Difference between revisions of "American Journal of Anatomy 26 (1919-20)"

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PEOPLE Full text of "The American journal of anatomy" See other formats THE AMERICAN JOURNAL
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OF
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ANATOMY
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Charles R. Bardebn
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University of Wisconsin
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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
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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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J. Playfair McMtjrrich
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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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Henry McE. Knower, Secretary
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University of Cincinnati
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VOLUME 26 SEPTEMBER, 1919— JANUARY, 1920
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THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY
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PHILADELPHIA, PA.
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CONTENTS
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No. 1. SEPTEMBER, 1919
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H. E. Jordan. The histology of the umbilical cord of the pig, with special reference to the vasculogenic and hemopoietic activity of its extensively vascularized connective tissue. Fifteen figures 1
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Henry A. Murray Jr. The development of the cardiac loop in the rabbit, with especial reference to the bulboventricular groove and origin of the interventricular septum. Seven figures 29
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Frank Blair Hanson. The ontogeny and phylogeny of the sternum. Twelve plates
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(forty-nine figures) 41
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George W. Corner. On the origin of the corpus luteum of the sow from both granulosa and theca interna. Twenty-six figures 117
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No. 2. NOVEMBER, 1919
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Harold J. Cooper. The hypophysis cerebri of the California ground-squirrel, Citellus
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beechyi (Richardson). Eleven figures 185
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Ralph Dougall Lillie. The early histogenesis of the blood in Bufo halophilus Baird
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and Girard. Seven figures 209
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Henry H. Donaldson. Quantitative studies on the growth of the skeleton of the albino
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rat. Twenty-three charts 237
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No. 3. JANUARY, 1920
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Leslie B. Arey. The origin, growth, and fate of osteoclasts and their relation to bone resorption. Twenty-four figures 315
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S. Sagtjchi. Studies on the glandular cells of the frog's pancreas. Five plates 347
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Frederic T. Lewis. The course of the Wolffian tubules in mammalian embryos. Thirteen figures 423
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William H. F. Addison. Histological study of the spleen of the rabbit under heightened phagocytic activity. Six figures (one plate) 437
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author's abstract of this paper issued BV THE bibliographic SERVICE, JULY 7
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THE HISTOLOGY OF THE UMBILICAL CORD OF THE PIG, WITH SPECIAL REFERENCE TO THE VASCULOGENIC AND HEMOPOIETIC ACTIVITY OF ITS EXTENSIVELY VASCULARIZED CONNECTIVE TISSUE
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H. E. JORDAN
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Department of Anatomy, University of Virginia
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FIFTEEN FIGURES
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The umbilical cord of the pig differs markedly from the human cord in that it is extensively vascularized, its connective tissue maintains throughout the gestation period largely its original embryonal character, and vasculogenic and hemopoietic activity persist to full term.
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The material upon which this investigation is chiefly based is a complete cord, 4 cm. in length, from a fetus of 16-cm. length. This fetus was secured in utero at the abattoir and preserved in 10 per cent formalin. Reckoned by length, it lacked between one and two weeks of full term. Portions of the cord from the proximal end, the middle, and the distal end, were imbedded both in celloidin and in paraffin. Serial sections were cut from the paraffin blocks and stained with hematoxylin and eosin. Some of the celloidinsections were similarly stained ; others were stained with resorcin-fuchsin and counterstained with picricacid-fuchsin, for a study of the elastic and collagen fiber content. A second specimen of a nearly full-term cord, for which I am indebted to Prof. George S. Huntington, was used for comparison. Cords of pig embryos from 9 to 21-mm. length and five full-term and three fetal (three to seven months) human cords, variously fixed and stained, were also employed for comparative study.
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Though primary interest centers upon the vasculogenic and hemopoietic activity of the connective tissue, it seems desirable to preface the description and discussion of these phenomena
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2 H. E. JORDAN
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with a brief description of the comparative histology of this cord. Compared with the human cord it is very short, of considerably lesser girth, and only slightly twisted. It has the same light gray, pearly appearance, and feels of about the same consistency. In transverse section it has an irregularly oval shape (fig. 1), measuring 5 by 7 mm. Its three main bloodvessels have an approximately equal caliber and thickness of wall. It contains a large open allantoic duct and remnants of the occluded yolk-stalk. The connective tissue contains many arterioles, venules, and capillaries. Only one of the post-embryonic human umbilical cords in my collection, a full-term specimen, contains any blood-vessels besides the usual umbilical arteries and vein. In this cord occurs a venule of considerable size, lying near the surface and completely filled with red bloodcorpuscles. The human umbilical cord is typically non-vascular except for an occasional capillary at the extreme proximal end. None of my sections of these eight human cords contains any vestige of the allantois. One cord contains a small, still patent yolk-stalk; three contain a double, occluded yolk-stalk remnant. One of the full-term cords of the pig also contains a double occluded yolk-stalk (figs. 1 and 3), the other only a single, small, occluded remnant in only a few sections. In one of the human cords the persistent, double, occluded yolk-stalk is enveloped by a double layer of smooth muscle, an inner longitudinal and an outer thinner circular layer.
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THE UMBILICAL ARTERIES AND VEIN
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In the cord of the pig the wall of the umbilical arteries only contains circularly disposed smooth-muscle cells, more compact centrally; the vein in one of the two specimens contains also scattered, longitudinally placed cells beneath the intima. The disposition of the muscle differs from that in the human cord where the arteries and the vein contain, in addition to the chief circular layer, also a thin internal longitudinal layer and scattered bundles of longitudinally arranged cells externally. With regard to the elastic tissue content also, the pig's cord differs sharply from the human cord. The arteries of the latter lack
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UMBILICAL CORD OF THE PIG 6
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an internal elastic membrane, but several muscle layers beyond the tunica intima the elastic tissue forms complete fenestrated membranes through many layers; toward the periphery of these vessels the elastic tissue only occurs as scattered delicate fibrils. The vein, on the contrary, contains a very robust internal elastic membrane, while through the central half of the wall occur relatively coarse scattered fibers. In the pig's cord neither arteries nor vein contain an internal elastic membrane. The elastic fibers are practically limited to the inner half of the wall, only very delicate and widely scattered fibrils occurring peripherally. In the arteries the elastic tissue forms membranes for from three to five layers beyond the central two or three layers. Considerable variation occurs with regard both to the amount and the disposition of both the smooth muscle and the elastic tissue constituents of the wall of the umbilical vessels both in the pig's cord and the human cord.
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ECTODERM
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The covering ectoderm constitutes a stratified epithelium of generally four layers of cells (fig. 2) . In certain restricted areas the epithelium only consists of three layers of cells, in others of as many as eight layers of cells. This epithelium resembles the transitional rather than the stratified squamous type. It is comparable to the thicker portions of the epidermis of the threemonth human fetus, which consists of from four to six layers of cells, including a superficial periderm. It differs from the ectoderm of the full-term human cord, which includes only two or three layers of flattened keratized cells. It differs also from the continuous abdominal ectoderm in that the latter includes about eight layers of cells, all of which, except the basal cuboidal layer, consist of greatly flattened cells. Small patches of partially fused, keratized cells occur in five different regions of the section here shown. The lowermost layer of the epithelium consists of cuboidal cells; the outermost variously of thick, rectangular, flattened or dome-shaped peridermal cells ; the intervening layers include polyhedral and stout fusiform types. In those portions
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4 H. E. JORDAN
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where the epithelium consists of more than four layers, the one next the basal cuboidal layer is generally composed of more flattened cells. The basal layer seems to rest directly upon the adjacent connective tissue, without the intervention of a definite basement membrane. After picric-acid-fuchsin counterstain, however, a narrow subepithelial layer of the connective tissue stains more deeply red.
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None of the cells contains mitotic figures. An occasional cell of the superficial layer contains two or even four nuclei or a nucleus in process of fission. The nuclei of the cells of the basal layer are of spheroidal shape and contain a clear, lightly staining nucleoplasm and a distinct granular reticulum. These cells are completely filled with a slightly basophilic cytoplasm. The nuclei of the several outer layers are irregular in shape and they have a homogeneous, cloudy, more deep-staining character, and the nuclear wall is generally wrinkled. The cytoplasm of the cells of the intermediate layers is aggregated next the cell wall so that the cell appears hollow. This condition is probably a fixation artifact. The nucleus almost invariably lies next the outer wall, as if moved by currents passing toward the surface of the ectoderm. The cells of the outermost layer are again completely filled with an acidophilic cytoplasm. The latter is keratized to an extent w 7 hich could resist the action of the fixation currents that caused the peripheral shrinkage and central excavation of the cells of the intermediate layers.
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YOLK-STALK
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The yolk-stalk remnant of one of the two practically full-term specimens of the pig's cord, and of four of the specimens of human cord used for comparative study, is a double structure. In the specimen of the pig's cord the two portions are of nearly equal size (fig. 3). They are approximately circular in outline and perfectly solid. The cells of the peripheral layer are squamous or very low cuboidal. The more central cells are polyhedral «r spheroidal. The cells next the outermost layer are generally flattened and appear , fusiform in longitudinal section. About
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UMBILICAL CORD OF THE -PIG 5
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twelve cells occupy the diameter of each of the two elements. Most of the central cells appear hollow, the nuclei having become moved to one side, almost invariably to that side nearest the center of the structure. These cells have a superficial resemblance to fat cells. The hollow condition is probably the result of the coagulative action of the fixing fluid upon the delicate cytoplasm. A few of the cells appear keratized, and are acidophilic in staining reaction, and solid. The nuclei of the outermost cuboidal cells are deep-staining, granular, greatly elongated bodies. Those of the more central cells have a generally irregular shape with wrinkled contour and a generally non-granular homogeneous nucleoplasm. No mitotic or amitotic figures can be detected. Each member of the double structure is enveloped by a very thin inner connective-tissue theca, forming a delicate, fibrillar, nucleated basement membrane. Both are inclosed in a common, more external theca. The intervening partition only consists of the fused basement membranes.
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The fact that four of the five specimens of human cords used in connection with this study also contain a double yolk-stalk indicates that this doubling is a common condition. Two explanations suggest themselves: 1) That the doubling is due to the partition of the originally single stalk by the ingrowth of a connective-tissue septum related to the regressive changes by which the stalk becomes obliterated. 2) That the 'doubling' is only apparent, it being due in section to a sharp turning of the stalk in certain regions. The latter interpretation is supported by the evidence from one of the human cords: here one of the 'halves' is cut transversely, the other half is very obliquely cut, and the two are in continuity. In other words, the condition is such as would result if the section passed obliquely through the proximal end of one of the limbs and the connecting loop of a U-shaped structure. Opposed to the latter interpretation, however, is the fact that in the specimen of the pig's cord here described a common connective-tissue sheath envelops both moieties.
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O H. E. JORDAN
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ALLANTOIC DUCT
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The allantoic duct (fig. 1, All.) is shown highly magnified in figure 4. It has an irregularly oval shape in cross-section. Its wall is thrown into deep folds. Within the depth of the folds the epithelium consists of a single layer of cuboidal or even squamous cells, comparable to its condition throughout in the 21-mm. fetus; over the crests the epithelium is of the stratified columnar type, consisting of from three to four layers of cells. No distinct basement membrane can be discerned other than as indicated by the deeper red color of the immediately subjacent connective tissue after picric-acid-fuchsin counterstain. The nuclei have an irregularly oval form; their wall is wrinkled, and the nucleoplasm generally lacks a distinct network or granules. A number of the cells are hollowed out centrally. The cells next the lumen have a more condensed, probably slightly keratized, broad distal border. The enveloping connective tissue is less differentiated and denser than in any other portion of the section. It resembles early embryonic connective tissue or young mesenchyma. It contains many vasofactive cells ('angioblasts'), and one large blood-island (B. I.), which will be described below.
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THE CONNECTIVE TISSUE
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The character of the connective tissue varies in different portions of the transverse section (compare figs. 2,3,4, and 5). In the region immediately surrounding the allantoic duct (fig. 1) it is least differentiated. Here it is compact and resembles embryonic connective tissue or young mesenchyma (fig. 4). In this region also are numerous vasofactive cells (fig. 15). Along the peripheral border of the specimen the connective tissue is somewhat more differentiated and represents an older type of mesenchyma (fig. 2). Here the cells have generally a stellate or fusiform shape and are more widely separated. In this region also the capillaries are most abundant; these for the most part lie along the radii of the section and appear to be growing towards the ectodermal covering. Here also are found abundantly initial stages in the formation of the blood channels (figs. 8, 9, and 10).
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UMBILICAL CORD OF THE PIG 7
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In the area between the lower umbilical artery on the left and the vein on the right, the connective tissue is in part of the type characteristic of the human cord; that is, it is typical mucous connective tissue, but with an occasional capillary. In this region also occur small bundles of collagen fibrils. The area around the yolk-stalk contains vascular connective tissue of an intermediate type, with occasional collagen fibers (fig. 5). In the narrow space between the two umbilical arteries, extending to a point below the allantoic duct, there occur several bloodislands (figs. 6 and 7). About midway between the central umbilical blood-vessels and the periphery occur numerous arterioles and venules. Occasionally these are arranged in pairs (fig. 5). These vessels terminate in capillaries. The arterioles are enveloped by a thin layer of smooth muscle; the wall of the venule only consists of endothelium resting upon the slightly more condensed enveloping connective tissue.
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Certain of these smaller blood-vessels can be traced into connection with the umbilical arteries and the vein at their proximal (fetal) end. This is true both in the case of the younger cords (of embryos from 9 to 21 mm.) and in those near full term. In the 21-mm. fetus branches from the proximal end of the umbilical vein can be seen entering the body wall as well as the connective tissue of the cord. It may be confidently assumed that all of the blood-vessels, including the numerous capillaries, connect with vessels which ultimately connect with the main umbilical vessels proximally. But not all of these vessels are properly interpreted as vasa vasorum. Undoubtedly many function thus, as is indicated by the numerous capillaries directed towards the walls of the umbilical arteries and vein; but others are equally certainly nutrient vessels for the allantoic duct, the ectodermal covering, and the general connective tissue. Nor can the assumption be properly made that all of these vessels arise by sprouting from originally direct umbilical branches. If the capillaries grew exclusively by sprouting, their tips should show mitotic figures. Mitotic figures are practically absent in these capillary terminals. On the contrary, these tips seem to fuse with the general connective tissue, the cells of which become hollowed out, arrange
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8 H. E. JORDAN
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themselves in line with the capillaries and ultimately become incorporated as part of the capillary network. In the full-term condition of the cord, blood-vessels arise in a manner identical with their primary origin in the original body-stalk, and become secondarily connected with the preexisting vascular net. The connective tissue of the full-term cord maintains the primitive vasculogenic mode by which the primitive blood-vessels were formed. The full-term cord is relatively much more extensively vascularized, and it consists of connective tissue largely of a less differentiated type than the cord of the 21-mm. fetus. Since the larger blood-vessels extend to the distal end of the cord, it may be inferred that they supply also the proximal pole of the allantois.
