish both mitoses owing to the following characteristics. The first spermatocytes are larger than the second ones. The spindle at the stage of metaphase is more slender in the second division. The number of chondriosomes decreases conspicuously. Finally the size and shape of the chromosomes as observed in Benda's material present a most distinctive character: in the first division they are unmistakably heterotypic.
 +
 
 +
The spermatids, which are exceedingly small, very soon form an axile filament. At first the mitochondria are scattered all around the nucleus but only for a short time. In the succeeding stage which is very characteristic and which, judging from its frequent occurrence in the preparations, lasts apparently a considerable period, all the mitochondria are found accumulated in one heap at the posterior pole of the nucleus where they surround the proximal part of the axile filament (fig. 13). A glance at these cells readily gives the impression that the number of their mitochondria is constant. When one attempts to count them however, one realizes that to obtain exact figures is almost impossible, for the granules are very small and not all in the same level. The numbers I found in the most favorable cases came very close to eight.
 +
 
 +
Further stages of spermiogenesis are characterized by changes in the mitochondria (which will be described below), the growth of the tail and the following modifications of the nucleus. First, the posterior side, which is in close contact with the mitochondria, becomes flattened or even somewhat concave (fig. 14). Its chromatic content then gradually accumulates at the periphery, with the exception of the posterior or flattened side, a process whose occurrence has been described several times in invertebrates and which begins in Fundulus at the stage represented by figure 14. The crust of chromatin thus formed assumes the outline of a horse-shoe, the space existing between the free ends of its branches being occupied by the mitochondria; from the same space emerges the axile filament (fig. 15 et seq.). Later,
 +
 
 +
 
 +
 
 +
142 J. DUESBERG
 +
 
 +
the head becomes somewhat elongated and the branches of the horse-shoe are by the same process brought nearer together (fig. 16, 17 and 18). At the same time the head loses its symmetry inasmuch as it becomes somewhat curved along its antero-posterior axis and its posterior facet becomes oblique, instead of being perpendicular, to the same axis. From this time on we can distinguish what I have, arbitrarily of course, termed face- views (figs. 16 and 21) and side-views (figs. 17, 18 and 20) of the spermatozoon.
 +
 
 +
All the modifications of the head are more easily followed on acid fuchsin-methylgreen preparations than on Benda's, for methylgreen gives a sharper stain for chromatin than sodiumsulfalizarinate. In material fixed with Regaud's fluid the clear middle-space of the head appears very conspicuous even in the last stages; but curiously enough, as soon as the spermatozoa have reached the excretory ducts, the staining reaction changes and the head takes up acid fuchsin instead of methylgreen. In preparations made from material fixed with Benda's fluid the ripe spermatozoa, that is, those which have reached the excretory ducts, appear somewhat different from those fixed in Regaud's fluid. In a side-view (fig. 20) the clear middle-space appears only indistinctly. In face- views (fig. 21) on the other hand, the same space is very conspicuous and sharply delimited, and has the appearance of a canal running from the posterior to the anterior extremity of the head.
 +
 
 +
During this period changes take place in the mitochondria also. Their number decreases and their size increases: in other words, there is a fusion of granules. This process can be best followed in Regaud's preparations for the reason that the thin sheet of protoplasm which keeps the mitochondria in place (figs. 14 and 15) and which is hardly visible in Benda's preparations, swells in Regaud's fluid as do also the mitochondria themselves. Consequently, the cells and the granules are somewhat larger than in Benda's preparations and they are more scattered. These differences are well illustrated by figures 15 and 19, which represent approximately the same stage, after Regaud's and Benda's fixation respectively. Thus in figure 15 we can count
 +
 
 +
 
 +
 
 +
TESTICLE-CELLS OF FUNDULUS 143
 +
 
 +
exactly six granules while Benda's preparations of the same stage (fig. 19) show an undecipherable heap of mitochondria. Later when the asymmetry of the head has become conspicuous, we find almost invariably four mitochondria (fig. 16), disposed with remarkable regularity upon the posterior facet of the head. Finally in the ripe spermatozoon the number is still more reduced, usually to three. Here Benda's material is more serviceable than Regaud's owing to the change in the staining reactions of the head mentioned above. A comparison of the different stages of this evolution, as they appear after fixation in Regaud fluid, shows that the increase in volume of the mitochondria is not directly proportional to their decrease in number (figs. 13 to 18) ; and, as there is no evidence of an elimination of mitochondria, one would be led to believe in a strong condensation of the chondriosomal substance. This conclusion is however not supported by Benda's preparations and I am forced to admit that the swelling produced by the formalin-bichromate mixture is greater in the first stages of spermiogenesis than in the later ones.
 +
 
 +
As stated above, the average number of mitochondria in the ripe spermatozoon, as counted in Benda's preparations, is three. They are especially conspicuous in face- views (fig. 21), where they are found regularly disposed on the posterior facet of the head. Occasionally spermatozoa are found with four, five or even six granules taking the chondriosomal stain. The majority of these granules are undoubtedly mitochondria and in such cases the fusion has, for some unknown reason, apparently not proceeded normally. Whether it is completed later is difficult to say. It is probable also that occasionally the centrioles are stained, for in certain cases it was possible to recognize a relationship between the proximal extremity of the axile filament and a small granule stained like a chondriosome (fig. 20). I cannot give any definite information about the behavior of the centrioles during the spermiogenesis of Fundulus,^ but there is no doubt that they are located in that region.
 +
 
 +
One thing however is certain: that their behavior is very different from the
 +
same in selachians (Suzuki, '98).
 +
 
 +
 
 +
 
 +
144 J. DUESBERG
 +
 
 +
Again as in many and perhaps all cases, the last stages of spermiogenesis bring about a change in the behavior of the chondriosomes towards reagents. It is well known that, in the mammalian testicle for example, the chondriosomes become more and more resistant to acetic acid as spermiogenesis progresses.^ The test of this resistance was not made here, but it was found that the chondriosomes of the last stages are structures much less labile than the chondriosomes of the early stages and are consequently much easier to bring into evidence. «
 +
 
 +
The preceding description of the spermatozoon of Fundulus agrees in the main with Retzius' observations on the spermatozoon of other teleosts, though differing in the details. It helps at the same time to emphasize the similarity in structure between these spermatozoa and those of a large number of invertebrates, while the spermatozoa of selachians and of the higher vertebrates are widely different.
 +
 
 +
From the same description it also appears very probable that the male chondriosomes, owing to their close contact with the nucleus, are carried into the egg at the time of fertilization. Though this can be ascertained only by the study of the fertilizing process, the evidence accumulated by an imposing number of observations made upon almost all classes of animals, especially in recent years, is certainly very much in favor of the theory according to which the penetration of the male chondriosomes into the egg is a general phenomenon. Shaffer who mentions only Meves' observations on Ascaris and Vander Stricht's on the bat, overlooks the largest part of this evidence. That Lillie ('12) found in Nereis that the 'middle-piece' and the tail of the spermatozoon do not enter the egg does not prove that the chondriosomes are not carried into it.
 +
 
 +
I still believe, as in 1915, that the real objection to the admission that the male chondriosomes play a role in heredity is to be found in Meves' observations on the echinoderm-embryo ; why their admitted chemical composition should plead against
 +
 
 +
^ I found recentl}^ that the same changes take place in the spermatids of opossum.
 +
 
 +
 
 +
 
 +
TESTICLE-CELLS OF' FUNDULUS 145
 +
 
 +
such a role, as Cowdry ('16, p. 437) seems to believe, I fail entirely to see. Concerning the hypothesis of their motile function which, first formulated by Benda, reappears occasionally in the literature, I do not see that any arguments have been brought forward in its favor, nor is there any clear expression of how we should imagine this function. Benda considered his 'mitochondria' as contractile bodies : how can this conception be applied to the spherical chondriosomes of the spermatozoa of so many invertebrates and of Fundulus? Furthermore, those who advocate this hypothesis entirely overlook two groups of observations, which we have to accept as long as their inexactitude has not been demonstrated : first, Meves' experiments on the spermatozoon of Salamandra and second, the observations of a number of authors, lately Koltzoff's, on the spermatozoa of decapods (see Duesberg, '12, p. 687).
 +
 
 +
Finally a few words concerning the occurrence of a constant number of chondriosomes in male germ-cells.
 +
 
 +
The first indication of this was given by Meves ('00) who found that the small spermatocytes (i.e., as the apyrene generation) of Paludina vivipara contain on the average eight loopshaped chondriosomes. Numerations made on spermatids of the same generation a short time after the second division likewise revealed an almost unvarying number of chondriosomes,. this time four.
 +
 
 +
Two other cases, much more striking, have been described lately, both in arachnoids, the first one by Sokolov ('13), the other by Wilson ('16).
 +
 
 +
In the spermatogonia and in the young spermatocytes of Euscorpius carpathicus Sokolov describes mitochondria which soon by confluence form filaments. Later rings appear, which are probably formed by fusion of the free ends of the filaments of the preceding stages. The average number of these rings is twentyfour. During mitosis they are not divided as is the case in the small spermatocytes of Paludina, but are segregated into two equal groups between the daughter-cells. Thus each spermatid contains one quarter of the number of rings, on the average six.
 +
 
 +
 
 +
 
 +
146 J. DUESBERG
 +
 
 +
The result of this process is an obvious and measureable reduction of the chondriosomal mass at the end of the divisions of maturation and Sokolov sees in it a confirmation of the views I have expressed as the result of my study of the behavior of the chondriosomes in the spermatocyte-divisions of the rat ('07) .
 +
 
 +
Wilson has studied the chondriosomes in the spermatogenesis of two other species of scorpions, Opisthacanthus elatus (Southern California) and Centrums oxilicauda (Southern Arizona). The results obtained from the study of the first named species are very similar to those of Sokolov. Each spermatocyte contains about twenty-four hollow spheroidal bodies, which are segregated by the spermatocyte-divisions into four approximately equal groups. Each spermatid thus receives as a rule six chondriosomes (in 73 per cent of the cases, on 200 numerations), sometimes five (in 16 per cent of the cases) or seven. No other numbers were observed. In the Arizona-scorpion, the process is quite different. All the chondriosomal material becomes concentrated in a single definite body in the form of a ring. This ring divides during mitosis in such a way that each spermatid receives exactly one-fourth of its substance, the process taking place with a precision that is comparable to that seen in the distribution of the chromosome material."
 +
 