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VASCULOGENESIS AND HEMOPOIESIS
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In the description of the mode of vasculogenesis illustrated in this cord, we may begin most conveniently with the stage represented by the cell of figure 8. This cell has the general characteristics of an irregular mesenchymal cell. Three vacuoles can be seen to the right of the nucleus. A later stage may be represented by the upper cell of figure 9. Here the cell is binucleated, and the originally smaller discrete vacuoles have presumably coalesced to form a single large vacuole, the precursor of the initial capillary lumen. The appearance of the lumen has effected a modification of one of the nuclei so that it begins to assume endothelial features. In figure 10 is shown a still later phase of vasculogenesis. Here, moreover, the more typical endothelial 'cell' has taken on hemoblast features and has differentiated an erythroplastid (ep.) intracellularly. Figure 11 may be conceived to represent a transection of the cell of figure 9 or 10. Cells like those of figures 8, 9, 10, and 11 are very numerous in the more peripheral regions of the cross-section. Figure 12 illustrates a binucleated cell with essentially mesenchymal features, which has differentiated an erythroplastid and a lumei^ centrally. The cell shown in figure 13 has the nuclear and cytoplasmic characteristics more of a young hemoblast. This cell represents a mesenchymal cell which has rounded up and differen
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UMBILICAL CORD OF THE PIG 9
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tiated into a hemoblast, and subsequently differentiated an erythroplastid intracellularly.
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The mesenchymal cells of this primitive 'mucous' connective tissue may apparently undergo any one of several types of differentiation: 1) They may separate from the mesenchymal syncytium, round up and differentiate into potential hemoblasts, which may lie freely among the undifferentiated mesenchymal cells, but apparently never in this condition directly metamorphose into erythroplastids ; but grouped into blood-islands, about which the adjacent mesenchyme differentiates into endothelium, they develop into erythroblasts (fig. 7). 2) They may become bi- or multinucleated and, as hemogenic giant-cells, differentiate erythrocytes intracellularly (figs. 6, e, and 15, /). 3) A mesenchymal cell may acquire a lumen and join with other cells to form an initial capillary, incidentally differentiating also erythroplastids intracellularly (fig. 12). Erythroplastids may originate intracellularly also in young endothelial cells (fig. 10). Hemoblasts can, therefore, apparently differentiate into erythrocytes only when inclosed by endothelium; or in the multinucleated condition, hemoblasts can differentiate intracellular erythrocytes. The latter phenomenon is essentially like that where a hemoblast is inclosed by endothelium.
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Figures 6 and 7 illustrate an earlier and later stage, respectively, in the differentiation of a blood-island. In figure 6 the bloodisland is still largely a syncytium. However, endothelium can be seen forming on its surface, and several of its cells are taking on erythroblast ('megaloblast') features (a, b, and d). The cell d has developed a large vacuole at one pole; this vacuole may form part of the subsequent lumen. Cell e has differentiated an erythrocyte. Several intercellular spaces have appeared in the syncytium; these are the forerunner of the subsequent lumen, to which certain intracellular spaces may also contribute. In figure 7 the endothelium and the lumen are developed further, and the hemoblasts are mostly in the erythroblast stage and are generally separated from each other by cell membranes.
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It seems desirable at this point to indicate the chief differences, nuclear and cytoplasmic, between the young mesenchymal
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10 H. E. JORDAN
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cell, young endothelial cell, hemoblast, erythroblast ('megaloblast'), and the erythrocyte. The mesenchymal cell has in general a light-staining, spheroidal or oval nucleus with a delicate reticulum; its cytoplasm contains delicate fibrillae. The young endothelial cell has a similarly light-staining, but generally more elongated nucleus; and its cytoplasm is less distinctly fibrillar. The hemoblast is generally spheroidal in shape, but it may assume various forms due to its ameboid capacity; its nucleus also generally has a spheroidal shape, but it contains a more distinct and more granular network, and the cytoplasm appears homogeneous. The nucleus of the young erythroblast ('megaloblast') has a spheroidal shape, a robust chromatic membrane, a generally deeper-staining nucleoplasm; and it contains one or several small nucleoli and numerous granules scattered over its delicate reticulum. Its cytoplasm is distinctly granular, and it stains deeply in eosin. The granular condition of the cytoplasm of the erythroblast is the most distinctive mark of this cell. This cell corresponds to the megaloblast of certain writers. The erythrocyte has a considerably smaller, generally deeperstaining, spherical nucleus, and a clear cytoplasm delimited by a distinct membrane (figs. 6, e, and 15,6 ande). Theerythroplastid has in contrast a brownish-yellow color.
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In figure 15 (a to i) are illustrated various vasofactive and hemogenic cells. In fact, as these figures clearly indicate, vasofactive and hemogenic activities are intimately associated. The cell a may be regarded as at the stage of a late hemoblast or a young erythroblast. The cell b is similar, but has produced an intracellular erythrocyte. Cell c has developed a lumen, and it has differentiated an inclosed erythroplastid. Cell d is essentially a young endothelial cell with vestiges of cytoplasmic hemoblast features. Cell e has become essentially a young endothelial cell with an included small erythrocyte and an erythroplastid. Cells g and i are essentially hemoblasts ('angioblasts') which have become differentiated into binucleated endothelial cells. Cells/ and h should be interpreted together; h is essentially a multinucleated hemoblast or small 'giant-cell/ one of whose nuclei is apparently undergoing amitotic division;
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UMBILICAL CORD OF THE PIG 11
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/ may be regarded as a later stage in the intracellular erythrocytogenic function of h, in which two of the nuclei and their enveloping cytoplasm have differentiated into erythroplastids (ep). Cells h, f, b, and e of figure 15 and e of figure 6, when considered in common, demonstrate that erythroplastids in these vasofactive cells do not arise as such out of the cytoplasm, but under the direct influence of a nucleus of a bi- or multinucleated hemoblast (memogenic giant-cell'). The absence of free erythrocytes and erythroplastids in the regions from which these cells are taken contravenes any suggestion that the intracellular red blood-corpuscles should be interpreted in terms of phagocytosis. Figures 12 and 14 supply similar evidence. The erythroplastid of figure 12 may appear to have arisen directly from the cytoplasm of this vasofactive cell. But figure 14 shows an essentially similar cell at a slightly earlier stage of differentiation, in which the chief nucleus has liberated a small bud. About this bud an erythrocyte and a lumen may be conceived to originate in the manner shown in e of figure 15, and so lead to a condition like that of figure 12. In other words, the cells of figures 12 and 14 are essentially multinucleated hemoblasts. In this sense multinucleated hemoblasts, hemogenic giant-cells, and blood-islands ('angioblasts') are fundamentally and potentially alike ; that is, they are essentially multiple ery throblasts enveloped by a layer of potential endothelium.
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DISCUSSION
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From the foregoing description it will be clear that the connective tissue of the full-term umbilical cord of the pig is extensively vascularized and that it is actually for the most part still in the condition of young mesenchyma or embryonal connective tissue. The conditions are essentially similar to those described for the body-stalk of very young human embryos. The question arises whether the connective tissue of this cord is in the primitive mesenchymal condition because it is vascularized or whether it is vascularized because the connective tissue is in the condition of undifferentiated mesenchyma. Since the bloodvessels have apparently arisen to a considerable extent in situ,
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12 H. E. JORDAN
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t he latter interpretation would seem to be the correct one; that is, that this cord has maintained early embryonic conditions, like that of its anlage, the body-stalk, and in consequence retained its original capacity for vasculogenesis and erythrocytogenesis. The cause of this maintainance of early embryonic vasculogenic and hemopoietic potentialities, especially singular in connection with the advanced developmental condition of the umbilical arteries and vein, and the several small areas of fully differentiated mucous connective tissue, remains for the present undertermined. It is most probably associated with the large functional allantois, but the nature of this association is not clear. The relatively highly developed character and healthy condition of the covering ectoderm may be secondary to the presence of the large number of capillaries in the subjacent connective tissue.
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Though this study can throw no light on the cause of the vascularized condition of the umbilical cord of the pig, the intense hemopoietic activity of its connective tissue supplies valuable data with respect to the initial steps in vasculogenesis. This is the chief point of value in this specimen. In this connection interest centers upon the mesenchymal cell, which becomes hollowed out to form an endothelial cell and at the same time differentiates erythrocytes (figs. 6, 12, 13, and 15). This cell combines the functions of an endothelioblast and an erythroblast. The process appears to be quite similar to that first described by Ranvier 8 (74) in the mesentery of the seven-day rabbit and in the great omentum of the cat, and independently by Schaefer 10 (74) in the subcutaneous tissue of the new-born rat, and subsequently confirmed by other workers on other forms. Ranvier named the cells concerned in the process 'vasoformative cells.' According to these investigators, mature (non-nucleated) 'erythrocytes' of greatly varying sizes are formed directly within the protoplasm of connective-tissue cells (vasoformative cells) by a process involving the coalescence of scattered granules of hemoglobin into condensed globules, which then come to lie in vesicles within the cells, the precursors of the capillary lumen.
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UMBILICAL CORD OF THE PIG 13
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The method of erythrocytogenesis here described for this specimen of umbilical cord of the pig, however, differs radically from that described by Ranvier and Schaefer, in that the erythroplastid in this case differentiates from a nucleated portion of a vasofactive cell (figs. 6, e; 15, b and e, and 14). That is, the erythroplastid differentiates from a typical erythroblast in the usual mode. The nucleus of the erythrocyte disappears by karyolysis (fig. 15, e). This is apparently a very rapid process, since it can be detected in only relatively few cells. If one considered only cells like those of figures 12 and 13, the process would appear to be identical with that described by Ranvier and Schaefer; but figures 15, b, e, h, and/ demonstrate the essential difference.
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Certain investigators (Spulef, 12 Fuchs, 3 et al.) have expressed dissent from Ranvier's and from Schaefer's interpretation of their observations; they explain these phenomena, the occurrence of which they confirm, in terms of regressive changes and phagocytosis. They believe that the so-called Vasoformative cells' are either isolated portions of a disintegrating embryonic vascular plexus or erythrophagic connective tissue cells. It is obvious that since the vasofactive phenomena here described for the umbilical cord of the pig are fundamentally different, while superficially apparently identical with those described by Ranvier and Schaefer, the criticisms of Spuler and Fuchs have no pertinancy to this case. Moreover, the red cells involved in this process in the umbilical cord of the pig show no distinct nuclear or cytoplasmic marks of degeneration. This cord, except for the almost complete absence of mitotic figures, appears in perfectly healthy condition. No free erythrocytes are available for phagocytosis in the regions here described. There is no indication of a disintegration of blood-vessels; on the contrary, the full-term cord is relatively more extensively vascularized than the cord of the 21-mm. fetus. Finally, and most significantly, this intracellular mode of erythrocytogenesis is strictly comparable to that described for other hemopoietic organs, e.g., yolk-sac of 10-mm. pig embryo, 5 yolk-sac of mongoose embryos, 6 and red bone-marrow. 7
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14 H. E. JORDAN
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The matter may be summed up with figures 15, a, b, and e, and 15, h and /. A connective-tissue cell becomes transformed into a hemoblast (erythroblast) with vasofactive capacity. This cell may become bi- or multinucleated. One or several of the nuclei with their enveloping cytoplasm may differentiate into erythrocytes. Meanwhile a lumen appears within the cell, and one or two of the original nuclei may persist as the nuclei of the peripheral cytoplasm of the differentiating cell, which now forms the endothelial wall of the initial capillary. In later stages in the yolk-sac and in the red bone-marrow generally, the peripheral 'endothelial' layer of the original 'vasofactive' cell disappears, thus freeing the intracellularly differentiated erythrocytes into the confining blood spaces. The mesenchymal cell thus appears endowed with divers hemogenic "potentialities: it may become an endothelial cell or a hemoblast (erythroblast) . The endothelial cell may secondarily differentiate into a hemoblast. These hemoblasts may differentiate into erythroblasts or, as multinucleated cells, they may differentiate both intracellular erythrocytes and a potential endothelial cell. These facts demonstrate the very close relation between mesenchyme, endothelium, and hemoblasts.
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Sabin 9 records a similar vacuolization of mesenchymal 'angioblasts' in the living blastoderm of the two-day chick embryo grown in Locke's solution, by which the blood-vessel lumen forms. But these observations do not justify her conclusion that they prove "that the lumen of a blood-vessel is intracellular" (p. 200). The data supplied by the umbilical cord of the pig show that the definitive lumen of the blood-vessel derived from a blood-island is of both inter- and intracellular origin.
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This brings us to the matter of the factors which determine whether the mesenchymal cell shall become an endothelial cell or a hemoblast, and relates this investigation to the discussion regarding theories of hemogenesis, that is, whether blood development proceeds according to the monophyletic or the polyphyletic mode. This much seems certain regarding this tissue: single hemoblasts, freed from the mesenchyme and wandering within its meshwork, d*o not differentiate into erythrocytes. It
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UMBILICAL CORD OF THE PIG 15
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is only when such a cell becomes inclosed by endothelium that it differentiates into a red blood-cell. Thus a group of such cells may form and in consequence produce pressure upon the surrounding mesenchyme, which then becomes transformed into endothelium. Under these conditions the enveloped hemoblasts become erythrocytes (figs. 6 and 7). Such endothelium is simply an adaptive form of mesenchyme, as originally maintained by Huntington 4 and by Schulte, 11 and it may subsequently return to mesenchyme, remain as endothelium, or differentiate hemoblasts either intra- or extraluminally. Endothelium, accordingly, develops originally by two different methods, both clearly represented in the specimen under consideration: 1) By adaptation of mesenchyme about a blood island; 2) by vacuolization of vasofactive mesenchymal cells ('angioblasts').