 +
As Wilson points out the body in question represents a hitherto undescribed type of chondriosome. The occurrence of this interesting process makes one speculate as to what the field of spermatogenesis, though so widely explored, still has in store for the investigator. It appears to me that conditions similar to those found in scorpions, at least to those found in Euscorpius and in Opisthacanthus, could be expected in the histogenesis of these spermatozoa in which, as stated above, the chondriosomes are represented by a constant or approximately constant number of well-defined granules. There is some indication of a similar process in Fundulus, but the small size of the cells unfortunately makes an exact numeration impossible. The same difficulty would certainly be met with in the study of the seminal cells of other teleosts as well as of echinoderms and celenterates ; molluscs and worms, however, would probably be a favorable material.
 +
 
 +
 
 +
 
 +
TESTICLE-CELLS OF FUNDULUS 147
 +
 
 +
BIBLIOGRAPHY
 +
 
 +
BoNNEViE, Kr. 1907 Untersuchungen iiber Keimzellen. 2. Physiologische
 +
 
 +
Polyspermie bei Bryozoen. lenaische Zeitschr., Bd. 42. CowDRY, E. V. 1916 The general functional significance of mitochondria.
 +
 
 +
Am. Jour. Anat., vol. 19. DuESBERG, J. 1907 Der Mitochondrialapparat in den Zellen der Wirbeltiere
 +
 
 +
und Wirbellosen. I. Arch, flir mikr. Anat., Bd. 71.
 +
 
 +
1910 Nouvelles recherches sur I'appareil mitochondrial des cellules
 +
 
 +
seminales. Arch, fiir Zellf., Bd. 4.
 +
 
 +
1912 Plastosomen, "Apparato reticolare interno" und Chromidial apparat. Ergeb. der Anat. und Entwickl., B. 20.
 +
 
 +
1915 Recherches cytologiques sur la fecondation des Ascidiens et sur
 +
 
 +
leur developpement. Contr. to Erabr. Carnegie Inst., 223.
 +
 
 +
1917 Chondriosomes in the cells of fish-embryos. Am. Jour.
 +
 
 +
Anat., vol. 21. Field, G. W. 1895 On the morphology and physiology of the echinoderm spermatozoon. Journ. INIorph., vol. 2. Friedmann, Fr. 1898 Beitriige zur Kenntniss der Anatomic und Physiologic
 +
 
 +
der mannlichen Geschlechtsorgane. Arch. fiir. mikr. Anat., Bd.
 +
 
 +
52. Ganfini, C. 1902 Struttura e sviluppo delle cellule interstitiali del testicolo.
 +
 
 +
Arch. ital. di Anat. e di Embr. vol. I. Jordan, H. E. 1911 The spermatogenesis of the opossum (Didelphys virgini ana), with special reference to the accessory chromosome and the
 +
 
 +
chondriosomes. Arch, fiir Zellforsch., Bd. 7. Lewis, M. R. 1917 The effect of certain vital stains upon the development of
 +
 
 +
the eggs of Cerebratulus lacteus, Echinorachnius parma and Lophius
 +
 
 +
piscatorius. Anat. Rec, vol. 13. LiLLiE, F. R. 1912 Studies of fertilization in Nereis. Jour. Exp. Zool., vol.
 +
 
 +
12. Meves, Fr. 1891 Ueber amitotische Kernteilung in den Spermatogonien des
 +
 
 +
Salamanders und Verhalten der Attraktionsphare bei derselben.
 +
 
 +
Anat. Anz., Bd. 6.
 +
 
 +
1900 Ueber den von la Valette St. George entdeckten Nebenkern
 +
 
 +
(Mitochondrienkorper) der Samenzellen. Arch, fiir mikr. Anat., Bd.
 +
 
 +
56.
 +
 
 +
1903 Ueber olygopyrene und apyrene Spermien und ihre Entstehung,
 +
 
 +
nach Beobachtungen an Paludina und Pygeara. Arch, fiir mikr.
 +
 
 +
Anat., Bd. 61.
 +
 
 +
1912 Verfolgung des sogenannten Mittelstiickes des Echiniden spermiums im befruchteten Ei bis zum Ende der ersten Furchungsteil ung. Arch, fiir mikr. Anat., Bd. 80. Payne, F. 1917 A study of the germ-cells of Gryllotalpa borealis and Gryl lotalpa vulgaris. Jour. Morph., vol. 28. PiCTET, C. 1891 Recherches sur la spermatogenese chez quelques inverte bres de la Mediterranee. Mitt, aus der zool. Station zu Neapel, Bd.
 +
 
 +
10.
 +
 
 +
 
 +
 
 +
148 J. DUESBERG
 +
 
 +
PrenaNT, A. 1911 Pr(?parations relatives aux mitochondries. Comptes-Rendus
 +
 
 +
Assoc. Anat. Paris. Retzius, G. 1904 a Zur Kenntnis der Spermien der Evertebraten. I. Biol.
 +
 
 +
Unters., N.F., Bd. 11.
 +
 
 +
1904 b Zur Kenntnis der Spermien der Evertebraten. Verhdl. Anat. Gesellsch. Jena.
 +
 
 +
1905 a Zur Kenntnis der Spermien der Evertebraten. 2. Biol. Unters, N.F., Bd. 12.
 +
 
 +
1905 b Zur Kenntnis der Spermien der Leptokardier, Teleostier und Ganoiden. Ibid.
 +
 
 +
1906 a Die Spermien der Gastropoden. Biol. Unters, N.F., Bd. 13. 1906 b Die Spermien der Enteropneusten und der Nemertinen. Ibid.
 +
 
 +
1906 c Die Spermien der Turbellarien. Ibid.
 +
 
 +
1906 d Die Spermien der Bryozoen. Ibid.
 +
 
 +
1909 a Die Spermien von Aurelia aurita (L). Biol. Unters., N.F.,
 +
 
 +
Bd. 14.
 +
 
 +
1909 b Die Spermien der Nereiden. Ibid.
 +
 
 +
1909 c Zur Kenntnis der Spermien der Elasmobranchier. Ibid.
 +
 
 +
1910 a Zur Kenntnis der Spermien der Echinodermen. Biol. Unters., N.F., Bd. 15.
 +
 
 +
1910 b Weitere Beitrage zur Kenntnis der Spermien mit besonderer
 +
 
 +
Beriicksichtigung der Kernsubstanz. Ibid. ScHREiNER, A. AND K. E. 1905 Ueber die Entwicklung der miJnnlichen Ge schlechtszellen von Myxine glutinosa. Archives de Biologic., vol. 21.
 +
 
 +
1908 Zur Spermienbildung der Myxinoiden. Arch, fiir Zellf., Bd. I. Shaffer, E. L. 1917 Mitochondria and other cytoplasmic structures in the
 +
 
 +
spermatogenesis of Passalus cornutus. Biol. Bull., vol. 32. SoKOLOV, I. 1913 Untersuchungen tiber die Spermatogenese bei den Arachni den. I. t'ber die Spermatogenese der Skorpione. Arch, fiir Zellf. ,Bd.
 +
 
 +
9. Suzuki, B. 1898 Notiz uber die Entstehung des Mittelstlickes der Samenfaden
 +
 
 +
von Selachiern. Anat. Anzeiger, Bd. 15. Wilson, E. B. 1916 The distribution of the chondriosomes to the spermatozoa
 +
 
 +
in scorpion. Science, N.S., 43 and Proceedings of Nat. Acad, of
 +
 
 +
Sciences.
 +
 
 +
 
 +
 
 +
PLATES
 +
 
 +
 
 +
 
 +
149
 +
 
 +
THE AMERICAN JOURNAL OF ANATOMY, VOL. 23, NO. 1
 +
 
 +
 
 +
 
 +
EXPLANATION OF FIGURES
 +
 
 +
All figures were outlined with a Zeiss camera-lucida, at the level of the stage of the microscope. Lens used: Zeiss apochr. imm. 1 m.m., 5; ocular 12. Artificial light (gas).
 +
 
 +
PLATE 1
 +
 
 +
EXPLANATIOX OF FIGURES
 +
 
 +
1 Fundulus majalis. Fixation: Benda, without acetic acid. Stain: Benda. Epithelium of an excretory duct.
 +
 
 +
2 Fundulus heteroclitus. Fixation: Benda. Stain: Benda. Supptised interstitial cells.
 +
 
 +
3 Fundulus majalis. Fixation: Benda, without acetic acid. Stain: Benda. Group of primary and secondary spermatogonia.
 +
 
 +
 
 +
 
 +
150
 +
 
 +
 
 +
 
 +
TESTICLE-CELLS OF FUNDULUS
 +
 
 +
J. DUESBERG
 +
 
 +
 
 +
 
 +
PLATE 1
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
151
 +
 
 +
 
 +
 
 +
PLATE 2
 +
 
 +
EXPLANATION OF FIGURES
 +
 
 +
4 and 5 Fundulus heteroclitus. Fixation: Benda. Stain: Benda. Metaphase and anaphase of the mitotic division of primary spermatogonia.
 +
 
 +
6 Same material. Tertiary spermatogonium.
 +
 
 +
7 Same material. Tertiary spermatogonium : metaphase.
 +
 
 +
8 Fundulus majalis. Fixation: Benda, without acetic acid. Stain: Benda. First spermatocyte.
 +
 
 +
9 and 10 Same material. ^Nletaphase and anaphase of first division of maturation.
 +
 
 +
11 Fundulus heteroclitus. Fixation: Regaud. Stain: acid fuchsin-methjdgreen. Anaphase of first division of maturation.
 +
 
 +
12 Same material. Anaphase of second division of maturation.
 +
 
 +
13 to 18 Same material. Six stages of spermiogenesis; in none is the tail represented in its full length. 16 and 17 are respectively face-view and side-view of approximatively the same stage.
 +
 
 +
19 Fundulus heteroclitus. Fixation: Benda. Stain: Benda. Group of spermatids in a cyst.
 +
 
 +
20 and 21 Fundulus majalis. Fixation: Benda, without acetic acid. Stain: Benda. Spermatozoa from the excretory ducts (the tail is not represented in its full length). 20: side-view; 21: face-view.
 +
 