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A point of special interest concerns the fact that the nucleated periphery of a multinucleated hemoblast supplies the same favorable conditions or factors for determining erythrocytogenic differentation as an endothelial wall. This phenomenon becomes intelligible when we consider that both endothelial cells and hemoblasts are only slightly modified mesenchymal cells, and that the latter, as vasofactive cells, may become hollowed out to form the lumen of an original capillary or differentiate intracellular erythrocytes. The central fact here pertains to the obviously very minute difference between the environmental conditions or stimuli that determine whether the same cell (the potential hemoblast, 'vasoformative cell', or 'angioblast') shall become an endothelial cell or an erythroblast. This suggests that also the factors which determine whether the hemoblast shall become a leucocyte or an erythrocyte, in accordance with the monophyletic theory of blood-cell origin, are similarly relatively subtle and of minute degree. Original confinement by endothelial walls furnishes the stimulus which determines erythrocytogenesis; extra vascular differentiation leads to granulopoiesis. As shown by the recent experiments of Danchakoff, 1,2 the original poly valency of the hemoblast, however, is lost by the erythroblast, and this degree of differentiation is irreversible. An erythroblast freed from its endothelial confines and
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THE AMERICAN JOURNAL OF ANATOMY, VOL. 26, NO. 1
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16 H. E. JORDAN
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thrown into the surrounding mesenchyme, as in the allantoic spleen grafts of DanchakofT, will not differentiate into a leucocyte, but into an erythrocyte. A mature endothelial cell does not normally, as originally, differentiate into a hemoblast. And an extra vascular hemoblast which is in process of differentiation into a granulocyte apparently cannot redifferentiate into an erythrocyte after it has wandered into the blood-vessel lumen.
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SUMMARY
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The results of this study of the umbilical cord of the pig, which maintains to full-term largely the embryonic condition of the original body-stalk, emphasize the polyvalent capacity of the mesenchymal cell and its hemoblast derivative, and supply further evidence in agreement with the monophyletic view of hemogenesis. The multinucleated hemogenic giant-cell furnishes the same essential stimuli for the differentiation of erythrocytes as does an inclosing endothelium. It is comparable to a blood-island, and produces erythrocytes intracellularly in a manner similar to that by which erythrocytes separate out of a blood-island syncytium. This tissue demonstrates also the origin of endothelium both by adaptation of mesenchyme about a blood-island and by vacuolization and fusion of vasofactive mesenchymal cells. It shows, moreover, that the lumen of the original blood-vessels includes both inter- and intracellular contributions.
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UMBILICAL CORD OF THE PIG 17
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LITERATURE CITED
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1 Danchakoff, Vera 1918 Cell potentialities and differential factors con sidered in relation to erythropoiesis. Am. Jour. Anat., vol. 24, p. 1.
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2 1918 Equivalence of different hematopoietic anlages (by method of stimu lation of their stem-cells). II. Grafts of adult spleen on the allantois and response of the allantoic tissues. Am. Jour. Anat., vol. 24, p. 127.
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3 Fuchs, H. 1903 Uber die sogcnannte "intracellulare" Entstehung der
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roten Blutkorperchen junger und erwachsener Sauger. Anat. Hefte, Bd. 22, S. 95.
 +
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4 Huntington, G. S. 1914 The development of the mammalian jugular
 +
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lymph sac, of the tributary primitive ulnar lymphatic and of the thoracic ducts from the viewpoint of recent investigations of vertebrate lymphatic ontogeny, together with a consideration of the genetic relations of lymphatic and hemal vascular channels in embryos of Amniotes. Am. Jour. Anat., vol. 16, p. 259.
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5 Jordan, H. E. 1916 The microscopic structure of the yolk-sac of the pig
 +
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embryo, with special reference to the origin of the erythrocytes. Am. Jour. Anat., vol. 19, p. 277.
 +
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6 1917 Hemopoiesis in the mongoose embryo, with special reference to the
 +
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activity of the endothelium, including that of the yolk-sac. Pub. 251 of the Carnegie Institution of Washington, p. 291.
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7 1918 A contribution to the problems concerning the origin, structure,
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genetic relationship and function of the giant-cells of hemopoietic and osteolytic foci. Am. Jour. Anat., vol. 24, p. 225.
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8 Ranvier, L. A. 1874 De dcveloppement et de 1'accroisement des vaisseaux
 +
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sanguins. Arch, de Physiol., T. 6, p. 429.
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9 Sarin, F. R. 1917 Preliminary note on the differentiation of angioblasts
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and the method by which they produce blood-vessels, blood plasma and red blood cells as seen in the living chick. Anat. Rec, vol. 13, p. 199.
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10 Schaefer, E. A. 1S74 The intracellular development of blood corpuscles
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in mammals. Mon. Micr. Jour., vol. 11, p. 261.
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11 Schulte, H. vox W. 1914 Early stages of vasculogenesis in the cat (Felis
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domestica) with special reference to the mesenchymal origin of endothelium. Memoir, Wistar Inst. Anat. and Biol., no. 3, pp. 1-92.
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12 Spuler, A. 1S92 Uber die "intracellulare" Entstehung roter Blutkor perchen. Arch. f. mikr. Anat., Bd. 40, S. 530.
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PLATE 1
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EXPLANATION OF FIGURE
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1 Photomicrograph of transverse section of umbilical cord of pig. The cord is covered with a stratified epithelium from three to eight (generally four) layers thick, resembling somewhat the transitional type. Tufts of keratized cells occur at certain points (E). To the right of the allantoic duct (All) are the umbilical arteries. The umbilical vein lies below the double remnant of the occluded yolk-stalk (Y.S.). At B.V. is one of the larger arterioles of the extensively vascularized connective tissue. In the area between the lower umbilical artery and the vein, the connective tissue resembles the mucous type; elsewhere it resembles more young mesenchyme, and contains many capillaries, arterioles and venules (A.V.), and also numerous hemoblasts and several typical bloodislands. (Photos, by W. S. Dunn, Cornell University Medical College, N. Y. City. The illustrations were made from the Columbia specimen.) X 18.
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18
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UMBILICAL CORD OF THE PIG
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H. E. JORDAN
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PLATE 1
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E.
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y.s.
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E.
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19
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PLATE 2
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EXPLANATION OF FIGURES
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2 Photomicrograph of the ectodermal covering of the cord in the region, Som., of figure 1. It includes four or five layers of cells and resembles transitional epithelium. The lowermost layer is composed of cuboidal cells; the outermost layer includes dome-shaped, rectangular, and flattened peridermal cells; the intermediate layers include spheroidal and polyhedral cells. X 300.
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3 Photomicrograph of remnant of yolk-stalk (fig. 1, Y.S.). It is double and occluded, and each moiety includes about twelve cells in its diameter. Art., arteriole. X 300.
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20
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UMBILICAL CORD OF THE PIG
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PLATE 2
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H. E. JORDAN
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.- •
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V *i
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21
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PLATE 3
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EXPLANATION OF FIGURES
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4 Photomicrograph of allantoic duct. The lining epithelium is thrown into folds. In the troughs of the folds the epithelium consists of a single layer of cuboidal or flattened cells ; over the crests, of from three to four layers, constituting a stratified columnar epithelium. Art., arteriole; B. I ., 'blood-island' of erythroblastic X 235.
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5 Photomicrograph of pair of blood vessels (the branching venule cut obliquely) and the surrounding mesenchyme (region A.V. of fig. 1). X 300.
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22
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UMBILICAL CORD OF THE PIG
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H. E. JORDAN
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PLATE 3
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Art:
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ML ■ *
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LP"
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**r
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23
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PLATE 4
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EXPLANATION OF FIGURES
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6 Drawing of blood-island, from region just below the allantoic duct (fig. 1, All.). Peripherally the cells are becoming differentiated into an endothelium. Centrally the syncytial mass is becoming vacuolated through the appearance of intercellular spaces, and certain of the cells have entered the early erythroblast ('megaloblast') stages (a and b). One erythroblast (d) contains a large vacuole. A hemoblast (e) has differentiated an intracellular erythrocyte. A hemoblast (h) is separating from the differentiating endothelium. Between the two endothelial cells below, appears another hemoblast. Figures 6 to 9 are magnified 1500 diameters.
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7 Blood-vessel in process of differentiation from the mesenchyme. This drawing is from the region to the right of the allantoic duct between the two umbilical arteries (fig. 1), and includes approximately the middle third of this entire vascular anlage. The forming lumen contains seven young erythroblasts ('megaloblasts'), separating out of an originally syncytial mass.
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8 Young vasofactive cell, with generally mesenchymal features and three vacuoles, the precursors of a capillary lumen.
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9 Slightly older vasofactive cell with large vacuole, the underlying nucleus assuming endothelial features.
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24
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UMBILICAL CORD OF THE PIG
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H. E. JORDAN
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PLATE 4
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h
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<&k'8
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6
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-A <f£;
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w
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&
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§ #
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8
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/
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9
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25
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PLATE 5
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EXPLANATION OF FIGURES
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10 Vasofactive cell with three nuclei. The nucleus at the right has original mesenchymal features; the endothelial 'cell' below the lumen has assumed hemoblast features, and has differentiated an intracellular erythroplastid (ep). Figures 10 to 15 are magnified 1500 diameters.
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11 Vasofactive cell differentiating a lumen. This figure corresponds to a transverse section of figure 10.
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12 and 13 Vasofactive cells with an erythroplastid in the lumen.
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14 Vasofactive cell in early stage of differentiation from mesenchyme. The cell has in general hemoblast features. The larger nucleus has produced a small bud at the left. From such nuclear buds and their enveloping cytoplasm develop intracellular erythroplastids.
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15 (a to i) Vasofactive mesenchymal cells at various stages of differentiation: a) Typical young erythroblast ('megaloblast'). h) Hemoblast that has differentiated an erythrocyte intracellularly. c) Cell with vascular lumen and an intracellularly differentiated erythroplastid. d) Cell with lumen, having assumed endothelial features, e) Cell with lumen, containing an . erythrocyte (ec, 'normoblast') and an erythroplastid (ep.). f) Binucleated cell with two intracellularly differentiated erythroplastids (ep). g) Cell with lumen and two nuclei, both with endothelial features, h) Cell with four nuclei, one apparently in process of amitotic division. The centrally located nuclei with their enveloping cytoplasm may differentiate into erythrocytes. ?') Binucleated cell, endothelial in character, with lumen containing an erythroplastid.
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26
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IMBILICAL CORD OF THE PIG
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H. E. JORDAN
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PLATE 5
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et?
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10
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II
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eR
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a
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12
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ev.
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13
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m
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14
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H
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/.T>
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b
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ec.
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<=P
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f
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15
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27
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l>
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Resumen por el autor, Henry Alexander Murray, Jr.
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Colegio de Medicos y Cirujanos, Columbia University.
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El desarrollo del asa cardiaca en el conejo, con especial mention
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del surco bulbo-ventricular y el origen del
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tabique inter-ventricular.
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El autor ha estudiado varios embriones de conejo para determinar si la formacion del asa cardiaca en esta especie es semejante a la descrita por Schulte en el gato. Los procesos de formacion son semej antes en ambas especies en lo referente al origen del asa primaria, que se produce a consecuencia del hundimiento de la hendidura bulbo-ventricular izquierda, y la fusion de los tubos miocardicos se verifica por la intervencion de la placa cardiaca media, que esta representada temporalmente por una cresta bien distinta situada en la pared interna del miocardio. En las series de embriones de conejo, sin embargo, esta elevation se oblitera algunas veces por completo y no susministra prueba alguna sobre su intervencion en la formacion del surco inter-ventricular primitivo.
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Translation by Jose F. Nonidez Carnegie Institution of Washington
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author's abstract of this paper issued by the bibliographic service, july 7
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THE DEVELOPMENT OF THE CARDIAC LOOP IN THE RABBIT, WITH ESPECIAL REFERENCE TO THE BULBOVENTRICULAR GROOVE AND ORIGIN OF THE INTERVENTRICULAR SEPTUM
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HENRY A. MURRAY, JR.
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College of Physicians and Surgeons, Columbia University, N. Y.
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SEVEN FIGURES
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The process of union of the two lateral cardiac vessels to form the heart has recently been described in detail by Doctor Schulte as exemplified in the series of young cat embryos of the Columbia Collection. It was at Doctor Schulte's suggestion and under his supervision that I undertook to ascertain whether fundamentally the same processes took place in the embryo rabbit.
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Earlier investigators had described how the lateral plexuses of blood-vessels, forming into two longitudinal endothelial tubes in the splanchnopleure, unite in the midline to create a cylindrical median structure — the heart; and they considered that during subsequent growth the heart became coiled to accommodate itself within the pericardium. Doctor Schulte's investigation, previously mentioned, showed that it was not such a simple process, but that a number of very interesting factors were responsible for the changes that took place. Through the kindness of Dr. F. T. Lewis in putting at my disposal the beautiful Harvard series of rabbit embryos and by his guidance and suggestions, I was able to study the fusion and subsequent history of the heart in another species. Approximately forty embryos from the Harvard rabbit and Columbia cat series were examined under the microscope and a dozen and a half models were constructed in wax, according to the Born method. The cat embryos were cut in 13.3/x sections, whereas the rabbits were cut in 6/x or 10/z sections, mostly transverse. The thicker sections are less apt to be damaged, pile into better models, and under
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29
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30
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HENRY A. MURRAY, JR.
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some conditions are to be recommended. For the finer details, however, thin sections are naturally the more desirable. The embryos were variously stained. My observations lead me to believe that the development of the rabbit heart tallies in every important respect with that of the cat.
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In the accompanying diagram (fig. 1), the schema usually presented to portray the formation of the cardiac loop may be contrasted with a parallel series of figures representing Doctor Schulte's findings. Note that the initial and final stages in each
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A
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B
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Fig. 1 A. Schema of cardiac loop formation; as presented in most modern text-books, based on the His models. B. Schema representing the same period of development, as observed by Doctor Schulte in the Columbia Laboratory. (Note that the bulboventricular clefts are formed in both hearts before fusion takes place, that the left groove together with the left shoulder of the ventricle becomes accentuated, that there is a corresponding obliteration of the right groove and shoulder, and that the venous end of the heart migrates to the left. These are the principal factors in the formation of the loop.)
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case are the same, but that the intervening processes are dissimilar. Instead of reiterating Doctor Hchulte's conclusions, I will ask the reader to examine carefully figure 2 before reading the following explanation. It is a model of the endocardial cavity in a nine-day rabbit. The myocardial mantles, not represented in the model, have completely fused, but the endothelial tubes have not as yet entirely coalesced. The picture presents a condition previous to the complete amalgamation of the vessels to form a common cavity. Those points where the endocardia
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CARDIAC LOOP IN RABBIT
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31
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are still separate mark out the line of fusion of the two primitive tubes, and it is thus quite evident what portions of the cavityare derived from the right cardiac vessel and what portions from the left. 1
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The elements represented are: a) the sinus venosus at the confluence of the vitelline veins; b) the canal between the sinus venosus and the ventricle, which, as the atrium develops at this point, we may call the atrial canal; c) the common ventricle,
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Fig. 2 Model representing a cast of the cavities within the heart and the connecting vessels in a rabbit embryo of nine days. Ventral view. Harvard Embryological Collection, Series 619. 1, aortic branches; 2, right and left bulbs; 3, apertures formed by the septum dividing the bulboventricular canal; 4, right shoulder; 5, left shoulder; 6, aperture formed by the septum dividing the common ventricle; 7, atrial canal; 8, right vitelline vein; 9, left vitelline vein. X 100.