 +
 
 +
 
 +
152
 +
 
 +
 
 +
 
 +
TESTICLE-CELLS OF FUNDULUS
 +
 
 +
J. DUESBERG
 +
 
 +
 
 +
 
 +
PLATE 2
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
.^4^
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
%'
 +
 
 +
 
 +
 
 +
i'^i^
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
p
 +
 
 +
 
 +
 
 +
0- 13
 +
 
 +
 
 +
 
 +
2C> 21
 +
 
 +
 
 +
 
 +
t^
 +
 
 +
 
 +
 
 +
153
 +
 
 +
 
 +
 
 +
author's abstract of this paper issued by thk bibliookaphic service, decemher 1
 +
 
 +
 
 +
 
 +
THE POSITION OF THE INSERTION OF THE PECTO RALIS MAJOR AND DELTOID MUSCLES ON
 +
 
 +
THE HUMERUS OF MAN
 +
 
 +
ADOLF H. SCHULTZ
 +
 
 +
Carnegie Institution of Washington
 +
 
 +
THREE FIGURES
 +
 
 +
The metrical determination of the position of the attachments of muscles to bones is a problem which affords a contribution to topographicaJ anatomy. It is also of importance from the standpoint of musculo-mechanics, because measuring the insertions of muscles is analogous to the determination of the lengths of leverage of the body. Since such an investigation promises to give information regarding differences or equalities of race or sex, as well as of the two halves of the body, it is of no less interest to the anthropologist. As far as the author can determine from a study of anatomical and anthropological literature, no measurements of the insertions of muscles have as yet been undertaken. In approaching this problem one is working in a new field of osteometry, where it becomes necessary to treat the bone not separately but in conjunction with the associated muscles, W'hich have been so neglected in anthropometry.
 +
 
 +
The following study deals with the insertion of the pectoralis major and deltoid muscles. The measurements were made on the right and left arms of one hundred and five bodies. Fortysix of these bodies were obtained from the University of Maryland in Baltimore, forty from the Jefferson Medical College in Philadelphia and nineteen from the Johns Hopkins Medical School in Baltimore. The author wishes here to express his appreciation to r)rs. W. H. Lewis, J. P. Schaeffer, J. Holmes Smith and J. W. Holland for their kindness in permitting the use of this material. All of the subjects measured were adults; juvenile and senile ones were excluded. It is regrettable that the sexes
 +
 
 +
155
 +
 
 +
 
 +
 
 +
156 ADOLF H, SCHULTZ
 +
 
 +
were very unequally represented, for the females numbered only twenty-seven, as against seventy-eight male subjects. A greater uniformity occurred in race, as there were fifty-one white and fifty-four colored bodies. The author wishes to call attention to the fact that the term race is used in its widest sense in the present paper, because both the white and colored inhabitants of America have originated from nmnerous races in a limited sense. In negroes one frequently witnesses a more or less extensive admixture of white blood; in cases where there was evidence of a too great intermingling with the white element the material was discarded.
 +
 
 +
The position of the muscle insertion was compared with the length of the humerus by measuring the distance of the most proximal and the most distal point of attachment from the proximal end of the bone, and further by determining the arithmetical mean of these distances in percentage of the length of the humerus. For this purpose we need first of all six exact points of measurement, a proximal and a distal point on the humerus, two corresponding points on the pectoralis major and two more on the deltoid. The two points on the humerus are found by measuring the length of the bone, choosing the distance of the highest point of the caput humeri from the lowest point of the capitulum and measuring parallel to the axis of the bone (fig. 1, points I and II). The points of measurement for the pectoralis major muscle are the most proximal and the most distal points of its insertion on the crista tuberculi majoris (fig. 1, points III and IV) ; as a rule they are readily determined. Occasionally the distal portion of the insertion is intimately connected with the tendon of the deltoid muscle and the distal point can only be obtained after careful separation of these structures. In a, limited number of cases the dorsal reflected portion of the muscle was observed to form a narrow tendinous band in the region where it spreads out proximally to join the tendinous lining of the sulcus intertubercularis (in figure 1 such an instance is indicated at a). In such cases this prolongation was ignored and the point of measurement taken at its distal end. The lower point of measurement of the deltoid is comparatively easy to
 +
 
 +
 
 +
 
 +
PECTORALIS MAJOR AND DELTOID INSERTION
 +
 
 +
 
 +
 
 +
157
 +
 
 +
 
 +
 
 +
 
 +
Fig. 1 Diagram of the points of measurement and distances on a right humerus seen from in front.
 +
 
 +
 
 +
 
 +
158 ADOLF H. SCHULTZ
 +
 
 +
ascertain, namely as the most distal point of the insertion on the tuberositas deltoidea (fig. 1, point VI). The most proximal point of insertion is frequently concealed by the body of the muscle and it is necessary therefore to remo\'e it partially. In doing this great care should i^e exercised as the deltoid is usually attached at its uppermost end by very delicate strands (fig. 1, point V). The distance between each of these four points and the highest point of the head of the humerus was measured parallel to the axis of the bone, similar to the longitudinal measurement of the humerous mentioned above and therefore these measurements are projections.
 +
 
 +
The measuring instrument employed was a modified small anthropometer of Martin (Stangenzirkel). This instrument is composed of a ruled metal bar or beam, possessing two arras at right angles to it, one of which is firmly attached to the end, the other movable in the direction of the bar, while both are movable at right angles to the latter. The modification consists merely in the addition of a third arm from another instrument of the same kind, which can also be moved both in the same direction and at right angles to the main axis (fig. 2).
 +
 
 +
First one measures the length of the humerus with the two outermost arms of the instrument holding the bar parallel to the axis of the bone, then the middle arm is approximated in turn to the four points of muscle insertion as defined above. This is performed by moving the arm up and down as required, shortening or lengthening it, simultaneously rotating the entire instrument around the axis of the humerus if necessary. Readings are taken each time on the ruled bar and correspond with the measurements two, three, four and five in figure 1. Indices for the relative position of the middle point of each muscle insertion were obtained b}- the following formulae:
 +
 
 +
nieasurcniont 2 + measurement 3
 +
 
 +
2
 +
 
 +
X 100 lor (he pectoralis major
 +
 
 +
measurement 1
 +
 
 +
measurement 4 + measurement 5
 +
 
 +
 
 +
 
 +
measurement 1
 +
 
 +
 
 +
 
 +
X 100 for the deltoid
 +
 
 +
 
 +
 
 +
PECTORALIS MAJOR AND DELTOID INSERTION
 +
 
 +
 
 +
 
 +
159
 +
 
 +
 
 +
 
 +
The greater these indices of position, the more distal, the smaller, the more proximal is the insertion of the muscle. Following is a short description of the mathematical treatment of the length of the humerus and the indices which have been used in this paper. A more detailed explanation of these methods, which are absolutely necessary for an understanding of the
 +
 
 +
 
 +
 
 +
Fig. 2 Small anthropometer with three parallel movable arms.
 +
 
 +
measurements on a large number of individuals, is to be found in the Textbook of Anthropology by R. Martin, Jena, 1914, p]). 63-103.
 +
 
 +
The average (M) is the arithmetical mean of the individual
 +
 
 +
values (F) of a group (/?. = number of individuals): M = -i;F.
 +
 
 +
 
 +
 
 +
The standard deviation (a) is the square root of the average of
 +
 
 +
 
 +
 
 +
160 ADOLF H. SCHULTZ
 +
 
 +
the squares of the deviations of the individual values from the average of the row and expresses the absolute variability:
 +
 
 +
(T = W-S(F — M)-. The variation coefficient (v) expresses the
 +
 
 +
standard deviation in percentage of the average, whereby a
 +
 
 +
criterion for the relative variability is obtained : v = -—-- . The
 +
 
 +
correlation coefficient (r) affords a means of determining the law,
 +
 
 +
according to which two characteristics combine. It is the sum
 +
 
 +
of the products of the deviations of the two characteristics from
 +
 
 +
the corresponding averages taken for each individual, divided by
 +
 
 +
the product of the number of individuals and the two standard
 +
 
 +
2 (x — X) (y — Y)
 +
 
 +
deviations : r = — — ~ . A complete correlation
 +
 
 +
na^ ay
 +
 
 +
exists when r = 1. If r = 0, no relation prevails between the two characteristics. A positive correlation coefficient indicates a change of the characteristics in the same direction, a negative one, in the opposite direction. Finally, to test the degree of exactness of the above formulae, the probable error (E) was determined by the following formulae:
 +
 
 +
 
 +
 
 +
EiM) = ± 0.6745 -J- for the average.
 +
 
 +
■\ n
 +
 
 +
E {a) = ±0.674:5—; — •- for the ptaudard deviation.
 +
 
 +
V2ft
 +
 
 +
V
 +
 
 +
E (v) = ±0.6745— — for the variation coefficient.
 +
 
 +
V2//.
 +
 
 +
 
 +
 
 +
If r > 10, the hist forinuhi must be multiplied bv -v'l + 2 ( — - )
 +
 
 +
- \ VlOO/
 +
 
 +
E (r) = ±0.6745 — r^— for the correlation coefficient
 +
 
 +
■\ n
 +
 
 +
The relation of the insertion of the muscles to the length of the humerus makes a short prehminary discussion of this absolute measurement necessary. Table 1 is a compilation of the averages and the conditions of variability of the length of the two hiuidred and ten humeri, which were measured. The extremes of these measurements range from 260 to 367 mm. The humerus in male whites is on the average 26 mm., in male negroes 31.8 mm.
 +
 
 +
 
 +
 
 +
PECTORALIS MAJOR AND DELTOID INSERTION
 +
 
 +
 
 +
 
 +
161
 +
 
 +
 
 +
 
 +
TABLE 1
 +
 
 +
Averages, standard deviations, variation coefficients, their probable errors and ranges of variation for the length of the humerus
 +
 
 +
 
 +
 
 +
RACE
 +
 
 +
 
 +
SEX
 +
 
 +
 
 +
NUMBER
 +
 
 +
 
 +
SIDE
 +
 
 +
 
 +
M ± E (M)
 +
 
 +
 
 +
a ± B (o-)
 +
 
 +
 
 +
V d= E (r)
 +
 
 +
 
 +
Minimum
 +
 
 +
 
 +
Maximum
 +
 
 +
 
 +
 
 +
 
 +
cT
 +
 
 +
 
 +
40
 +
 
 +
 
 +
Y.
 +
 
 +
 
 +
316.3±1.71
 +
 
 +
 
 +
15.94±1.22
 +
 
 +
 
 +
5.04±0.38
 +
 
 +
 
 +
283
 +
 
 +
 
 +
347
 +
 
 +
 
 +
 
 +
 
 +
o^
 +
 
 +
 
 +
40
 +
 
 +
 
 +
1.
 +
 
 +
 
 +
316.5±1.82
 +
 
 +
 
 +
17.05±1.29
 +
 
 +
 
 +
5.40±0.41
 +
 
 +
 
 +
283
 +
 
 +
 
 +
352
 +
 
 +
 
 +
Whites <
 +
 
 +
 
 +
d^
 +
 
 +
 
 +
80
 +
 
 +
 
 +
r. 1.
 +
 
 +
 
 +
316. 4± 1.25
 +
 
 +
 
 +
16.50±0.87
 +
 
 +
 
 +
5.22±0.27
 +
 
 +
 
 +
283
 +
 
 +
 
 +
352
 +
 
 +
 
 +
9
 +
 
 +
 
 +
11
 +
 
 +
 
 +
r.
 +
 
 +
 
 +
293.1±2.58
 +
 
 +
 
 +
12.65±1.81
 +
 
 +
 
 +
4.32±0.62
 +
 
 +
 
 +
269
 +
 
 +
 
 +
309
 +
 
 +
 
 +
 
 +
 
 +
9
 +
 
 +
 
 +
11
 +
 
 +
 
 +
1.
 +
 
 +
 
 +
287.7±2.02
 +
 
 +
 
 +
9.92±1.42
 +
 
 +
 
 +
3.44±0.50
 +
 
 +
 
 +
267
 +
 
 +
 
 +
303
 +
 
 +
 
 +
 
 +
 
 +
9
 +
 
 +
22
 +
 
 +
 
 +
r. 1.
 +
 
 +
 
 +
290.4±1.67
 +
 
 +
 
 +
11.69±1.19
 +
 
 +
 
 +
4.03±0.41
 +
 
 +
 
 +
267
 +
 
 +
 
 +
309
 +
 
 +
 
 +
 
 +
 
 +
c^
 +
 
 +
 
 +
38
 +
 
 +
 
 +
Y.
 +
 
 +
 
 +
326.2±1.87
 +
 
 +
 
 +
17.18±1.34
 +
 
 +
 
 +
5.27±0.41
 +
 
 +
 
 +
290
 +
 
 +
 
 +
367
 +
 
 +
 
 +
 
 +
 
 +
&
 +
 
 +
 
 +
38
 +
 
 +
 
 +
1.
 +
 
 +
 
 +
323. 5 ±2. 02
 +
 
 +
 
 +
18.50±1.44
 +
 
 +
 
 +
5.71±0.44
 +
 
 +
 
 +
283
 +
 
 +
 
 +
365
 +
 
 +
 
 +
Negroes . . . <
 +
 
 +
 
 +
c^
 +
 
 +
 
 +
76
 +
 
 +
 
 +
Y. 1.
 +
 
 +
 
 +
324 8±1.39
 +
 
 +
 
 +
17.89±0.98
 +
 
 +
 
 +
5.50±0.30
 +
 
 +
 
 +
283
 +
 
 +
 
 +
367
 +
 
 +
 
 +
9
 +
 
 +
 
 +
16
 +
 
 +
 
 +
r.
 +
 
 +
 
 +
294.6±2.45
 +
 
 +
 
 +
14.51±1.74
 +
 
 +
 
 +
4.92±0.59
 +
 
 +
 
 +
266
 +
 
 +
 
 +
321
 +
 
 +
 
 +
 
 +
 
 +
9
 +
 
 +
 
 +
16
 +
 
 +
 
 +
1.
 +
 
 +
 
 +
291.5±2.51
 +
 
 +
 
 +
14.84±1.78
 +
 
 +
 
 +
5.10±0.61
 +
 
 +
 
 +
260
 +
 
 +
 
 +
312
 +
 
 +
 
 +
 
 +
 
 +
9
 +
 
 +
 
 +
32
 +
 
 +
 
 +
r. 1.
 +
 
 +
 
 +
293.0±1.77
 +
 
 +
 
 +
14.75±1.24
 +
 
 +
 
 +
5.03±0.42
 +
 
 +
 
 +
260
 +
 
 +
 
 +
321
 +
 
 +
 
 +
 
 +
longer than in females. The averages in negroes exceed in both sexes the corresponding values for whites. The division of table 1 into separate rows for the right and left humerus shows that the variability is greater on the left side except in the group of white females of which the number measured was quite small. Furthermore it shows that the white males, who possess the same average length of the humerus on both sides, form an exception to the rule of the greater length of the humerus on the right side. Table 2, which gives a survey of the absolute and
 +
 
 +
 
 +
 
 +
TABLE 2
 +
 
 +
 
 +
 
 +
Absolute and relative numbers of individuals uiith equal and different lengths of the humeri and average differences of the individual asymmetries (mm.)
 +
 
 +
 
 +
 
 +
 
 +
 
 +
SEX
 +
 
 +
9 9
 +
 
 +
 
 +
BOTH SIDES EQITAL
 +
 
 +
 
 +
RIGHT SIDE LONGER
 +
 
 +
 
 +
 
 +
 
 +
AVERAGE DIFFERENCE IF
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
Right
 +
 
 +
side
 +
 
 +
longer
 +
 
 +
 
 +
Left
 +
 
 +
side
 +
 
 +
longer
 +
 
 +
 
 +
Whites 1
 +
 
 +
Negroes <
 +
 
 +
 
 +
10=25.0% 0= 0.0%
 +
 
 +
8 = 21.0%
 +
 
 +
7 = 43.8%
 +
 
 +
 
 +
17 = 42.5%
 +
 
 +
9 = 81.8%
 +
 
 +
22 = 58.0% 9 = 56.2%
 +
 
 +
 
 +
13 = 32.5%
 +
 
 +
2 = 18.2%
 +
 
 +
8 = 21.0% 0= 0.0%
 +
 
 +
 
 +
3.88 7.67
 +
 
 +
5.73 5.44
 +
 
 +
 
 +
5.62 5.00
 +
 
 +
3.25
 +
 
 +
 
 +
 
 +
 
 +
162
 +
 
 +
 
 +
 
 +
ADOLF H. SCHULTZ
 +
 
 +
 
 +
 
 +
relative number of cases possessing humeri of equal and different lengths and the average differences of the individual asymmetries, shows that 32.5 per cent of white males have a longer left humerus. It also demonstrates that in white males the differences in favor of the left side are on the average greater than those on the right, which is not the case in the other groups. The greatest absolute asymmetry occurred in a negro whose right humerus exceeded the left in length by 23 mm.
 +
 
 +
TABLE 3
 +
 
 +
Averages, standard deviations, variation-coefficients, their probable errors and ranges of variation for the position index of the insertion of the pectoralis major m uscle
 +
 
 +
 
 +
 
 +
Whites
 +
 
 +
 
 +
 
 +
Negroes
 +
 
 +
 
 +
 
 +
SEX
 +
 
 +
 
 +
XUMBER
 +
 
 +
 
 +
SIDE
 +
 
 +
 
 +
f ^
 +
 
 +
 
 +
40
 +
 
 +
 
 +
r.
 +
 
 +
 
 +
cf
 +
 
 +
 
 +
40
 +
 
 +
 
 +
1.
 +
 
 +
 
 +
d^
 +
 
 +
 
 +
80
 +
 
 +
 
 +
Y. 1.
 +
 
 +
 
 +
9
 +
 
 +
 
 +
11
 +
 
 +
 
 +
r.
 +
 
 +
 
 +
9
 +
 
 +
 
 +
11
 +
 
 +
 
 +
1.
 +
 
 +
 
 +
L 9
 +
 
 +
 
 +
22
 +
 
 +
 
 +
r. 1.
 +
 
 +
 
 +
I ^
 +
 
 +
 
 +
38
 +
 
 +
 
 +
Y.
 +
 
 +
 
 +
d"
 +
 
 +
 
 +
38
 +
 
 +
 
 +
1.
 +
 
 +
 
 +
cf
 +
 
 +
 
 +
76
 +
 
 +
 
 +
r. 1.
 +
 
 +
 
 +
9
 +
 
 +
 
 +
16
 +
 
 +
 
 +
r.
 +
 
 +
 
 +
9
 +
 
 +
 
 +
16
 +
 
 +
 
 +
1.
 +
 
 +
 
 +
L 9
 +
 
 +
 
 +
32
 +
 
 +
 
 +
r. 1.
 +
 
 +
 
 +
 
 +
M ± E {M)
 +
 
 +
 
 +
 
 +
a± E (o-)
 +
 
 +
 
 +
 
 +
28. 37 ±0 17 1.5o±0.12
 +
 
 +
■28.50±0.16 1.49±0.11
 +
 
 +
28 43±0 11 1 o2±0.08
 +
 
 +
26 37±0.35 1.74±0 25
 +
 
 +
26 35 ±0.56 2.73±0.39
 +
 
 +
26.36±0.33 2.29±0 23
 +
 
 +
28 27±0.19 1.75±0.14
 +
 
 +
27.99±0.17 1 57±0.12
 +
 
 +
28.13±0.13 1 67±0 09
 +
 
 +
26 82 ±0 32 1 89±0 23
 +
 
 +
26 45±0 3l| 1.82±0 22
 +
 
 +
26.6]±0 22 1.86±0.16
 +
 
 +
 
 +
 
 +
E (v)
 +
 
 +
 
 +
 
 +
5.46±0.41
 +
 
 +
5 23±0.40 5.35±0.28 6.59±0.94
 +
 
 +
10.34±1.48 8.67=t0.88
 +
 
 +
6.18±0.48 5.61=t0.44 5.94±0.33 7.05±0 85
 +
 
 +
6 89±0.83 6.99±0.59
 +
 
 +
 
 +
 
 +
MINI- MAXMUM MUM
 +
 
 +
 
 +
 
 +
24.3 25.3 24.3 21.9 21.3 21.3
 +
 
 +
26.1 25.1 25.1 23.1 22.3 22.3
 +
 
 +
 
 +
 
 +
31.3 31.8 31.8 28.3 29.5 29.5
 +
 
 +
35.2 32.6 35.2 30.9 30.3 30.9
 +
 
 +
 
 +
 
 +
The averages and the conditions of variability of the index of position for the middle of the insertion of the pectoralis major muscle are given in table 3. This index differs in the entire material between 21.3 and 35.2. Expressing this in terms of the mechanics of levers, one can state that in the adult the lifting arm of the musculus pectoralis major is related to the carrying arm — the length of the humerus — in a ratio varying from 21.3: 100 to 35.2: 100. In other words the relation of the lever arms may differ by almost 14 per cent of the length of the carrying arm, and this expressed in an absolute number equals on the average about 45 mm. A different proi:)()rtion of the lever arms influences not onlv the force of the muscle hut also the movement
 +
 