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x The valuable study of the development of the pericardium in ferrets by Professor Robinson (Journ. of Anat. and Phys., 1902, vol. 37) has recently been extended by his pupil, Doctor Wang, with results which deserve more critical consideration than can be given here. Being concerned especially with early stages, Wang does not discuss the questions raised by Schulte, and his interpretation of the model shown in figure 2 would differ from ours, as may be seen by comparing it with his figure 31 representing the heart of a ferret of 13 to 14 segments. Wang's most interesting observation is of a 'primary heart rudiment,' a vessel crossing the median line and subsequently dividing into two endothelial tubes. The lateral vessels thus formed, or others somewhat posterior and leading to them, then reunite to make a 'secondary' heart. In the rabbits of the Harvard Collection, as Doctor Lewis informs me, there may be seen a strand of presumably angioblastic tissue in the region of the primary heart of Wang, but nothing which should be interpreted as a heart. At present, therefore, we are not inclined to recognize a heart previous to the union of the lateral cardiac vessels.
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THE AMERICAN JOURNAL OF ANATOMY, VOL. 26, NO. 1
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32 HENRY A. MURRAY, JR.
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with a right and left shoulder; d) the restricted portion between ventricle and bulb, which may be called the bulbo ventricular canal; e) the bulb, and /) the arterial branches. The following points should be particularly noted: 1) the atrial canal has been forced to the left, and that portion of the canal contributed by the right heart has become relatively much reduced; 2) the left shoulder of the ventricle is elevated, the right depressed; 3) the left bulboventricular cleft is pronounced, the right is obscure, and 4) on account of the greater impression made by the left bulboventricular cleft that portion of the bulbar canal contributed by the left heart is diminished. Later, the right bulboventricular cleft disappears, the left becomes more pronounced and vertical, the atrium develops from the atrial canal growing cephalad behind the ventricles, and the cardiac loop is then complete. My observations commence at a stage when the original lateral tubes have become ventrally placed and are united through the intervention of a middle cardiac plate. The process by which this change is effected — a subject upon which Wang speculates at some length — will not be discussed. Attention will be focused on what may be considered the most fundamental aspects of the succeeding modifications, namely, 1) the middle cardiac plate with a consideration of its future history and possible connection with the interventricular septum, and 2) the bulboventricular groove.
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MIDDLE CARDIAC PLATE AND INTERVENTRICULAR SEPTUM
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In figure 3 observe the middle cardiac plate connecting the two hearts. It will be noticed that it is narrow cephalad, connecting the bulbs, and broad caudad between the ventricles. This mesothelial element gradually becomes incorporated into the myocardial walls of the enlarging ventricular cavity and is later represented in the rabbit by a ridge or series of ridges marking the original line of fusion. The ridges which are quite apparent on the inner aspect of the ventral wall are well shown in figure 4; they are placed opposite the septa which still remain between the two endothelial tubes (compare fig. 2). The early embryos of the cat in the Columbia series show similar ridges
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CARDIAC LOOP IN RABBIT
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33
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and also some intermediate stages. The rabbit differs from the cat in that this ental protrusion of the myocardia is a simple ridge and is not surmounted by a groove. Has this ridge any definite relation to the future interventricular septum? I think not. After modeling a number of rabbit and cat hearts between this stage an,d the stage when the interventricular septum is first apparent, I find no connection between the two. As shown in figure 4, when last observed the median ridge is directed towards the atrial canal. As the latter does not change its position until a later date, if the interventricular septum were a product of this ridge we should expect to find it at first obliquely placed
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Fig. 3 Model of the myocardium (ventral view) from a rabbit embryo of nine days; H. E. C, Ser. 620. 1, right bulb; 2, left bulb; 3, right bulboventricular groove; 4, left bulboventricular groove; 5, right ventricle and shoulder; 6, left ventricle and shoulder; 7, middle cardiac plate. X 100.
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and in line with the canal. This is not the case. On the contrary, we find a septum arising apparently as a thick muscular ridge from the most caudal portion of the ventricular loop, corresponding to a groove on the exterior. 2 Both septum and groove are sagittally placed and are not at this early stage directed towards the atrial canal (compare fig. 6). Furthermore, the septum appears at a considerably later date, after the common atrium is well formed and the ventricular wall has undergone great expansion and growth with considerable trabecular formation. In both the rabbit and cat embryos the increased
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2 As Mall says, it is more correct to speak of the downward growth of the apices of the two ventricles than the upward growth of the septum.
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34 HENRY A. MURRAY, JR.
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growth in thickness of the ventricular wall is first manifest on the ventral wall and gradually spreads laterally and dorsally. In my series, the actual height 3 of the heart in the rabbit when the last sign of a middle cardiac plate can be determined is 0.32 mm., whereas the earliest sign of an interventricular septum is found in an embryo with an actual heart length of approximately 0.75 mm. Doctor Schulte, in the latest stage in which a middle cardiac plate was present in his material, found it continued caudad by a sulcus which he regarded as the beginning septum ventriculorum. Beyond this stage there was a gap in his material to the period of a well-developed septum with no remnant of the middle cardiac plate. My reason for dissenting from his conclusion that the median plate gives rise to the septum is that in the rabbit models and in embryos of the cat subsequently obtained the sulcus mentioned is found to disappear and the ventricle becomes evenly convex without a trace of indentation referable to the middle cardiac plate. Only later does the ventricular septum arise in the manner I have described. The interior of the model portrayed in figure 5 shows a smooth wall with no sign of a ridge, although at two points the endothelial tubes have not yet completely united. Considerably later, as the atrial canal is moved to the right while the interventricular septum remains fixed, the canal comes to be immediately dorsal to the septum ; the septum will then be seen to extend toward the centre of the canal and still later to become fused to the endocardial cushion. The subsequent development of these parts is beyond the scope of this paper.
 +
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THE BULBOVENTRICULAR GROOVES
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These clefts first appear on each side before the lateral cardiac vessels have fused. They have been given this name because in their primary position they separate primitive bulb from primitive ventricle. Later, as we shall see, the bulb contributes to the right ventricle, and the left bulboventricular cleft may then be termed an 'interventricular groove.' In figure 3 the
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3 As measured from the most cephalic to the most caudal points, regardless of what portions of the heart these may be.
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CARDIAC LOOP IN RABBIT
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35
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Fig. 4 Model of the myocardium (dorsal view of the ventral wall) from a rabbit embryo of nine days; H. E. C, Ser. 619. 1, bulboventricular canal; 2, left shoulder; 3, left bulboventricular groove; 4, right shoulder; 5, right bulboventricular groove; 6, inward protrusions of the myocardium in a line directed toward the center of the atrial canal; 7, sinus venosus. X 100.
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Fig. 5 Model of the myocardium (ventral view) from a rabbit embryo of nine and one-half days; H. E. C, Ser. 565. 1, aortic branches; 2, bulb; 3, bulboventricular groove; 4, shoulder; 5, common ventricle; 6, right vitelline vein; 7, left vitelline. X 100.
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36 HENRY A. MURRAY, JR.
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grooves are horizontal. The left groove is already assuming a more significant r61e and the asymmetry forecasts future changes. In the cat this prominence of the left cleft is not accentuated until somewhat later. A subsequent stage in the rabbit is represented in the endothelial model, figure 2, and in figure 5 a fully developed cardiac loop is seen. The bulboventricular cleft (there is now only one, the right having become obliterated) is more oblique. From this stage onward there is a continual progressive extension of this furrow, and as it develops, its plane is modified so that in figure 6 we find it very nearly vertical. Notice in this drawing its relationship to the primitive septum. It is not in line with the latter structure. In the next period, however, the bulboventricular groove, formerly horizontal, is now vertical, protrudes into the ventricular chamber, becomes continuous with the septum, and together with it divides the cavity into right and left portions. This is well shown in figure 7. The division of the common ventricle then seems to be the result of four processes: 1) the interventricular septum growing cephalad from the floor of the loop; 2) the bulboventricular groove becoming vertical and forming the ventral portion of the septum; 3) the migration of the atrial canal to the right, allowing the endothelial cushions to play their part, and finally, as His has shown, 4) the downgrowth of the pulmonoaortic septum which fuses with the above-mentioned elements so as to form a continuous partition between the right and left hearts.
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Fig. 6 Model of the myocardium (dorsal view of the ventral wall) from a rabbit embryo of ten and one-half days; H. E. C, Ser. 559. 1, bulb; 2, ridge extending into the common ventricular cavity and corresponding to the bulboventricular cleft; 3, shoulder; 4, interventricular septum (this is the first indication of the ridge found at the apex of the ventricular loop). X 100.
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Fig. 7 Model of the endocardial cavity (ventral view) from a cat embryo of 7 mm. ; Columbia Collection, Series 266. By kind permission of Dr. A. J. Brown. In the Harvard Laboratory there is a very similar model of the cavities in the heart of a 4.4-mm. pig embryo, made in 1909, under Dr. Minot's direction, by Mr. A. E. Meyers. 1, cleft made by the ridge growing upward from the caudal extremity of the loop, which is continuous with 2, the impression made by the bulboventricular groove. Together they partially subdivide the ventricular cavity; 3, atrial canal; 4, left atrium. X 50.
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CARDIAC LOOP IN RABBIT
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37
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.*.»>oe s
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38 HENRY A. MURRAY, JR.
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SUMMARY
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The processes in the early development of the rabbit's heart are fundamentally the same as in the cat. 1) The primary loop is due to the deepening of the left bulboventricular cleft and a disappearance of the right, accompanied by a reduction on the part of the right shoulder of the ventricle and a very marked growth of the left. 2) The middle cardiac plate, marked temporarily after myocardial fusion by a distinct ridge which corresponds precisely to the line of fusion of the endothelial tubes, eventually becomes entirely obliterated. 3) The primitive interventricular septum arises de novo from the floor of the loop in a sagittal plane. 4) The left bulboventricular cleft, at first horizontal, becomes oblique and then vertical; protruding into the common ventricular cavity as a well-marked ridge, it meets the septum developing in the apical portion of the heart and contributes to the formation of the interventricular septum of the adult.
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■*
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CARDIAC LOOP IN RABBIT 39
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BIBLIOGRAPHY
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Bremer, J. L. 1912 Development of the aorta and aortic arches in rabbits.
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Am. Journ. Anat., vol. 13, no. 2. Lewis, F. T. Intraembryonic blood vessels of rabbits from 8| to 13 days. Am.
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Journ. Anat., vol. 3. Mall, F. P. 1912 On the development of the human heart. Am. Jour. Anat.,
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vol. 13.
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1912 Bifid apex of human heart. Anat. Rec, vol. 6.
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1912 Aneurysm of membranous septum. Anat. Rec, vol. 6. Parker, K. M. The early development of the heart and anterior vessels in
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marsupials with special reference to Berameles. Proc. Zool. Soc,
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London, 1915, Pt. 3. Robinson 1902 Early stages of development of the pericardium. Journ. of
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Anat. and Physiol., vol. 37. Rouviere, H. 1904 Etudes sur le developpement du pericarde chez le lapin.
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Jour, de l'anat et de la physiol., vol. 11. Schtjlte, H. W. 1916 The fusion of the cardiac anlages and the formation of
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the cardiac loop in the cat. Am. Journ. Anat., vol. 20, no. 1. . 1914 Early stages of vasculogenesis in the cat with especial reference
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to the mesenchymal origin of endothelium. Memoirs of Wistar Institute of Anat. and Biol., no. 3. Strahl and Caritjs 1899 Beitrage zur Entwickelungsgeschichte des Herzens
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und der Korperhohlen. Arch. f. Anat. und Physiol., Bd. 15. Tandler, J. Keibel and Mall. Manual of human embryology, vol. 2. Wang, Chtjng-Ching 1917 Earliest stages of development of the blood-vessels
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and of the heart in ferret embryos. Journ. of Anat., vol. 52, part 1,
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Resumen por el autor, Frank Blair Hanson. Universidad Washington, San Luis.
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La ontogenia y filogenia del esternon.
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Existen en la literatura tres teorias diferentes sobre el origen del esterndn, suponiendose generalmente que este hueso no es homologo en los amniotos e ictiopsidos. El autor demuestra que el estern6n de los vertebrados esta mas intimamente ligado con la cintura escapular que con las costillas, y describe diferentes estados de desarrollo en los embriones de un cierto numero de mamiferos — gato, rata, raton, cerdo y hombre — en los cuales las barras esternales son estructuras bien manifiestas antes de su uni6n con las costillas, cuyo hecho es contrario a la teoria de Ruge sobre el origen costal des esternon. El presternon esta intimamente asociado con los coracoides en todas las clases de los vertebrados, incluso los monotremas. En los embriones jovenes del rat6n y el hombre se forma una cintura mesenquimatosa continua comparable a la cintura pectoral del tiburdn, de la cual se derivan la cintura escapular y el manubrio. Por consiguiente, el elemento anterior del esternon tiene un origen comun con la cintura escapular y, en el embri6n o durante toda la vida del animal, esta en intima relacion con los coracoides. Las bandas esternales son derivados del rudimento anterior medio, pudiendose asociar secundaria pero no geneticamente con las costillas. El estern6n es una estructura hom61oga en todos los grupos de vertebrados y se presenta en las formas comprendidas entre el tibur6n Hexanchus hasta el hombre.
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Translation by Josd F. Nonidez Carnegie Institution of Washington
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AUTHOR « ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, AUGUST 4
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THE ONTOGENY AND PHYLOGENY OF THE STERNUM
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FRANK BLAIR HANSON
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Zoological Laboratory of Washington University, St. Louis, Missouri
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TWELVE PLATES (FORTY-NINE FIGUKES)
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CONTENTS
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I. Introduction 41
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II. Critical estimate of existing theories 42
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1. Ruge's theory of costal origin 42
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2. Paterson's coracoidal theory 43
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3. Work of Parker and Howes 45
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4. Whitehead and Waddell's 'in situ' theory 47
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5. Work of Rathke, Kravetz, Mueller, etc 52
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III. The ontogeny of the sternum 58
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1. The sternal bands 58
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2. The anterior median sternal rudiment 60
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3. The sternebrae 61
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4. Stages in the ontogeny of the mammalian sternum 63
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5. Conclusions 63
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IV. The phylogeny of the sternum 64
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1. Fishes 64
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2. Amphibia 66
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3. Reptiles 70
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4. Birds 76
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5. Monotremes 78
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6. Marsupials 78
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7. Aquatic mammals 81
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8. The adult human sternum 83
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9. Conclusions 87
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V. Summary 88
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I. INTRODUCTION
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The shoulder-girdle complex presents some of the most fascinating and difficult problems of vertebrate morphology. In all
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the lower vertebrates the sternum, because of its intimate relation to the coracoids, enters into and constitutes one of these problems. Its origin, development, and homologies have been
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41
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42 FRANK BLAIR HANSON
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the subject of numerous contributions for a century past, yet to-day there is no general agreement upon many of the points involved.