 +
 
 +
 
 +
PECTORALIS MAJOR AND DELTOID INSERTION 163
 +
 
 +
of the lever, if the shortening of the muscles is equal. This can be readily seen from the diagram (fig. 3) A —B represents the humerus, its caput at A. C and D correspond to the two most extreme points of insertion of the pectoralis major. 1 and 2 indicate the two appertaining muscles. Should the latter shorten by the same amounts C — C' = D' — D' , than the lower end of the humerus B is moved more extensively by muscle 1 (to B") than by muscle 2 (to B'), for instance the humerus is turned through a greater angle when the muscles are contracting equally by the more proximal one, and consequently also more quickly, A more distally situated pectoralis major would have to contract more in order to pull the arm forward to a certain
 +
 
 +
 
 +
 
 +
D'-/--..C'
 +
 
 +
 
 +
 
 +
B ' * i — '^'-^^-'- A
 +
 
 +
D C
 +
 
 +
Fig. 3 Diagram of the movements of the humerus with equal shortening of the pectoralis major muscle at different points of attachment.
 +
 
 +
angle than would be necessary if it were more proximally attached. With increased contraction, however, a muscle loses in tension and consequently the greater shortening of the more distal muscle diminishes the advantage of its favorable lever arm.
 +
 
 +
In the group of whites as well as in the negroes, the averages of the index of position in the males exceed those of the females, the differences being 2.07 and 1.50 respectively. Since the probable errors of these averages are only small, this sexual difference, such as that the female possesses a more proximally attached pectoralis major muscle, must be considered as a rather essential and definite one. It is not only of interest in connection with the above mentioned consideration of musculo-mechaniscs, but also
 +
 
 +
 
 +
 
 +
164
 +
 
 +
 
 +
 
 +
ADOLF H. SCHULTZ
 +
 
 +
 
 +
 
 +
indicates that the female arm is looser, that is the perpendicular diameter of the axilla in the female is relatively shorter since the caudal edge of the pectoralis major at its lateral end represents the lower border of the axilla. The average of both sexes in the whites hardly differs from that found in the negroes; the two races are alike therefore in regard to the position of the insertion of the pectoralis major. The variation coefficient of the index is always rather high and even exceeds 10 in one case. The conclusion can be drawn from this that the position of the attachment of the pectoralis major can be only slightly influenced by the length of the humerus, since the index connecting these two
 +
 
 +
TABLE 4 Absolute and relative numbers of individuals ivith symmetrical and asymmetrical
 +
 
 +
 
 +
 
 +
position of the pectoralis major insertion and aver
 +
 
 +
 
 +
jges of the indi
 +
 
 +
 
 +
vidual
 +
 
 +
 
 +
differ
 +
 
 +
ences of the position index
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
AVER.A.GE
 +
 
 +
 
 +
RACE
 +
 
 +
 
 +
SEX
 +
 
 +
 
 +
THE SAME POSITION ON BOTH
 +
 
 +
 
 +
POSITION ON THE RIGHT MORE
 +
 
 +
 
 +
POSITION ON THE LEFT MORE
 +
 
 +
 
 +
DIFFERENCE IF MORE DIST.\.LLT
 +
 
 +
 
 +
 
 +
 
 +
&
 +
 
 +
 
 +
SIDES
 +
 
 +
 
 +
DISrAL
 +
 
 +
 
 +
DIST.U,
 +
 
 +
 
 +
on the right
 +
 
 +
 
 +
on the left
 +
 
 +
 
 +
Whites 1
 +
 
 +
 
 +
1 = 2.5%
 +
 
 +
 
 +
17 = 42.5%
 +
 
 +
 
 +
22=55.0%
 +
 
 +
 
 +
1.38
 +
 
 +
 
 +
1.29
 +
 
 +
 
 +
9
 +
 
 +
 
 +
= 0.0%
 +
 
 +
 
 +
4 = 36.4%
 +
 
 +
 
 +
7 = 63.6%
 +
 
 +
 
 +
2.17
 +
 
 +
 
 +
1.21
 +
 
 +
 
 +
Negroes <
 +
 
 +
 
 +
9
 +
 
 +
 
 +
2 = 5.2% = 0.0%
 +
 
 +
 
 +
18 = 47.4% 13 = 81.2%
 +
 
 +
 
 +
18 = 47.4% 3 = 18.8%
 +
 
 +
 
 +
1.70 62
 +
 
 +
 
 +
1.10 0.73
 +
 
 +
 
 +
 
 +
is quite variable. In order to throw some light on the question concerning the relationship of the point of attachment and the strength of the muscle, the individuals of each group were divided into four subgroups, namely into weak, medium, strong and very strong ones. Positive and negative variants of the index of position were found to be indiscriminately distributed among the four subgroups, in whites as well as in negroes and in males as well as in females. The strength of the muscle has therefore no influence on the position of the attachment. Table 4 shows a grouping of the absolute and relative number of individuals with symmetrical and asymmetrical position of muscle attachment, and the averages of the individual differences in the index of position. The position of attachment of
 +
 
 +
 
 +
 
 +
PECTORALIS MAJOR AND DELTOID INSERTION 165
 +
 
 +
the pectoralis major muscle was the same on both sides in onlythree out of one hundred and five individuals. The unusually large percentage of asymmetrical cases is on the average equally distributed on the two sides. One finds only small differences between the averages on the right and left side in table 3. The largest individual asymmetry was found in a negro whose index of position was 6.0 greater on the right than on the left side.
 +
 
 +
The index which has just been discussed gives a clear idea of the position of the median point of the muscle insertion, and it will therefore be of interest to devote our attention briefly to the length of the insertion of the pectoralis major, from which the median point was obtained. The absolute value of this insertion length is represented by the difference between measurement 2 and 3 in figure 1. In order to make this measurement independent of the individual size of the upper arm it has been expressed in percents of the humerus length. The formula of this relative measurement is as follows :
 +
 
 +
measurement 3 — measurement 2
 +
 
 +
XlOO
 +
 
 +
measurement 1
 +
 
 +
The averages and the conditions of variability of this index are tabulated in table 5. One notices a tremendous range of variation from 8.8 to 23.1, and the variation coefficients also are unusually large. It seems inadvisable therefore to attach any particular significance to the slight differences in sex and race, such as the relatively longer attachment of the muscle in females and in whites. There is no correlation between the relative insertion length and the muscle strength nor the position of the insertion. The measurement which has just been discussed is somewhat longer in whites on the right side, and in negroes on the left. There is a very marked tendency to asymmetry in the relative insertion lengths in the different individuals, as has already been found to be the case for the position of the insertion. The relative attachment length was equal on both sides in only four cases, and in only one case did the absolute length of attachment show no asymmetry.
 +
 
 +
 
 +
 
 +
THE AMERICAN JOUBMAL OF ANATOMY, VOL. 23, NO. 1
 +
 
 +
 
 +
 
 +
166
 +
 
 +
 
 +
 
 +
ADOLF H. SCHULTZ
 +
 
 +
 
 +
 
 +
TABLE 5
 +
 
 +
Averages, standard deviations, variations-coefficients, their probable errors and ranges of variation for the relative length of the insertion of the pectoralis major mtiscle
 +
 