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For several years the writer has been studying the shouldergirdle region in the vertebrates. This included also a restudy of the origin of the sternum, the results of which are embodied in this paper. The fact that my conclusions are at variance with the usually accepted theory of sternum origin only adds to the interest of the undertaking. If this paper settles the points at issue or stimulates further investigation upon the part of others, it will in either case not have been in vain.
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One of the recognized deficiences of much of the previous work is that general and far-reaching conclusions have been deduced from the study of only one or two forms, and these most often the more highly specialized ones. The author has attempted herein to bring together corroborating lines of evidence from both ontogeny and phylogeny, believing that a theory of sternal origin only so demonstrated can command consideration.
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I desire to express my deep appreciation for the constructive criticisms and helpful suggestions given by Prof. J. Sterling Kingsley during the course of this investigation.
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II. CRITICAL ESTIMATE OF EXISTING THEORIES
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1 . Ruge 's theory of costal origin
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Ruge ('80) was the first investigator to work up and present a well-developed and illustrated paper containing a theory of the origin of the sternum. His fifty pages of text and twenty-two figures gave his theory a commanding place in the literature. Most books on human and comparative anatomy, until the present day, copy his figures and accept his view that the sternum arises as a product of the ventral costal cartilages. As an example of this, Keibel and Mall in their two-volume Embryology give the following statement concerning the origin of the sternum: "The cartilage of the sternum arises mainly from the cartilage of the ribs, from which it is secondarily separated by the formation of the costosternal joints." So completely has Ruge's
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ONTOGENY AND PHYLOGENY OF THE STERNUM 43
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work dominated the field that even in the latest editions of Human Anatomy texts his work is alone mentioned, or at most, a footnote is added to the effect that this view has of late been questioned by some.
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However, there are at least two later views as to the origin of the sternum. Pater son ('00, '02, '04), on the one hand, and Whitehead and Waddell ('11), on the other, have propounded theories which are at once contradictory of Ruge 's view and also antagonistic to each other. Thus there are at the present time three distinct and opposing theories concerning the origin of the sternum, and it was with a view to clearing up this confusion and also to give the prominence deserved to this later work that the present investigation was undertaken. For while a study of the papers representing these theories may not convince one of the validity of any one of them as opposed to the others, the latter two mentioned do point out very clearly that there are data which Ruge did not consider; and further, that our commonly accepted view concerning the origin of the sternum, held for nearly forty years, must be greatly modified and possibly cast aside altogether.
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2. Pater son's coracoidal theory
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Professor Paterson in a series of papers ('00, '02, '04) was the first to attempt to overthrow Ruge's theory of the origin of the sternum from the ventral ends of the ribs. His observations were made upon the rat, rabbit, and man. He describes a single median rudiment which is directly continuous with the mass of cells destined to form the shoulder-girdle. From this median mass two strands of cells grow caudally to form the sternal bands. Whitehead and Waddell ('11) say, "thus in the final analysis, according to Paterson 's view, the sternum is derived from the shoulder-girdle." This seems an unwarranted statement. It would be as correct to say that, according to Paterson, the shoulder-girdle is derived from the sternum as to say, as do Whitehead and Waddell, that Paterson makes the sternum to be a derivative of the shoulder-girdle. What Paterson does succinctly say is, "that the presternum and shoulder-girdle are
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44 FEANK BLAIR HANSON
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originally derived from the same (italics mine) element; a primitive band of cellular tissue which crosses the midline. " Paterson has no interest in deriving the sternum from the shoulder-girdle or vice versa; his contention being, first, that the sternum is not a product of the costal cartilages, and, second, that it is yielded from a common, continuous, mesenchymatous element which gives rise to the shoulder-girdles and the sternum.
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Paterson ('02) compares this continuous cellular element in the rat to the girdle in the elasmobranchs. He exhibited before the British Medical Association his sections of rat embryos side by side with embryos of Acanthias vulgaris to demonstrate that essentially the same method of development occurs in the dogfish and in the rodent. But a marked difference is produced in the process of development. Instead of a jointed and highly differentiated structure such as is characteristic of mammals, a simple continuous bar of cartilage is formed, across the middle line and below the heart, which gives rise laterally to the primitive shoulder-girdle."
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Paterson ( '00) also points out, and gives several figures in substantiation, that the parts of the sternum opposite the costal attachments remain longest in a cellular condition. His point being, of course, that if the sternum were ossified from the ribs, these regions should ossify first, and not last as is actually the case.
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This comparison of the girdles in the shark and rat embryos is very suggestive. Many other structures of present-day mammals may be traced directly back to homologous structures in the elasmobranchs, and since in the cartilaginous girdle of the shark we have all the necessary material and in proper position for differentiation into scapulae, coracoids, and sternum, we might even upon a priori grounds expect to find in the higher groups of vertebrates an embryonic stage in which the rudiment of the girdles and sternum might be represented by such a "continuous bar .... reaching across the middle line " as Paterson found in the rat.
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ONTOGENY AND PHYLOGENY OF THE STERNUM 45
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3. Work of Parker and Howes
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Parker ('91) claims to have found a sternum in the shark Notidanus indicus. A small blunt process is set in between the two cartilages which unite later to form the girdle. This structure was earlier described in the same shark by Haswell ( '84) who says "the intercepted cartilage is temptingly like a presternal, but the absence of such an element in the skeleton of any group nearer than the Amphibia seems to preclude this explanation." Parker's ('91) figures 1 and 2 would indicate that this was a presternum, and that Haswell was more nearly correct in his observation than in his deduction therefrom. Had Paterson used Hexanchus rather than Acanthias, he might have found an even more striking resemblance of stages between the rat and shark than he did.
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By the courtesy of the officials of the U. S. National Museum, I was permitted to examine their type specimen of Hexanchus. The body wall had been laid open along the ventral side to allow the preserving fluid to bathe the viscera. By means of a short anterior and two lateral incisions I was enabled to lay bare the median ventral portion of the pectoral girdle without otherwise disturbing the value of the specimen as a type. The girdle (fig. 1) was exactly as described by Haswell ('84) and Parker ('91). The median cartilage in a young specimen was distinctly marked off from the coracoids and in general appearance was not unlike the fetal girdle of the marsupial (fig. 35). Later I dissected two specimens of Acanthias that measured 4 inches and 7 inches, respectively, in order to confirm Paterson's statement of its likeness to the early embryonic girdle of the rat. In both these specimens the girdle was approximately the same as in the adult. Figure 2 shows the girdle of the 7-inch specimen, and if compared to the marsupial girdle (fig. 35) and the mouse girdle (fig. 5), the morphological relations are apparent. In these early stages of the marsupial and mouse no suture has as yet appeared between coracoids and presternum, giving the resulting shark-like girdle, complete across the midventral line.
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46 FRANK BLAIR HANSON
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Parker ('91), accepting in common with others the validity of Ruge's theory of a costal sternum in reptiles, birds, and mammals, but being unable to relate a sternum of such derivation with the sterna of the Ichthyopsida, suggested that there must be two distinct types of sterna: 1) a costal sternum, characteristic of the Amniota, and 2) a coracoidal or clavicular sternum, characteristic of the Ichthyopsida.
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This classification of the sternum was adopted by Howes ('91) who says, "the distinction indicated by the two terms 'costal sternum' and 'coracoidal sternum' is but the expression of a fundamental morphological difference between the two structures." Howes slightly altered the terminology of Parker. He would distinguish between a 'coracoidal archisternum'of the Ichthyopsida and a 'haemocoracoidal neosternum' of the Amniota. This latter term was based upon his idea that the " interclavicle may be, throughout, the vanishing vestige of the coracoidal sternum of the Ichthyopsida." The acceptance by Parker ('91) and by Howes ('91) of this division of two morphologically different sterna in the group of the vertebrates indicates how completely Ruge's theory dominated their thoughts, and the thought and teaching of that day concerning the origin of the sternum. If, however, the conclusions of later workers regarding Ruge's theory prove valid, and all the facts at the present time seem to substantiate their validity as we shall later see, it is no longer necessary to divide the sterna of the various classes of vertebrates into coracoidal and costal, for no sternum is costal in origin, the union of ribs and sternum being but a late and secondary stage in development. This is obviously an important item, if proved, for it enables us to homologize all vertebrate sterna. Heretofore it has been impossible to homologize the sterna of the Ichthyopsida and the Amniotes because of their supposed dual origin. One of the objects of the present paper is to determine this matter of a single or dual origin for the sternum and the solution of its homology throughout the vertebrates.
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ONTOGENY AND PHYLOGENY OF THE STERNUM 47
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4. Whitehead and Waddell's 'in situ' theory
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In 1911 Whitehead and Waddell undertook to settle the whole vexed question by a reexamination of all the evidence and by a study of younger stages than had hitherto been used. Their work was based on observations made upon three forms : the pig, cat, and man. These studies, however, instead of settling the dispute between the theories of Ruge and Paterson, led the authors to reject both of them and to propound an entirely new one. So, as a result of this latest paper, there are at the present time three, instead of two, rival theories of sternal origin. For neither of these two latter theories had been able of its own weight effectually to settle the points in question ; Ruge's theory of costal origin still maintains its hold upon the minds of most morphologists ; but nevertheless, new evidence produced by later work throws very strong doubt upon the conclusions of Ruge.
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Ruge apparently had no stage prior to that in which the sternal bands were united with the costal cartilages; but it must be admitted, that his conclusions, based upon the material that passed through his hands, are clearly valid for the stages described — in fact, the only ones that could possibly be deduced therefrom. It is probably this fact that gave Ruge's theory its persistence through the years. The later workers, however, have had as their goal stages much earlier than Ruge's, and, while they have succeeded in finding them, are still very far apart in the interpretation thereof.
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As Whitehead and Waddell's paper is of considerable importance and has never been reviewed, a brief summary of its contents and conclusions is necessary here.
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They studied first the pig, because the absence of clavicles in this form tends to simplify matters at the cranial end of the sternal rudiment; next the cat, for here the clavicle is a rudimentary bone and does not articulate with the sternum, and finally the human embryo, where the clavicle reaches its fullest development.
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These authors began with pigs of 24 mm. and worked through successively smaller stages, until the sternal rudiment was very
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THE AMERICAN JOURNAL OF ANATOMY, VOL. 26, NO. 1
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48 FRANK BLAIR HANSON
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feeble, while in specimens smaller than 15 mm. no rudiment could with certainty be detected.
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In a pig 24 mm. long, to follow the description of Whitehead and Waddell, the sternal rudiment is an aggregation of mesenchymal cells lying transverse to the median plane of the body. In cross-section it is triangular, with the apex directed ventrally, and each lateral angle of the base connected with the corresponding first rib, there being a perfect and direct continuity of tissue between the rudiment of the first rib and that of the sternum. Proceeding backward in the series of sections two bands of mesenchymal cells separate from the mass and extend as far back as the level of the seventh rib, all seven ribs being connected with and shading off into the sternal bands without any definite demarkation. This is the stage in which Kravetz ('05) found that the first ribs did not reach the sternal rudiment, and the junction of the other six ribs was too feeble to have any morphological significance. Whitehead and Waddell think this is not a tenable conclusion in light of the intermediate position occupied by this specimen, i.e., between older stages in which no doubt of the absolute continuity of ribs and sternum exists, and younger stages in which they seek for new facts concerning the earliest relation between these two structures.
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In a 20-min. pig the pericardial cavity extends into the neck, the ventral ends of the ribs are wide apart, and, in the region anterior to the level of the first rib, the sternal rudiment is present and is composed of three parts : the two sternal bands and a plate of more diffused mesenchymal cells connecting the two across the middle line. This stage presents two facts worthy of note: the sternal bands are well defined separate structures at a level considerably anterior to that of the first rib, and, further, in this anterior region the two sternal bands are connected by a median aggregate of cellular tissue. Posteriorly, in the region of the ribs, there is the same continuity between costal extremity and sternal band as in the 24-mm. stage.
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Just as the older stage of Whitehead and Waddell (24-mm. pig) corresponded to the youngest studied by Kravetz, so does this 20-mm. stage in the pig correspond essentially to the youngest
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ONTOGENY AND PHYLOGENY OF THE STERNUM 49
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stage in man found by Mueller ('06), yet the interpretations of these stages by the three authors are totally at variance.
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The next stage was a 22-mm. pig, longer than the preceding by 2 mm., but distinctly a younger stage in so far as development of these parts was concerned. The sternal bands and connecting bridge of mesenchymal cells are still anterior to the first ribs; the pericardial cavity reaches far into the neck and separates the sternal band and first rib of each side. Passing caudally in the series of sections, the first rib falls just short of reaching the sternal band; the extremity of the second rib approaches more nearly to the sternal band, and the remaining five present the same continuity as before.
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In the next younger stage, the 18-mm. pig, the heart is far forward in the neck, with the consequent wide separation of the sternal bands in this region; the median connecting portion is now absent; the remaining parts of the sternal bands were traced from a point 150 ^ anterior to the level of the first ribs back to the level of the ventral extremity of the seventh; the first two pairs of ribs do not reach the sternal bands, but the other five are firmly fused with it.
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The earliest stages in which any sternal rudiment could be detected were in 15- and 16-mm. pigs. The bands are not very clearly defined and stop at the level of the third and fourth ribs, respectively; again the first and second ribs fail to reach the sternal bands, and it is the opinion of the authors that, "judging from the behavior of the ventral extremities of the first and second ribs in somewhat older stages, we think it probable that a stage exists in which no rib is connected directly with the sternal bands, but we were unable to detect such a stage."
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The cat embryos studied by Whitehead and Waddell (from the Princeton Embryological Collection) ranged in size from 25 mm. to 10 mm. From the description given, they are essentially the same as the several corresponding stages of the pig. In the 12-mm. cat the first three ribs clearly did not reach the sternal bands, the fourth was uncertain, while the three posterior pairs made the connection. Since, from the account given, the early stages in the pig and cat are practically identical, nothing further need be said of the sternum in the cat.
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50 FRANK BLAIR HANSON
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Several human embryos from the Johns Hopkins Embryological Collection, ranging in size from 17.2 to 10.5 mm. are described in the paper now under review. The 13-mm. human embryo corresponds essentially to the 18-mm. pig and 13-mm. cat. The pericardial cavity extends far forward; the sternal bands are traceable to the level of the ventral extremities of the fifth ribs; the ventral tips of the ribs are non-cartilaginous, and any possible continuity between them and the sternal bands is much too slight to suggest that the latter is a derivative of the former; neither median sternal rudiment nor clavicles were detected, though this was probably due to the loss of an entire slide of sections from the very region in which one would expect such a structure to occur, if present.
 +
 +
In a 10.5-mm. human embryo, the sternal bands reach posteriorly only as far as the fourth rib ; and although the cells composing the bands are not sharply differentiated from the surrounding tissue, they are still recognizable and ' ' it is evident that they are not continuous with the tips of the ribs, but are connected with them only by loose mesenchymal cells."
 +
 +
In these stages which are far earlier than any Ruge describes, there is a history quite different from that which later stages had led us to expect. In the first place, there was no indication of segmentation in the sternal bands, which might be expected were these bands due to proliferated cells from the tips of the costal cartilages, and, second, in the pig the first two ribs in the earliest stages studied did not reach the sternal bands at all; three did not do so in the cat, while in the 10.5-mm. human embryo none of the ribs reached the band.
 +
 +
These results also contradict the statement of Paterson that the median rudiment is a part of the scapular arch, and that the sternal bands are derivatives of the single median blastema. Whitehead and Waddell do not mention the condition or extent of the girdles in any of the forms studied by them, merely stating that at no point was there any connection discernible between sternum and girdles; and that in every case "the appearance of the paired portion of the rudiment, the sternal bands, antedates that of the median portion."
 +
 +
 +
 +
ONTOGENY AND PHYLOGENY OF THE STERNUM 51
 +
 +
Since the observations of Whitehead and Waddell accord with neither those of Ruge nor of Paterson, the formulation of a new theory of sternal origin was necessary.
 +
 +
Their discovery of the single median rudiment would not allow of Ruge's conclusion, for he thought the sternum a product of the costal extremities; neither would they square with Paterson's view, for he derived the sternal bands from the median rudiment; and further, it was apparently not a product of the sternal ends of the clavicles. So recourse was had to either of two other theories, each possible so far as their observations go: " first, it may be formed 'in situ,' or, second, it may be derived from the anterior ends of the sternal bands by each of them sending a prolongation medialward to join its fellow in the median plane. The fact that we never found this rudiment in a paired condition, but always as a single band of cells uniting the anterior ends of the sternal bands leads us to believe that the first interpretation is the more probable."
 +
 +
Thus Whitehead and Waddell say there are two possibilities remaining for the formation of the anterior median rudiment, either that it arises 'in situ' or as a derivative of the sternal bands. They overlooked another possibility, its derivation from and relation to the shoulder-girdle. This may not have been an unnatural error in view of the fact that they studied chiefly the pig where the clavicle and that associated coracoidal mesenchymatous material of the early embryo is lacking. Their view-point was derived from the developmental stages in one or two mammals only, and they paid little attention to the comparative anatomical and the phylogenetic side.
 +
 +
This median rudiment is considered by Whitehead and Waddell to be the homotype of the presternum of monotremes, but no reasons or arguments for such a belief are set forth. This conclusion, however, is clearly invalid, for it is impossible to reconcile the theory of 'in situ' for this anterior rudiment with their statement that it finds its homologue in the presternum of lower forms or the so-called prosternum of monotremes. If these be homologous, then the presternum and prosternum also arise 'in situ,' and no morphologist believes that they do.
 +
 +
 +
 +
52 FRANK BLAIR HANSON
 +
 +
The paired rudiments, or sternal bands, also arise very early, according to Whitehead and Waddell, 'in situ,' one on either side of the body and unattached in the earlier stages to the ventral extremities of the ribs. The paired rudiments antedate the appearance of the median rudiment.
 +
 +
While Whitehead and Waddell and Paterson are very far from being in accord as to the origin of the sternal rudiments, they agree in demonstrating that the attachment of ribs and sternum is a secondary fusion of parts, and that Ruge, while essentially correct in his description of the stages he had under observation, did not have the earlier stages and therefore was not in a position to frame a theory of sternal origin. Without stopping now to consider the relative merits of these two later theories, it must be pointed out that their united efforts in overthrowing the Ruge theory are of great value because of the hitherto widespread, almost universal acceptance of this view.
 +
 +
5. Work of Kravetz, Rathke, Mueller, etc.
 +
 +
Kravetz ('05) worked on the pig. His youngest stage (24 mm.) was also the oldest stage of Whitehead and Waddell. In the 24-mm. pig he found that the first ribs did not reach the sternal rudiment, and, from the conditions in a series of later stages, came to the conclusion that primarily there is no connection whatever between the sternal rudiment and the costal cartilages at their ventral ends.
 +
 +
Bruch ('52) describes the early stage of the sternum as two longitudinal rods, one on either side, which later unite with each other and with the ribs of their respective sides. He thus indirectly denies a costal origin, but fails to indicate just what his views were in this respect. It is highly probable that the question was never raised in his mind at that early date. Whitehead and Waddell add but little to the description of this early worker, except that they have a theory of 'in situ' origin for the structure in question.
 +
 +
Rathke ('48) has an early, but very important paper in connection with this discussion. His views are set forth in two short paragraphs which are quoted in full as follows :
 +
 +
 +
 +
ONTOGENY AND PHYLOGENY OF THE STERNUM 53
 +
 +
According to the researches I have made on the development of the sternum in mammals, birds, and batrachians, this bone (sternum) maybe formed in a two-fold fashion. In mammals and birds it occurs under the form of two very slender, long rods (italics mine), divided into two lateral halves, and already at an early period consisting of cartilaginous tissue, each of which rods unites itself with the extremities of several ribs of its own side (italics mine) when these project themselves through a small part of the lateral wall of the body. The two halves, therefore, at first, lie at a considerable distance from each other. Gradually, however, these two rods are approximated to one another by the extension and development of the ribs, until, at length, they come into contact throughout their whole length, and ultimately coalesce, forming the sternum.
 +
 +
As regards the Batrachia, even in those which possess ribs, there is never at any time two rods which unite the ribs and coalesce with one another to form the sternum, but in some of these Amphibia there originates a single cartilaginous lamina; in others a row of two or three such laminae quite independent of the lateral rays of the vertebral column
 +
 +
Rathke's account of the origin of the sternum in birds and mammals gives us a description of a stage far earlier than Ruge's youngest sternum. Ruge saw these 'rods, long and slender' only after they had been united with ribs, and therefrom made his deductions that they originated from ribs. Over thirty years before Ruge's paper, Rathke accurately described this very early stage, which had only been rediscovered in a few mammals very recently. Ruge mentions Rathke's work, but, strange to say, makes no reference to this earlier stage in his account.
 +
 +
This earlier account by Rathke is hidden in a discussion of the homologies of the Chelonian plastron, and has not been mentioned by any writer since Ruge. Its confirmation by Paterson, Whitehead and Waddell, as well as by my own observations, renders Ruge's theory of the sternum untenable.
 +
 +
However, accurate as this description of Rathke's is, we must not forget that he did not recognize its significance; in fact, it is introduced into a paragraph describing and maintaining that the sternum of the mammals is different in origin from that of batrachians, not realizing as we do to-day that in separating it completely in its genesis from the costal cartilages, he made unnecessary a dual theory of sternal origin.
 +
 +
 +
 +
54 FRANK BLAIR HANSON
 +
 +
Charlotte Mueller ('06) worked out the development of the thorax in a series of human embryos. She modeled the entire thoracic framework, including vertebral column, ribs, and sternum. The series of models as pictured in her paper are fine examples of the possibilities in careful modeling of large and complicated structures. However, her youngest stage had the sternal bands, though remote from each other, firmly fused on either side with the costal cartilages, and, following Ruge, she describes the sternal bars as arising from the ribs. She makes little other contribution to the subject of sternal genesis, for her material, like Ruge's, was far too advanced for the early history of this bone.
 +
 +
1 1 is at once apparent that the theory of Parker, Ruge, Mueller, etc., on the one hand, and those of Paterson, Whitehead and Waddell on the other are mutually exclusive. But it would seem that those of Paterson and Whitehead and Waddell have only an apparent incompatibility, and that at least in so far as their observations go, each was correct in reporting what he saw, but the fact that they worked on very different forms and interpreted their results in a widely different manner, leads to the belief that they were- looking on opposite sides of the same shield, and that it is possible to reconcile the two.
 +
 +
The 'in situ' theory of Whitehead and Waddell is hardly to be taken seriously. So ancient a structure as the sternum, dating back to the Elasmobranchs, as we have seen, and found in every higher group of vertebrates, including at least one or two teleosts, and having an enormous and highly complex development in many of the groups, can scarcely be accounted for in this simple fashion. The 'in situ' theory took form from the material upon which its authors worked. The pig is the basis of their main conclusions and this form is peculiarly unsuited for this work, because of the degenerate character of the shoulder-girdle, which is lacking at once in both clavicle and coracoid process (the socalled subcoracoid now being thought to be an epiphysis). For a proper interpretation of the sternum and shoulder-girdle in the mammals those groups with well-developed clavicles, episternals, suprasternal, and other appurtenances of this
 +
 +
 +
 +
ONTOGENY AND PHYLOGENY OF THE STERNUM 55
 +
 +
region are the only ones which can be lined up with the lower groups of vertebrates. The rat was used by Paterson and the mouse is the basis of the present paper, and this would seem to be the ideal form; for the rodents, while highly specialized in some respects, are primitive in others, and are to be grouped with the Edentates and Insectivores somewhere near the monotreme stem. They are also small enough so that when sectioned, comparatively high powers of the microscope may be used, and it is possible to section all stages up to the ripe fetus.
 +
 +
Discarding, then, Whitehead and WaddelFs theory of sternal origin, while retaining an appreciative memory for their valuable work in combating one of the remaining theories, we can reduce the great mass of papers and discussion on this subject to just two absolutely irreconcilable theories of sternal origin, which may for convenience in treatment be designated as Ruge's 'theory of costal origin,' and Paterson's 'theory of coracoidal origin.' All other workers, except Whitehead and Waddell, have supported one or other of these theories or modifications of them. 1
 +
 +
1 Through the courtesy of Doctor Kingsley, I have just received two papers on the sternum, one of which requires mention. This is by Albrecht: Sur les Copulae Intercostoidales et les Hemisternoides du Sacrum des Mammifers. Bruxelles, 1883. It contains a most curious modification of Ruge's theory of costal origin. Albrecht's idea is that the first and second ribs of each side at first are united by an arch of cartilage, giving, according to his schematic figures, a structure similar to a horseshoe magnet, the two arms of the magnet being the ribs, the arch connecting them the sternal band. Then by a union of the two sides in the midline, and the fusion of the consecutive pairs of such magnetshaped structures, a sternum is derived. This is ingenuous and is the only theory of costal origin which gives the sutures between the sternebrae their proper position, i.e., opposite the ends of the ribs, making the sternebrae intercostal as they actually are. However, the arguments used to overthrow Ruge's theory apply equally here. This theory does not account for the anterior and posterior extension of the sternal bands for a considerable distance beyond the region of ribs; it does not explain the appearance of the bands prior to their union with ribs; and fatal to Albrecht's hypothesis is the fact that the bars are continuous, unsegmented structures throughout their entire length from their earliest appearance in the mesenchyme, and never occur in short, semicircular segments connecting the ends of the ribs. Albrecht was evidently unable to find any stages in actual material in support of his theory, for his figures without exception are diagrammatic, and do not fit the observed facts of sternal development.
 +
 +
 +
 +
56 FRANK BLAIR HANSON
 +
 +
To sum up, then, there are extant at the present time in the literature three opposing theories as to the origin of the sternum in the Mammalia. The oldest and most generally accepted of these is that proposed by Ruge in 1880, which in substance states that the sternum is a direct derivative of the ventral ends of the costal cartilages.
 +
 +
In 1900 and more fully in 1902 and 1904, Paterson was led to doubt the validity of Ruge's theory, claiming that there was an earlier history than that of which Ruge was aware. Paterson derived the presternum from the same element which gives rise to the shoulder-girdle, describing a continuous cellular element crossing the midline in the rat. He derived the sternal bands from this presternum as backward prolongations, which later and secondarily are fused with the ventral ends of the ribs.
 +
 +
Whitehead and Waddell ('11) agree with Paterson that Ruge did not have the earliest stages, and that his theory is therefore untenable, but they disagree with Paterson as to the interpretation of these early stages. They deny any connection or relation between sternum and shoulder-girdle, believing that both presternum and sternal bands arise 'in situ.'
 +
 +
This discussion of the literature is one of selected papers which supports one or other of the different theories of sternal origin and is fairly representative of the literature. However, only a few papers are mentioned in comparison with the voluminous literature extant. The author has collected a bibliography of about one hundred titles on the sternum, but has considered it necessary to treat only a few of the more prominent ones in this connection, with the assurance that those omitted contain nothing new or affect the situation as outlined here.
 +
 +
It has added greatly to the confusion existing between these opposing theories that most of the more important papers (Ruge's excepted) are very inadequately illustrated. Paterson ('02) does not give a single figure in this paper and his figures in the 1900 paper are small and inadequate. Important stages are described in the Whitehead and Waddell paper, but those upon which they base their chief conclusions are not supported by any figures. If we had clear-cut drawings of Paterson's continuous
 +
 +
 +
 +
ONTOGENY AND PHYLOGENY OF THE STERNUM
 +
 +
 +
 +
57
 +
 +
 +
 +
cellular element extending across the middle line, and could compare this with equally well-drawn stages from the material of Whitehead and Waddell, an unprejudiced worker, from a study of the figures, supplemented by his own observations, might bring the whole tangled mass into harmony. As it is, the present author has but little to start with except the verbal statements of the opposing theorists.
 +
 +
Table 1 gives at a glance the position of several of the leading workers in their attitude toward the problem.