 +
 
 +
 
 +
RACE
 +
 
 +
 
 +
SEX
 +
 
 +
 
 +
'.NUMBER
 +
 
 +
 
 +
SIDE
 +
 
 +
 
 +
M ± E (M)
 +
 
 +
 
 +
o^ E (o-)
 +
 
 +
 
 +
V ^ E (lO
 +
 
 +
 
 +
MINIMUM
 +
 
 +
 
 +
MAXIMUM
 +
 
 +
 
 +
 
 +
 
 +
d'
 +
 
 +
 
 +
40
 +
 
 +
 
 +
r.
 +
 
 +
 
 +
16.84±0.25
 +
 
 +
 
 +
2.37^0.18
 +
 
 +
 
 +
14.11±1.09
 +
 
 +
 
 +
9.2
 +
 
 +
 
 +
23.1
 +
 
 +
 
 +
 
 +
 
 +
&
 +
 
 +
 
 +
40
 +
 
 +
 
 +
1.
 +
 
 +
 
 +
16.32±0,25
 +
 
 +
 
 +
2.35±0.18
 +
 
 +
 
 +
14.42±1.12
 +
 
 +
 
 +
11.5
 +
 
 +
 
 +
20.8
 +
 
 +
 
 +
Whites . . . . <
 +
 
 +
 
 +
&
 +
 
 +
 
 +
80
 +
 
 +
 
 +
r. 1.
 +
 
 +
 
 +
16.58±0.18
 +
 
 +
 
 +
2 36±0.12
 +
 
 +
 
 +
14.22±0.77
 +
 
 +
 
 +
9.2
 +
 
 +
 
 +
23.1
 +
 
 +
 
 +
9
 +
 
 +
 
 +
11
 +
 
 +
 
 +
r.
 +
 
 +
 
 +
16.95±0.39
 +
 
 +
 
 +
1. 94=^0.28
 +
 
 +
 
 +
11.48±1.66
 +
 
 +
 
 +
13 6
 +
 
 +
 
 +
20.3
 +
 
 +
 
 +
 
 +
 
 +
9
 +
 
 +
 
 +
11
 +
 
 +
 
 +
1.
 +
 
 +
 
 +
16. 53 ±0.50
 +
 
 +
 
 +
2.45±0.35
 +
 
 +
 
 +
14.85±2.16
 +
 
 +
 
 +
12 9
 +
 
 +
 
 +
21.5
 +
 
 +
 
 +
 
 +
 
 +
9
 +
 
 +
 
 +
22
 +
 
 +
 
 +
r. 1.
 +
 
 +
 
 +
16 74±0.32
 +
 
 +
 
 +
2 22±0.23
 +
 
 +
 
 +
13.29±1.38
 +
 
 +
 
 +
12.9
 +
 
 +
 
 +
21.5
 +
 
 +
 
 +
 
 +
 
 +
c^
 +
 
 +
 
 +
38
 +
 
 +
 
 +
r.
 +
 
 +
 
 +
16.13±0.38
 +
 
 +
 
 +
3.52±0.27
 +
 
 +
 
 +
21.86±1.77
 +
 
 +
 
 +
8.8
 +
 
 +
 
 +
22.7
 +
 
 +
 
 +
 
 +
 
 +
cf
 +
 
 +
 
 +
38
 +
 
 +
 
 +
1.
 +
 
 +
 
 +
16.30±0.24
 +
 
 +
 
 +
2.18±0.17
 +
 
 +
 
 +
13,37±1.06
 +
 
 +
 
 +
10.5
 +
 
 +
 
 +
20.2
 +
 
 +
 
 +
 
 +
 
 +
&
 +
 
 +
 
 +
76
 +
 
 +
 
 +
r. 1.
 +
 
 +
 
 +
16.21±0.23
 +
 
 +
 
 +
3.02±0.16
 +
 
 +
 
 +
18.64±1.02
 +
 
 +
 
 +
8.8
 +
 
 +
 
 +
22.7
 +
 
 +
 
 +
 
 +
 
 +
9
 +
 
 +
 
 +
16
 +
 
 +
 
 +
r.
 +
 
 +
 
 +
15.90±0.41
 +
 
 +
 
 +
2.42±0.29
 +
 
 +
 
 +
15.22±1.88
 +
 
 +
 
 +
12.3
 +
 
 +
 
 +
21.9
 +
 
 +
 
 +
 
 +
 
 +
9
 +
 
 +
 
 +
16
 +
 
 +
 
 +
1.
 +
 
 +
 
 +
16.88±0.38
 +
 
 +
 
 +
2. 27 ±0.27
 +
 
 +
 
 +
13.43±1.64
 +
 
 +
 
 +
12.0
 +
 
 +
 
 +
20.4
 +
 
 +
 
 +
 
 +
 
 +
9
 +
 
 +
 
 +
32
 +
 
 +
 
 +
r. 1.
 +
 
 +
 
 +
16.39±0.28
 +
 
 +
 
 +
2.36±0.20
 +
 
 +
 
 +
14.39±1.23
 +
 
 +
 
 +
12.0
 +
 
 +
 
 +
21.9
 +
 
 +
 
 +
 
 +
The averages and the conditions of variation of the index, which expresses the relative position of the middle point of the insertion of the deltoid muscle, are given in table 6. The variation extends from 34.0 — 46.5, that is it equals 31 per cent of the middle value of the two extremes and therefore remains
 +
 
 +
TABLE 6
 +
 
 +
Averages, standard deviations, variation-coefficients, their probable errors and ranges of variation for the position index of the insertion of the deltoid muscle
 +
 
 +
 
 +
 
 +
RACE
 +
 
 +
 
 +
SEX
 +
 
 +
 
 +
NUMBER
 +
 
 +
 
 +
SIDE
 +
 
 +
 
 +
M ^E (.W)
 +
 
 +
 
 +
<j ^E {(t)
 +
 
 +
 
 +
V ^ E (t))
 +
 
 +
 
 +
MINIMUM
 +
 
 +
 
 +
MAXIMUM
 +
 
 +
 
 +
[•
 +
 
 +
 
 +
' &
 +
 
 +
 
 +
40
 +
 
 +
 
 +
r.
 +
 
 +
 
 +
40.45±0.22
 +
 
 +
 
 +
2.03±0.15
 +
 
 +
 
 +
5.02±0.38
 +
 
 +
 
 +
37.2
 +
 
 +
 
 +
44 8
 +
 
 +
 
 +
 
 +
 
 +
d"
 +
 
 +
 
 +
40
 +
 
 +
 
 +
1.
 +
 
 +
 
 +
41.17±0.19
 +
 
 +
 
 +
1.80±0.14
 +
 
 +
 
 +
4.37±0.33
 +
 
 +
 
 +
37.5
 +
 
 +
 
 +
45.6
 +
 
 +
 
 +
Whites . . . . <
 +
 
 +
 
 +
d
 +
 
 +
 
 +
80
 +
 
 +
 
 +
r. 1.
 +
 
 +
 
 +
40.81±0.15
 +
 
 +
 
 +
1.94±0.10
 +
 
 +
 
 +
4.76±0.25
 +
 
 +
 
 +
37.2
 +
 
 +
 
 +
45.6
 +
 
 +
 
 +
9
 +
 
 +
 
 +
11
 +
 
 +
 
 +
r.
 +
 
 +
 
 +
39.35±0.52
 +
 
 +
 
 +
2.57±0.37
 +
 
 +
 
 +
6.52±0.93
 +
 
 +
 
 +
34.0
 +
 
 +
 
 +
42.5
 +
 
 +
 
 +
 
 +
 
 +
9
 +
 
 +
 
 +
11
 +
 
 +
 
 +
1.
 +
 
 +
 
 +
40.61±0.42
 +
 
 +
 
 +
2.06±0.29
 +
 
 +
 
 +
5.07±0.72
 +
 
 +
 
 +
36.4
 +
 
 +
 
 +
42.9
 +
 
 +
 
 +
 
 +
 
 +
9
 +
 
 +
 
 +
22
 +
 
 +
 
 +
r. 1.
 +
 
 +
 
 +
39.98±0.34
 +
 
 +
 
 +
2.41±0.24
 +
 
 +
 
 +
6.03±0 61
 +
 
 +
 
 +
34
 +
 
 +
 
 +
42.9
 +
 
 +
 
 +
r
 +
 
 +
 
 +
d
 +
 
 +
 
 +
38
 +
 
 +
 
 +
r.
 +
 
 +
 
 +
40.40±0.24
 +
 
 +
 
 +
2.22±0.17
 +
 
 +
 
 +
5.50±0.43
 +
 
 +
 
 +
34.0
 +
 
 +
 
 +
44.7
 +
 
 +
 
 +
 
 +
 
 +
d
 +
 
 +
 
 +
38
 +
 
 +
 
 +
1.
 +
 
 +
 
 +
40.52±0.22
 +
 
 +
 
 +
2.00±0.16
 +
 
 +
 
 +
4.94±0.38
 +
 
 +
 
 +
36.2
 +
 
 +
 
 +
44.6
 +
 
 +
 
 +
Negroes . . . <
 +
 
 +
 
 +
&
 +
 
 +
 
 +
76
 +
 
 +
 
 +
r. 1.
 +
 
 +
 
 +
40.46±0.16
 +
 
 +
 
 +
2.12±0.12
 +
 
 +
 
 +
5.24±0.29
 +
 
 +
 
 +
34.0
 +
 
 +
 
 +
44.7
 +
 
 +
 
 +
9
 +
 
 +
 
 +
16
 +
 
 +
 
 +
r.
 +
 
 +
 
 +
40.64±0.48
 +
 
 +
 
 +
2.86±0.34
 +
 
 +
 
 +
7 04±0.84
 +
 
 +
 
 +
34 3
 +
 
 +
 
 +
46.5
 +
 
 +
 
 +
 
 +
 
 +
9
 +
 
 +
 
 +
16
 +
 
 +
 
 +
1.
 +
 
 +
 
 +
41.11±0.42
 +
 
 +
 
 +
2 50±0.30
 +
 
 +
 
 +
6.08±0 73
 +
 
 +
 
 +
36.1
 +
 
 +
 
 +
45
 +
 
 +
 
 +
 
 +
 
 +
9
 +
 
 +
 
 +
32
 +
 
 +
 
 +
r. 1.
 +
 
 +
 
 +
40.87±0.32
 +
 
 +
 
 +
2 70±0.23
 +
 
 +
 
 +
6 60±0.55
 +
 
 +
 
 +
34 3
 +
 
 +
 
 +
46.5
 +
 
 +
 
 +
 
 +
PECTORALIS MAJOR AND DELTOID INSERTION 167
 +
 
 +
considerably less, than the variability of the position index of the pectoralis major, the variation of which equaled 49 per cent of its mean. The last named index shows in every group a greater variation coefficient than the corresponding ones in table 6. Therefore the deltoid possesses a more constant position of insertion than the pectoralis major muscle. Judging from the averages of the position index the attachment of the deltoid muscle must be relatively slightly more distal in the males of the white race, and slightly more proximal in the males of the negroes than in the females of either. There is no difference in the two races in the position of the insertion of the deltoid, similar to that found to be the case for the pectoralis major. The relative position of the deltoid insertion is also almost regularly unequal on both sides; more frequentl}^ the muscle is more proximally situated on the right side. In all the groups the averages of the position index of the deltoid are on the right — in part even considerably — smaller than those of the left side. A relationship between the strength of the deltoid muscle and its insertion position does not exist. The question as to what extent the positions of the insertions of the pectoralis major and the deltoid may change correspondingly is best answered by the following tabulation of the correlation coefficients with their probable errors for the two indices of position which have been previously used. White males + 0.52 ± 0.057, white females -f 0.37 ± 0.123, negro males + 0.29 ± 0.071, negro females + 0.70 ± 0.061. The coefficients, which are regularly positive, indicate that a shifting of one of the muscles is usually followed by a change to a greater or less extent of position of the other muscle in the same direction. This is very noticeable in female negroes and in male whites. In the material used the proximal point of measurement of the insertion of the deltoid was found above the distal point of measurement of the pectoralis major muscle insertion in one hundred and eighty-six cases; sixteen times t'le points referred to were at the same height and in only eight cases was the first point found below the latter.
 +
 