 +
 +
 +
 +
TABLE 1
 +
 +
 +
 +
Ruge, G. ('80)
 +
 +
 +
 +
Paterson ('00) Paterson ('02) Paterson ('04)
 +
 +
Kravetz ('95)
 +
 +
 +
 +
Mueller ('06)
 +
 +
 +
 +
Parker ('91)
 +
 +
 +
 +
Whitehead-Waddell ('11)
 +
 +
Rathke ('48)
 +
 +
 +
 +
OKIGIN OF STERNUM
 +
 +
 +
 +
Ventral ends of ribs
 +
 +
Shoulder girdle
 +
 +
 +
 +
Two longitudinal bars
 +
 +
Ventral ends of ribs
 +
 +
Coracoidal and costal
 +
 +
 +
 +
'In situ'
 +
 +
 +
 +
Two longitudinal bars
 +
 +
 +
 +
FORMS STUDIED
 +
 +
 +
 +
Man
 +
 +
 +
 +
Rat, rabbit, man, dogfish
 +
 +
 +
 +
Pig
 +
 +
 +
 +
Man
 +
 +
 +
 +
Hexanchus Apteryx
 +
 +
 +
 +
Pig, cat, man
 +
 +
Batrachia, chick, pig
 +
 +
 +
 +
HOMOLOGY OP PRESTERNUM
 +
 +
 +
 +
Middle part shark girdle
 +
 +
 +
 +
No morphological importance
 +
 +
Episternum
 +
 +
 +
 +
Omo-sternum in shark; omo-sternum in amphibia
 +
 +
Episternum; prosternum
 +
 +
 +
 +
58 FRANK BLAIR HANSON"
 +
 +
III. THE ONTOGENY OF THE STERN I'M
 +
 +
The earliest development of the sternum in a number of mammals has been worked out by Paterson, Kravetz, Whitehead and Waddell, and myself. An attempt is made to compare the steps of development in ontogeny with those in the phylogeny of the sternum, or in other words to make a practical demonstration of Haeckel's recapitulation theory as applied to sternal development.
 +
 +
1. The sternal bands
 +
 +
One decisive result of this investigation has been to demonstrate the existence of the sternal bands as independent structures far earlier in development than Ruge and the older workers suspected. Hence a new theory of sternal origin was demanded, and as above indicated, this has taken two directions: Whitehead and Waddell do not relate the sternal rudiment genetically to any preexisting structure, while Paterson identified it with the coracoidal girdle of lower forms.
 +
 +
The conclusive evidence against Ruge's theory of costal origin led the author to examine the material of Paterson and Whitehead and Waddell, in an effort to confirm one or other of these workers or reject both, as the case might be. Paradoxically enough, I have been able to corroborate Paterson's account of the shark-like girdle, found by him in the rat, both in the mouse and human embryos; and in the identical slides 2 of cat and human embryos used by Whitehead and Waddell have been equally able to confirm their observations. In pig embryos of 24-mm. length, Kravetz found that the first rib did not reach the sternal band, and the connections of the remaining six ribs were too feeble to have any morphological importance. Whitehead and Waddell studied a 24-mm. pig in which the first rib did just reach the sternal rudiment and the union of the other six ribs was marked. My 24-mm. stage agrees with that of Kravetz in that the first rib does not reach the band, and with Whitehead and Waddell's in that the connection of the other
 +
 +
2 My sincere thanks are tendered Dr. C. W. F. McClure, of Princeton University, and Dr. Oeorge L. Streeter, of The Johns Hopkins University, for the loan of the series of cat and human embryo studied by Whitehead and Waddell.
 +
 +
 +
 +
ONTOGENY AND PHYLOGENY OF THE STERNUM 59
 +
 +
six is marked. No special significance attaches to these variations since individual differences in development as well as in measurements and amount of shrinkage gives form for considerable variation. However, in stages of the pig 22 mm., 20 mm., 18 mm., and smaller, there is no doubt of the failure of several pairs of the anterior ribs to meet the sternal rudiment.
 +
 +
Figures 3 and 4 are of two cat embryos from the Princeton Embryological Collection. Camera-lucida drawings were made of the sternal bands and ventral ends of the ribs. These structures were then plotted upon millimeter-ruled paper, which gives a graphic reconstruction made to scale. Several wax models were also made from the early pig and mouse embryos. Figure 3, of the 12-mm. cat embryo, shows clearly that in this stage the first three pairs of ribs do not extend to and unite with the sternal bands. It is also apparent that no anterior sternal rudiment or presternal rudiment is present at this age. It is undoubtedly true, as claimed by Whitehead and Waddell, that in the ontogeny of the mammalian sternum the two sternal bands antedate in appearance the median and anterior rudiment. However, in the phylogeny of the sternum, as will be shown further on, the presternum is the first to arise, and from this come the sternal bars. I am unable to account for this discrepancy by any observed facts, but think the history from phylogeny must take precedence over that from ontogeny; explaining the rise of the sternal bands in the mesenchyme of the mammal as the result of protoplasmic memory, which dates back to the early reptilian ancestor in which the presternum grew backward as two prolongations that became the mesosternum and the xiphisternum,
 +
 +
In human embryos from the F. P. Mall Collection studied by Whitehead and Waddell and myself, is found the best evidence of the complete separation of ribs and sternal bars in the early stages of development. In embryos 10.5 mm. and 13 mm. long none of the ribs reaches the sternum, the presternum has not yet appeared, and no clavicles are apparent. These stages, if graphically represented, would appear similar to figure 3 of the cat, except that all the ribs would be in the same relation to the sternum as are the first three in the stage of the cat figured.
 +
 +
 +
 +
60 FRANK BLAIR HANSON
 +
 +
2. The anterior median sternal rudiment
 +
 +
In the mouse, rat, and human embryos occurs a stage in which a mesenchymatous girdle appears, in shape and relations comparable to the pectoral girdle of the shark. Figures 5 and 6 show this girdle in the mouse and human embryos. It is composed throughout of mesenchyme cells, and the structural development of each part may be followed in later stages. In the mouse girdle the two dorsally extending wings on either side are the rudiments of the scapulae; the medial and ventral extensions are the coracoids and clavicles; the enlarged portion in the ventral midline is the fundament of the presternum. This is conclusive evidence that the presternum is intimately associated with the shoulder-girdle in the earliest ontogenetic stages in the mammals, just as they are also phyletically bound together in the evolution of the vertebrate shoulder-girdle (infra).
 +
 +
The mesenchymatous material extending from the scapulae to the presternum (fig. 5) is the track in which the clavicles will soon develop. In the human embryo (fig. 6, cl.) this has already commenced on one side. According to Gegenbaur and his followers, the core of the clavicles is the old cartilaginous precoracoid of the Amphibia. If it be true that the clavicles do have a precoracoidal core of cartilage, as Gegenbaur thought, here is the coracoidal extension in the human embryo reaching the presternum in the ventral midline, just as it does in Hexanchus, Amphibia, Reptilia, Aves, Monotremes, and fetal Marsupialia (infra).
 +
 +
Gegenbaur's clavicle containing a precoracoidal cartilaginous core has been attacked in several papers by Broom, who denied the presence of any cartilage in the earliest stages of the clavicle. However, Broom admits that cartilage does appear at a later stage in the development of the clavicle, and it may be assumed that cartilage appearing either as a clavicular basis (Gegenbaur) or at some later stage (Broom) would in this region in highest probability be coracoidal tissue. This position is strengthened when it is recalled that in the Anura a precoracoid actually functions as the core of the dermal clavicle. Huntington 3 is
 +
 +
3 From a private communication containing Huntington's views on several shoulder-girdle problems, kindly prepared and sent to the author October 30, 1918.
 +
 +
 +
 +
ONTOGENY AND PHYLOGENY OF THE STERNUM 61
 +
 +
the latest defender of the Gegenbaurian hypothesis, and, going a step further even than Gegenbaur, declares that in the case of the frog's girdle there is a " preparatory action on the part of the coracoid cartilage directed toward the reception and assimilation of the corresponding dermal accession of the clavicle."
 +
 +
In later stages of the mouse and human embryos, after the mesenchymatous girdle has become broken up into its component parts, the coracoid process is relatively much larger than in the adult and has a medial and ventral extension. I have observed this repeatedly in embryos of pigs, mice, cats, and man. In one mouse embryo, 7.75 mm., there seemed to be a distinct thickening of the mesenchyme between the very large coracoid process and the yet partly mesenchymatous clavicle. This was a striking spectacle in the pig, because of the well-known fact that in the adult no coracoid process is present, but only the subcoracoid, glenoid-sharing portion.
 +
 +
3. Sternebrae
 +
 +
The segmentation of the sternum into sternebrae occurs late both in phylogeny and ontogeny. A glance through the figures in the literature assures one that sternebrae are unknown in four of the five classes of vertebrates, being found only in the mammals. Hence they play little or no part in the origin or development of the sternum phyletically.
 +
 +
Likewise, they are a secondary and- acquired character in ontogeny. It is contrary to all expectation, if sternal bands are derived from ribs, to find that the sternebrae are invariably intercostal, and not at the point opposite the ends of the ribs. Figures 7 to 10 show what is the true condition in all fetal mammals as regards the formation and ossification of its sternebral elements. The center of ossification always occurs at a point midway between two ribs, while the line of transverse division crosses the sternum exactly in the center of the area of union of ribs and sternum. According to Ruge's theory, this should be just the reverse. The sutures between the sternebrae should be intercostal, the sternebrae themselves opposite the costal cartilages.
 +
 +
 +
 +
62 FRANK BLAIR HANSON
 +
 +
Furthermore, if the sternum is ossified from the ribs, segmentation of that structure should be apparent at its earliest appearance. On the contrary, however, the sternal bands exhibit no trace of segmentation until a late period of development. The bands may be followed carefully from section to section, or the parts reconstructed in wax, but no one has ever reported the slightest indication of an early division of the bands into segments.
 +
 +
The sternebrae may be interpreted as arising by a process of segmentation in response to the demand for as great a measure of elasticity on the ventral side of the animal as is allowed by the more or less flexible vertebral column on the dorsal side.
 +
 +
Sutures arising in this manner, as a response to strain, Avill naturally appear at the weakest parts along the sternum. At the points of attachment for the ribs the sternum is often deeply notched, weakening this region, and here, as expected, occur the lines of divisions of the sternum into segments or sternebrae. That this is the cause and manner is indicated by the fact that there are always the same number of sutures as there are pairs of ribs attached to the sternum. By cutting a typical sternum out of cardboard or a wax plate, and notching the sides for the reception of ribs, it is possible by applying a lateral strain fo produce sutures or cracks across the cardboard or wax sternum, dividing it into sternebrae exactly as in the actual sternum.
 +
 +
In many of the reptiles and such animals as the cats among the mammals, where a long, lithe body in making its way through thick undergrowth or over rough ground is often twisted into almost an S-shape, the advantage of a segmented sternum is obvious. How large a part this plays in breathing is not so apparent, but doubtless has some bearing.
 +
 +
In the Primates where the semi- and upright position obtains, there is less need of flexibility, and the sternebrae tend to become fused into the three typical parts of the primate sternum. That the entire sternebral development is a secondary and late acquisition and has no bearing on the origin of the sternum is quite apparent.
 +
 +
 +
 +
ONTOGENY AND PHYLOGENY OF THE STERNUM 63
 +
 +
So far as I am aware, this will constitute the only explanation in the literature of the rise of the sternebrae, other than the statement that they represent the original costal contributions, which is, as we have shown, absolutely untenable.
 +
 +
4. Stages in the ontogeny of the mammalian sternum
 +
 +
1. Appearance of two laterally situated sternal bands, independent of ribs.
 +
 +
2. Appearance of a single median anterior rudiment, intimately associated with the shoulder-girdle.
 +
 +
3. Gradual approximation and union of sternal bands with the anterior sternal rudiment on the one hand, and with the ventrally growing tips of the ribs upon the other.
 +
 +
4. Gradual approach and fusion of sternal bands in midline of body to form a sternum.
 +
 +
5. Division of sternum into a number of sternebrae. Lines of division (sutures) always appearing opposite the ends of each pair of ribs.
 +
 +
6. Ossification of the intercostal sternebrae by the appearance of one or more centers for each segment.
 +
 +
7. Fusion of the sternebrae in Primates into three parts: manubrium, gladiolus, and xiphisternum.
 +
 +
5. Conclusions
 +
 +
1. That the sternal bands arise and remain as two unsegmented structures until the relatively late process of ossification
 +
 +
begins.
 +
 +
2. That the sternebrae are invariably intercostal; arise by reason of functional demands for greater freedom of movement, and play no part in the origin of the sternum.
 +
 +
3. That in the mouse and rat embryos a mesenchymatous horseshoe-shaped girdle extends across the ventral midline; from this material are derived presternum, coracoids, and scapulae; this girdle being the homologue of the adult cartilaginous pectoral girdle of Hexanchus.
 +
 +
THE AMERICAN JOURNAL OF ANATOMJf, VOL. 26, NO 1
 +
 +
 +
 +
64 FRANK BLAIR HANSON
 +
 +
4. That in early stages of the cat, pig, mouse, and human embryos, the sternal bands exist as well-defined, separate, mesenchymatous entities, prior to their union with the costal cartilages, thus indicating their independence of, and the secondary nature of their relation to, the ribs.
 +
 +
[V. THE PHYLOGENY OF THE STERNUM
 +
 +
Paterson's comparison of the " continuous bar .... across the middle line" in the rat with the cartilaginous scapular arch of Acanthias, suggested the idea of following this structure found by Haswell, and later independently by Parker, in the middle ventral line of the shark Notidanus and identified by them us a presternum, up through the various groups of vertebrates to see how nearly it could be carried up in a phylogenetic series to the rodents, in which Paterson thought he had detected it again. For this purpose recourse was had to all available figures extant in the literature, and especially to that monumental monograph on the shoulder-girdle and sternum, by Parker ('68). From these sources a series of figures has been adapted, beginning with Parker's ('91) figures of the presternum in the shark, and including one figure from the Ganoids, and one each from the Teleosts and Dipnoi; then numerous figures from the Amphibia, Reptilia, Birds, and Mammals. Such a search through the literature, though wearisome, has rewarded the labor far beyond any expectations.
 +
 +
It is believed that the evidence presented in this phylogenetic survey would alone go far toward convincing any one of the truth of the conclusions arrived at in this paper, even though it were not preceded by the corroborating evidence of the section on the ontogeny of the sternum. It is the hope of the author that it may lead to a general agreement as to the homology and origin of the mammalian sternum.
 +
 +
1. Fishes
 +
 +
It is pretty clearly demonstrated by Haswell ('84) and also by Parker ('91) that at least in one shark there is a presternum
 +
 +
 +
 +
ONTOGENY AND PHYLOGENY OF THE STERNUM 65
 +
 +
derived from the ends of the coracoidal portions of the usually single continuous pectoral girdle characteristic of sharks. Figure 11 shows the girdle of Notidanus from the ventral side with the " intercepted cartilage .... temptingly like a presternal" (Parker, '91). Figure 43, the first of the series of girdles shown in plate 12, is another drawing from the same form. It would seem that here is the initial material from which all later presterna might possibly be derived.