 +
The most proximal region of insertion of the deltoid is much more variable than the most distal. In order to free the index
 +
 
 +
 
 +
 
 +
168
 +
 
 +
 
 +
 
 +
ADOLF H. SCHULTZ
 +
 
 +
 
 +
 
 +
of position, which has just been discussed and which uses the mesLU value depending on the two terminal points of the insertion, from the great variability of the upper point it was found necessary to calculate a second position index for the deltoid, employing only the most distal point of insertion. This new index gives information as to how far down the deltoid extends upon the humerus. The formula for this index, using the measurements of figure 1, reads as follows:
 +
 
 +
 
 +
 
 +
measurement 5 measurement 1
 +
 
 +
 
 +
 
 +
X 100
 +
 
 +
 
 +
 
 +
The averages and conditions of variability of the index of the relative position of the most distal point of attachment of the deltoid are given in table 7. The entire variation reaching from 44.8 to 57.5 comprises 25 per cent of the mean obtained fromthe end values just cited; it is therefore relatively much smaller than the variation of the preceding index, which employed the middle point of insertion. The variation coefficients in table 7 lie in all the groups below those in table 6 and should be considered as relatively small. The position of the most distal point
 +
 
 +
 
 +
 
 +
TABLE
 +
 
 +
 
 +
 
 +
Averages, standard deviations, variation-coefficients, their probable errors and ranges of variation for the position index of the most distal point of insertion of the deltoid muscle
 +
 
 +
 
 +
 
 +
RACE
 +
 
 +
 
 +
SEX
 +
 
 +
 
 +
NUMBER
 +
 
 +
 
 +
iSIDE
 +
 
 +
 
 +
A/ ± £; (iW)
 +
 
 +
 
 +
0- ± E (cr)
 +
 
 +
 
 +
t) ± £; 00
 +
 
 +
 
 +
MINIMUM
 +
 
 +
 
 +
MAXIMUM
 +
 
 +
 
 +
 
 +
 
 +
&
 +
 
 +
 
 +
40
 +
 
 +
 
 +
r.
 +
 
 +
 
 +
50.13±0.21
 +
 
 +
 
 +
1.98±0.15
 +
 
 +
 
 +
3.95=^0.30
 +
 
 +
 
 +
44.8
 +
 
 +
 
 +
55.0
 +
 
 +
 
 +
 
 +
 
 +
&
 +
 
 +
 
 +
40
 +
 
 +
 
 +
1.
 +
 
 +
 
 +
50.81±0.21
 +
 
 +
 
 +
1.96±0.15
 +
 
 +
 
 +
3.86±0.29
 +
 
 +
 
 +
47.9
 +
 
 +
 
 +
57.5
 +
 
 +
 
 +
Whites . .. . <
 +
 
 +
 
 +
&
 +
 
 +
 
 +
80
 +
 
 +
 
 +
r. 1.
 +
 
 +
 
 +
50.47±0.15
 +
 
 +
 
 +
1.97±0.10
 +
 
 +
 
 +
3.90±0 21
 +
 
 +
 
 +
44.8
 +
 
 +
 
 +
57.5
 +
 
 +
 
 +
9
 +
 
 +
 
 +
11
 +
 
 +
 
 +
r.
 +
 
 +
 
 +
49.88±0.35
 +
 
 +
 
 +
1.74±0.25
 +
 
 +
 
 +
3.49±0.50
 +
 
 +
 
 +
45.6
 +
 
 +
 
 +
52.5
 +
 
 +
 
 +
 
 +
 
 +
9
 +
 
 +
 
 +
11
 +
 
 +
 
 +
1.
 +
 
 +
 
 +
50.34±0.36
 +
 
 +
 
 +
1.76±0.25
 +
 
 +
 
 +
3.50±0.50
 +
 
 +
 
 +
47.3
 +
 
 +
 
 +
53.9
 +
 
 +
 
 +
 
 +
 
 +
9
 +
 
 +
 
 +
22
 +
 
 +
 
 +
r. 1.
 +
 
 +
 
 +
50.11±0.25
 +
 
 +
 
 +
1.76±0.18
 +
 
 +
 
 +
3.51±0.36
 +
 
 +
 
 +
45.6
 +
 
 +
 
 +
53.9
 +
 
 +
 
 +
 
 +
 
 +
&
 +
 
 +
 
 +
38
 +
 
 +
 
 +
r.
 +
 
 +
 
 +
49.59±0.16
 +
 
 +
 
 +
1.50±0.12
 +
 
 +
 
 +
3.02±0.23
 +
 
 +
 
 +
46.2
 +
 
 +
 
 +
52.8
 +
 
 +
 
 +
 
 +
 
 +
cf
 +
 
 +
 
 +
38
 +
 
 +
 
 +
1.
 +
 
 +
 
 +
50.18*0.15
 +
 
 +
 
 +
1.41±0.11
 +
 
 +
 
 +
2.81±0.22
 +
 
 +
 
 +
46.5
 +
 
 +
 
 +
52.9
 +
 
 +
 
 +
Negroes ... ■
 +
 
 +
 
 +
9
 +
 
 +
 
 +
76 16
 +
 
 +
 
 +
r. 1. r.
 +
 
 +
 
 +
49.88±0.12 48.96±0.39
 +
 
 +
 
 +
1.48±0.08 2.31±0.28
 +
 
 +
 
 +
2.97*0.16 4.71±0.56
 +
 
 +
 
 +
46.2 45.1
 +
 
 +
 
 +
52.9 54,8
 +
 
 +
 
 +
 
 +
 
 +
9
 +
 
 +
 
 +
16
 +
 
 +
 
 +
1.
 +
 
 +
 
 +
49.52±0.37
 +
 
 +
 
 +
2.21±0.27
 +
 
 +
 
 +
4.46±0.53
 +
 
 +
 
 +
45.0
 +
 
 +
 
 +
54.1
 +
 
 +
 
 +
^
 +
 
 +
 
 +
9
 +
 
 +
 
 +
32
 +
 
 +
 
 +
r. 1.
 +
 
 +
 
 +
49.24±0.27
 +
 
 +
 
 +
2.27±0.19
 +
 
 +
 
 +
4.61*0.39
 +
 
 +
 
 +
45.0
 +
 
 +
 
 +
54 8
 +
 
 +
 
 +
 
 +
PECTORALIS MAJOR AND DELTOID INSERTION
 +
 
 +
 
 +
 
 +
169
 +
 
 +
 
 +
 
 +
of the deltoid is accordingly the most constant of the points of muscle insertion which have been used, and can be located with considerable precision near the middle point of the humerus. Actually, this point lies slightly below the middle of the length of the humerus in whites, slightly above in negroes; a racial difference which is represented by the average difference of 0.73 of the averages of the index. In the female the most distal point of the deltoid is situated somewhat more proximal than in the male. It is also of interest to note, that the distal end point of the deltoid insertion, similarly to the middle point, is located on the average noticeably more distally on the left side than on the
 +
 
 +
TABLE 8
 +
 
 +
Absolute and relative numbers of individuals with symmetrical and asymmetrical
 +
 
 +
position of the most distal point of insertion of the deltoid and averages
 +
 
 +
of the individual differences of the position index
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
AVERAGE
 +
 
 +
 
 +
RACE
 +
 
 +
 
 +
SEX
 +
 
 +
 
 +
THE SAME POSITION ON BOTH
 +
 
 +
 
 +
POSITION ON THE RIGHT MORE
 +
 
 +
 
 +
POSITION ON THE LEFT MORE
 +
 
 +
 
 +
DIFFERENCE IF MORE DIST.'ULLY
 +
 
 +
 
 +
 
 +
 
 +
cf
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
on the right
 +
 
 +
 
 +
on the
 +
 
 +
left
 +
 
 +
 
 +
Whites 1
 +
 
 +
 
 +
= 0.0%
 +
 
 +
 
 +
13 = 32.5%
 +
 
 +
 
 +
27 = 67.5%
 +
 
 +
 
 +
1.28
 +
 
 +
 
 +
1.61
 +
 
 +
 
 +
9
 +
 
 +
 
 +
1 = 9.1%
 +
 
 +
 
 +
5 = 45.4%
 +
 
 +
 
 +
5 = 45.4%
 +
 
 +
 
 +
1.28
 +
 
 +
 
 +
2.28
 +
 
 +
 
 +
Negroes <
 +
 
 +
 
 +
9
 +
 
 +
 
 +
2 = 5.2% = 0.0%
 +
 
 +
 
 +
12 = 31.6% 6 = 37.5%
 +
 
 +
 
 +
24 = 63.2% 10=62.5%
 +
 
 +
 
 +
1.08 73
 +
 
 +
 
 +
1.47 1.34
 +
 
 +
 
 +
 
 +
right. Table 8 gives a view of the absolute and relative number of individuals with equal and unequal indices of position for the most distal point of the deltoid insertion and also the average differences of the unequal indices. It shows that the point referred to occupies the same relative position on both sides in a total of only three cases; furthermore that the point was in a greater number of instances more distally situated on the left side, and that the differences in favor of the left always exceeded on the average those of the right. The greatest individual difference of the index appears in a white male with 6.4 in iavor of the left side.
 +
 
 +
The exact determination of the position of the insertion of the pectoralis major and deltoid muscles on the humerus shows,
 +
 
 +
 
 +
 
 +
170 ADOLF H. SCHULTZ
 +
 
 +
when briefly summarized, the possibiUties of variation, the more constant position of the deltoid insertion compared with the pectoraUs major, the equahty of the positions of the two muscle insertions in whites and negroes, the relatively higher attachment of the pectoralis major in females, and lastly the surprisingly common asymmetry of the insertion positions. The author has noticed that asymmetries of position of the insertions occurred as early as birth, although they are not as frequent nor as marked in the newborn as in adults.
 +
 
 +
In conclusion, the individual relative measurements and humerus lengths which form the basis of this paper are tabulated, and are arranged according to increasing lengths of the right humerus.
 +
 
 +
 
 +
 
 +
PECTORALIS MAJOR AND DELTOID INSERTION
 +
 
 +
 
 +
 
 +
171
 +
 
 +
 
 +
 
 +
White males
 +
 
 +
 
 +
 
 +
HUMERUS LENGTH
 +
 
 +
 
 +
POSITION INDEX
 +
 
 +
FOR THE INSERTION
 +
 
 +
OF THE
 +
 
 +
PECTORALIS MAJOR
 +
 
 +
 
 +
REL.\TIVE LENGTH
 +
 
 +
OF THE
 +
 
 +
INSERTION OF THE
 +
 
 +
PECTORALIS
 +
 
 +
MAJOR
 +
 
 +
 
 +
POSITION INDEX
 +
 
 +
FOB THE
 +
 
 +
INSERTION OP THE
 +
 
 +
DELTOID
 +
 
 +
 
 +
POSITION INDEX
 +
 
 +
FOR THE
 +
 
 +
MOST DISTAL POINT
 +
 
 +
OF INSERTION
 +
 
 +
OF THE DELTOID
 +
 
 +
 
 +
Right
 +
 
 +
 
 +
Left
 +
 
 +
 
 +
Right
 +
 
 +
 
 +
Left
 +
 
 +
 
 +
Ri?ht
 +
 
 +
 
 +
Left
 +
 
 +
 
 +
Right
 +
 
 +
 
 +
Left
 +
 
 +
 
 +
Right
 +
 
 +
 
 +
Left
 +
 
 +
 
 +
283
 +
 
 +
 
 +
283
 +
 
 +
 
 +
31.3
 +
 
 +
 
 +
31.8
 +
 
 +
 
 +
16.6
 +
 
 +
 
 +
19.8
 +
 
 +
 
 +
41.7
 +
 
 +
 
 +
41.9
 +
 
 +
 
 +
49.8
 +
 
 +
 
 +
50.9
 +
 
 +
 
 +
286
 +
 
 +
 
 +
286
 +
 
 +
 
 +
28.5
 +
 
 +
 
 +
27.8
 +
 
 +
 
 +
13.6
 +
 
 +
 
 +
11.5
 +
 
 +
 
 +
42.3
 +
 
 +
 
 +
41.4
 +
 
 +
 
 +
52.4
 +
 
 +
 
 +
51.7
 +
 
 +
 
 +
289
 +
 
 +
 
 +
294
 +
 
 +
 
 +
28.4
 +
 
 +
 
 +
29.6
 +
 
 +
 
 +
15.2
 +
 
 +
 
 +
12.2
 +
 
 +
 
 +
44.8
 +
 
 +
 
 +
43.0
 +
 
 +
 
 +
55.0
 +
 
 +
 
 +
53.1
 +
 
 +
 
 +
296
 +
 
 +
 
 +
303
 +
 
 +
 
 +
27.2
 +
 
 +
 
 +
28.5
 +
 
 +
 
 +
20.6
 +
 
 +
 
 +
14.2
 +
 
 +
 
 +
42.6
 +
 
 +
 
 +
41.7
 +
 
 +
 
 +
47.6
 +
 
 +
 
 +
47.9
 +
 
 +
 
 +
299
 +
 
 +
 
 +
291
 +
 
 +
 
 +
27.9
 +
 
 +
 
 +
28.4
 +
 
 +
 
 +
18.4
 +
 
 +
 
 +
15.5
 +
 
 +
 
 +
42.3
 +
 
 +
 
 +
40.7
 +
 
 +
 
 +
50.2
 +
 
 +
 
 +
52.2
 +
 
 +
 
 +
299
 +
 
 +
 
 +
298
 +
 
 +
 
 +
29.9
 +
 
 +
 
 +
30.2
 +
 
 +
 
 +
23.1
 +
 
 +
 
 +
20.8
 +
 
 +
 
 +
41.8
 +
 
 +
 
 +
41.8
 +
 
 +
 
 +
50.5
 +
 
 +
 
 +
51.0
 +
 
 +
 
 +
299
 +
 
 +
 
 +
301
 +
 
 +
 
 +
27.4
 +
 
 +
 
 +
29.2
 +
 
 +
 
 +
17.4
 +
 
 +
 
 +
16.6
 +
 
 +
 
 +
37.6
 +
 
 +
 
 +
42.0
 +
 
 +
 
 +
47.2
 +
 
 +
 
 +
50.2
 +
 
 +
 
 +
300
 +
 
 +
 
 +
300
 +
 
 +
 
 +
29.0
 +
 
 +
 
 +
26.0
 +
 
 +
 
 +
16.7
 +
 
 +
 
 +
18.7
 +
 
 +
 
 +
39.3
 +
 
 +
 
 +
39.0
 +
 
 +
 
 +
48.3
 +
 
 +
 
 +
50.0
 +
 
 +
 
 +
302
 +
 
 +
 
 +
294
 +
 
 +
 
 +
29.0
 +
 
 +
 
 +
26.0
 +
 
 +
 
 +
12.3
 +
 
 +
 
 +
13.9
 +
 
 +
 
 +
42.9
 +
 
 +
 
 +
42.7
 +
 
 +
 
 +
50.7
 +
 
 +
 
 +
51.4
 +
 
 +
 
 +
302
 +
 
 +
 
 +
296
 +
 
 +
 
 +
28.5
 +
 
 +
 
 +
26.9
 +
 
 +
 
 +
15.9
 +
 
 +
 
 +
15.9
 +
 
 +
 
 +
42.7
 +
 
 +
 
 +
41.0
 +
 
 +
 
 +
51.3
 +
 
 +
 
 +
48.3
 +
 
 +
 
 +
302
 +
 
 +
 
 +
311
 +
 
 +
 
 +
29.5
 +
 
 +
 
 +
30.2
 +
 
 +
 
 +
15.9
 +
 
 +
 
 +
12.2
 +
 
 +
 
 +
41.1
 +
 
 +
 
 +
42.1
 +
 
 +
 
 +
49.7
 +
 
 +
 
 +
51.4
 +
 
 +
 
 +
304
 +
 
 +
 
 +
303
 +
 
 +
 
 +
25.0
 +
 
 +
 
 +
27.9
 +
 
 +
 
 +
18.4
 +
 
 +
 
 +
17.5
 +
 
 +
 
 +
37.3
 +
 
 +
 
 +
43.1
 +
 
 +
 
 +
48.4
 +
 
 +
 
 +
50.8
 +
 
 +
 
 +
306
 +
 
 +
 
 +
313
 +
 
 +
 
 +
27.8
 +
 
 +
 
 +
28.9
 +
 
 +
 
 +
15.7
 +
 
 +
 
 +
16.0
 +
 
 +
 
 +
37.4
 +
 
 +
 
 +
39.3
 +
 
 +
 
 +
49.6
 +
 
 +
 
 +
49.8
 +
 
 +
 
 +
307
 +
 
 +
 
 +
307
 +
 
 +
 
 +
28.7
 +
 
 +
 
 +
28.7
 +
 
 +
 
 +
20.8
 +
 
 +
 
 +
15.6
 +
 
 +
 
 +
36.5
 +
 
 +
 
 +
39.7
 +
 
 +
 
 +
50.5
 +
 
 +
 
 +
51.5
 +
 
 +
 
 +
311
 +
 
 +
 
 +
310
 +
 
 +
 
 +
29.7
 +
 
 +
 
 +
27.0
 +
 
 +
 
 +
17.0
 +
 
 +
 
 +
16.5
 +
 
 +
 
 +
40.4
 +
 
 +
 
 +
40.3
 +
 
 +
 
 +
50.2
 +
 
 +
 
 +
48.7
 +
 
 +
 
 +
314
 +
 
 +
 
 +
312
 +
 
 +
 
 +
30.1
 +
 
 +
 
 +
29.8
 +
 
 +
 
 +
9.2
 +
 
 +
 
 +
17.3
 +
 
 +
 
 +
38.5
 +
 
 +
 
 +
39.6
 +
 
 +
 
 +
51.3
 +
 
 +
 
 +
52.6
 +
 
 +
 
 +
315
 +
 
 +
 
 +
311
 +
 
 +
 
 +
26.8
 +
 
 +
 
 +
27.5
 +
 
 +
 
 +
15.0
 +
 
 +
 
 +
12 5
 +
 
 +
 
 +
40.3
 +
 
 +
 
 +
39.2
 +
 
 +
 
 +
49.8
 +
 
 +
 
 +
48.9
 +
 
 +
 
 +
315
 +
 
 +
 
 +
315
 +
 
 +
 
 +
27.5
 +
 
 +
 
 +
30.6
 +
 
 +
 
 +
17.5
 +
 
 +
 
 +
20.0
 +
 
 +
 
 +
43.0
 +
 
 +
 
 +
45.6
 +
 
 +
 
 +
51.1
 +
 
 +
 
 +
57.5
 +
 
 +
 
 +
315
 +
 
 +
 
 +
315
 +
 
 +
 
 +
26.7
 +
 
 +
 
 +
27.5
 +
 
 +
 
 +
19.0
 +
 
 +
 
 +
18.7
 +
 
 +
 
 +
39.7
 +
 
 +
 
 +
41.6
 +
 
 +
 
 +
51.1
 +
 
 +
 
 +
50.2
 +
 
 +
 
 +
317
 +
 
 +
 
 +
310
 +
 
 +
 
 +
28.2
 +
 
 +
 
 +
27.7
 +
 
 +
 
 +
19.2
 +
 
 +
 
 +
16.8
 +
 
 +
 
 +
38.6
 +
 
 +
 
 +
43.2
 +
 
 +
 
 +
51.7
 +
 
 +
 
 +
51.6
 +
 
 +
 
 +
319
 +
 
 +
 
 +
318
 +
 
 +
 
 +
27.0
 +
 
 +
 
 +
28.9
 +
 
 +
 
 +
16.9
 +
 
 +
 
 +
14.5
 +
 
 +
 
 +
40.6
 +
 
 +
 
 +
41.2
 +
 
 +
 
 +
50.8
 +
 
 +
 
 +
50.3
 +
 
 +
 
 +
319
 +
 
 +
 
 +
319
 +
 
 +
 
 +
28.1
 +
 
 +
 
 +
30.3
 +
 
 +
 
 +
17.9
 +
 
 +
 
 +
17.2
 +
 
 +
 
 +
40.4
 +
 
 +
 
 +
43.9
 +
 
 +
 
 +
50.2
 +
 
 +
 
 +
52.0
 +
 
 +
 
 +
320
 +
 
 +
 
 +
315
 +
 
 +