 +
 +
The author is of course aware that neither Notidanus nor any other living shark is the direct ancestor of the vertebrates, and that the following phylogenetic series of figures does not necessarily mean that the successively higher animals are direct descendants of those immediately lower which are used for illustrating the points.
 +
 +
Among the teleosts a complete girdle across the midventral line is ordinarily lacking. However, several do have the clavicles prolonged toward the center, and when so, there is quite uniformly a cartilaginous element in the midline which Parker ('68) calls the 'epicoracoid.' 4 This lies between the coracoids, in the identical position of a presternum. It is interesting to note from figures 12, 13, 14, and 15, that this condition is so closely alike in a ganoid, Polypterus (fig. 12) ; a teleost, Gobius niger Linn. (fig. 13) ; and a dipnoan, Lepidosiren (figs. 14 and 15). A description of the condition in these three fishes may be given in the account of Parker ('68) for that of Lepidosiren annectens: "Lepidosiren agrees with the elasmobranchs in a well developed epicoracoidal belt. Originally the epicoracoid mass must have been double, and perhaps in a very early stage each moiety was continuous with the coracoid proper, but a wide transverse cleft was soon formed which separates epicoracoid and coracoid."
 +
 +
4 In this paper the term 'epicoracoid' is employed to designate the cartilaginous ventral ends of the posterior coracoids. The author is following here the usage of Parker and others from whom many of the figures have been borrowed. That this is not the correct term, he is well aware, and in another paper is suggesting the term ' infracoracoid' as probably a more suitable one for these parts. The term 'epicoracoid' was applied to the anterior element of the monotremes bv Cuvier, and this use should be retained.
 +
 +
 +
 +
66 FRANK BLAIR HANSON
 +
 +
Figures 14 and 15 show the epicoracoid to have a large midventral portion, rounded out anteriorly and posteriorly (comparable directly with the so-called presternum of the shark and the omosternum and sternum of the Amphibia), while laterally extend two bars, the parts which originally were continuous with the coracoids. Here there is cut off from the coracoids that material which nature will use in all higher classes of vertebrates in the construction of a presternum. And if it be said that this dipnoid fish is not the direct ancestor of the Tetrapoda, it may be replied that every consideration points to the same condition in the direct ancestor, whatever form it was. Both Paterson, in the rat, and the author, in the mouse, found stages in early embryo of these mammals where the clavicles, extending toward the midline, are closely invested with mesenchymatous bars which unite into a single, median mass, comparable at once to this epicoracoid in the dipnoid fish and to the 'presternum' of Haswell's shark, and actually in the rodents uniting to form the manubrium.
 +
 +
2. Amphibia
 +
 +
A similar sternum is found in the Amphibia. As Howes remarks, "that the Amphibian sternum is for the most part, if not wholly, a derivative of the shoulder-girdle, there can be no longer a question; and although the researches of Goette ('77) leave us in doubt concerning the hypo (post-omosternum) they show that that can be no derivative of the costal apparatus."
 +
 +
Parker ('91), believing in the diverse origins of the sternum in the Ichthyopsida and Amniota, also describes a dual origin for the sternum in the Amphibia. He says, "a pair of narrow strips are separated off from the posterior borders of the coracoids," also "a pair of cartilaginous bands appear in the inscriptiones tendineae of the mm. recti abdominis. From these four elements the sternum is produced." Ruge considers that these cartilaginous bands are to be looked upon as vestigial ribs. The narrow strips are admitted to be from the shouldergirdle.
 +
 +
 +
 +
ONTOGENY AND PHYLOGENY OF THE STERNUM 67
 +
 +
This description of two cartilaginous bands appearing in the inscriptiones tendineae is strikingly similar to the account of the sternal bands in the pig by Whitehead and Waddell. There seems to be no antagonism anywhere to the view that in the Amphibia the anterior part of the sternum is the product of the shoulder-girdle.
 +
 +
Nor is it necessary to stretch any morphological relations unduly to see this coracoidal sternum reproduced in the Dipnoi, Gobius, Polypterus, and Notidanus. We must agree with Parker ('91), Has well ('84), and others, that the sternum in the Ichthyopsida is coracoidal in origin and homologous throughout that group.
 +
 +
From here on, however, we part company with all early workers, and most later ones as well, for all of them accept Ruge's conception of a costal sternum in the Amniotes, and deny any homology between the sterna of Ichthyopsida and Amniota. One of the designs of this present phylogenetic sketch is to show from figures and data already in the literature that the two are one and the same thing in origin and development, and therefore homologous.
 +
 +
As practically all are agreed that the sternum in the Amphibia is coracoidal in origin, while but few are agreed that it is so in the Amniotes, we are brought to the necessity of bridging in some manner the alleged gulf between the amphibian and reptilian sternum.
 +
 +
In order later to make direct comparisons between amphibians and reptiles, when treating the latter group, it is necessary to introduce here a number of figures and remarks thereon for several representative amphibians. Starting with the frog, figures 16 to 19, inclusive, give a very good idea of the development of the coracoids and sternum in this form. It is hardly necessary to point out the close relation between coracoid and sternum nor to suggest that the figures can lead only to one conclusion, that of a coracoidal origin for the sternum.
 +
 +
Pipa (fig. 20) has a sternum which, as Parker says, challenges attention. A little study of this figure will convince one that the sternal region must have been at one time a part of, and
 +
 +
 +
 +
68 FRANK BLAIR HANSON
 +
 +
continuous with the epicoracoids. The ossification in each is at exactly the same stage; the amount of soft cartilage around the edges is the same in coracoid and sternum. At the anterior end is differentiated a small region that corresponds to the larger omosternum in the frog. We shall come back to Pipa when treating some of the reptiles, and by a comparison attempt to show that this subreptilian creature has a sternum essentially like certain reptiles.
 +
 +
Does Bufo (fig. 21) have a sternum? Kingsley ('17) would say that since no ribs are in this region, no one can say. On the theory of Ruge, this would be true, but if we compare the sternum of Bufo with that of Pipa and the early stages in the frog, it is hard to believe that there is any essential difference between the two structures, although the connection of the sternum with the epicoracoids in Bufo is not so extensive as in many other Amphibia. The entire body of evidence in this paper and many others on the shoulder-girdle in the Amphibia can only lead to the conclusion that Bufo does have a sternum and that it can only be derived from the coracoidal portions of the shoulder-girdle.
 +
 +
Siredon (fig. 22) has large coracoids and a considerable overlapping of their epicoracoidal edges. The interesting feature to us is that the sternum lying immediately behind the overlapping epicoracoids also shows very distinctly two grooves corresponding exactly to those made by the overlying edges of the epicoracoids. It is the condition precisely to be expected of the sternum if it were in an early stage a posterior continuous extension of the cartilage, sharing in the overlapping, and then later had been cut off by sutures from the main element, but retaining these evidences of its formation from the two epicoracoids.
 +
 +
Little need be said concerning such a girdle as that of Dactylethra (fig. 23) . There is no overlapping of the coracoids and no sutural evidence in the sternum of the union of the two sides; however, as this is an adult specimen, none need be expected. Nevertheless, the intimate relation of sternum and girdle is evident.
 +
 +
In Calamites (fig. 24) we see a sternum that for the first time
 +
 +
 +
 +
ONTOGENY AND PHYLOGENY OF THE STERNUM 69
 +
 +
has two backward prolongations, or sternal bars. This sternum is very suggestive of several in the reptiles, and embryos of the mammals, wher.e a single median anterior rudiment is continued backward as two rods or bars. This is an adult specimen and therefore the permanent form in this species ; if, however, we compare this with several of the reptiles, such as shown in figures 26, 27, and 28, and with descriptions by Rathke, Bruch, Paterson, etc., of the early sternum in the mammals, there appears to be more than a mere resemblance — there is genetic relationship and homology. If in Calamites the coracoids were to retreat to a mere process attached to the scapula, leaving only the clavicle and sternum in this region, and the sternal bars of the latter fused together in the midline, leaving a fossa at the upper end of the union as in many reptiles, and the posterior ends were but incompletely fused leaving two small blunt laterally projecting horns, we would have a sternum such as is actually found in Chirotes (fig. 30) and the embryos of mammals.
 +
 +
Wilder ('03) describes several cartilaginous rudiments found in Necturus and related by him to the sternal apparatus. These are a series of thin cartilages located in the myocommas of the pectoral region. One of them is usually larger than the rest and situated near the posterior part of the overlapping coracoids. This element is identified by Wilder as the homologue of the sternum of the higher Urodeles.
 +
 +
If Wilder's theory be correct, Necturus presents an exception to the rule established in this paper that the presternum is a derivative of the coracoids, for obviously this element in Necturus could not possibly be derived from that source. My own dissections of Necturus, however, do not bear out Wilder's hypothesis. My interpretation of these cartilages is, that they are simply chrondifications of the outer part of the connective tissue of the intermuscular septa and have no relation whatever to the formation of the presternum in higher forms.
 +
 +
They are rather to be looked upon as subcutaneous splints and find their homologues in the inscriptiones tendineae of other forms, and also possibly in the abdominal ribs of Chamaeleo and Polychrus. It is significant to note in this connection that many
 +
 +
 +
 +
70 FRANK BLAIR HANSON
 +
 +
animals have both the inscriptiones tendineae and also a complete sternal and costal apparatus. If the latter be the derivative of the former, why this persistence of the two structures side by side in nearly all groups above the lower Urodeles?
 +
 +
3. Reptiles
 +
 +
Fossil reptiles constitute the next group in which we have looked for a sternum or any part of one which either originates independently of the ribs or which has intimate relations with the shoulder-girdles. The author has studied figures and plates of much of the recent work done on fossil reptiles, of which the investigations of Credner ('81-'93), Gregory ('15), Seeley ('92, '94), Woodard C98), and Zittell ('00, '13) are typical, and, in addition has examined a number of mounted specimens in the U. S. National Museum. It may be said in general that in those skeletons which have the shoulder-girdles preserved there is a strong tendency for the coracoids to grow around the side of the body ventrally, though never meeting in the midline, for the soft epicoracoids are not preserved, and that the coracoids are enormously developed in size.
 +
 +
Gunther ('67), Schauinsland ('00), and Howes and Swinnerton ('01) worked on the development and anatomy of that primitive and now almost extinct reptile from New Zealand, Hatteria punctata or Sphenodon. In one of Gunther 's figures the shoulder-girdle and sternal bars are shown in their natural relation at that stage. The coracoids do not have a large ventral extension, but are capped on their medial ends by slender processes which later unite to form the presternum. Three pairs of ribs reach each sternal bar, so that the sternal and costal connections are much too far advanced for any statement as to the origin of the sternal bars in Sphenodon. So far as I am aware, this is the youngest stage of the Sphenodon sternum figured in the literature.
 +
 +
From the condition in the embryos of living reptiles, as well as in many of the adult species, it may be assumed that in the early condition of fossil reptiles the coracoids were in intimate relation to the sternum.
 +
 +
 +
 +
ONTOGENY AND PHYLOGENY OF THE STERNUM 71
 +
 +
When we come to consider the living reptiles the material and evidence is abundant and of the greatest significance.
 +
 +
Among the recent forms, Goette (77) has studied Cnemidophorus. While he does not show conclusively that the two anterior triangular rudiments are products of the nearby coracoids, he demonstrates that only the first ribs have reached and attached themselVes to the sternum, while that part of it opposite the second and third ribs is formed independently of them, as a backward prolongation of the anterior paired rudiments.
 +
 +
These results of Goette showing that the sternum is the result of a backward growth of tissue from an anterior portion are in striking anticipation of what Paterson claimed to see in the rat, where it is stated that the anterior median portion of the sternum is derived from the same element as is the shoulder-girdle, and this in turn yields the sternal bands as posterior prolongations.
 +
 +
Anguis fragilis (fig. 25) has a sternum that in many respects is typically amphibian in character, as may be seen at once by a comparison of this form with that of Pipa. As in Pipa, there is only present an anterior or presternum, separated by sutures only from the epicoracoids, and as in Pipa, ossification and the amount of soft cartilage around the borders are the same for sternum and coracoids. Of this form Parker ('68) says, "there is a well developed sternum, not continuous with the ribs; " and Rathke, describing two embryos of Anguis, says, "with the coracoids the sternum was intimately united, but it was not very closely connected with the neighboring ribs, lying at a much greater distance from them than in adult Blindworms. There cannot be any doubt that in the Blindworms the two latter halves of the sternum do not .... originate under the ribs, and unite with them, but develop at a distance from the ribs. " Barring the presence of the interclavicle in Anguis, it would be difficult to recognize this shoulder-girdle as being that of a reptile, for in the relations of its girdle and presternum and in the absence of ribs it is characteristically amphibian.
 +
 +
Stellio cordylinus (fig. 26) has a sternum in which the anterior part is enormously enlarged; is in intimate relation to the epicoracoids, and in addition has two greatly extended xiphisternal
 +
 +
 +
 +
72 FRANK BLAIR HANSON
 +
 +
horns. No ribs are attached to these horns, but three pairs reach the anterior portion, to which they are but feebly attached. If it be assumed that the presternum here is of necessity derived either from the coracoids or from the ribs, the answer can only be that it musl have come from the former. This sternum with its great horns reappears in Manis longicauda (Parker, '68).
 +
 +
A scries of three figures (figs. 27, 28, and 29) gives an idea of how the mesosternum and xiphisternum are formed. In figure 27 the presternum is as before, and the two bars extend caudally. This is very similar to the amphibian Calamites (fig. 24), and the suggested series of changes outlined in the description of Calamites necessary to make of it a typical reptilian or mammalian sternum are progressively illustrated in these three reptiles.
 +
 +
In figures 28 and 29 the xiphisternal bars, by a fusion along their medial surfaces, have formed a middle sternal piece or mesosternum. The posterior ends of the coalesced bars remain apart in the xiphisternum.
 +
 +
In the mesosternum is a sternal fossa, where the union was not complete. This may persist throughout life in many forms (Varanus, Crocodilia) or, as in others, close up later, leaving a whitish streak to indicate the line of fusion. This fontanelle is also common in mammals, but there it is usually located in the xiphisternum, and I have also repeatedly observed the whitish streak of hyaline cartilage in the mesosternum in fetuses of pigs and mice. It would seem from this reptilian material, and avian and mammalian material agree, that the presternum is a product of the coracoids, and this in turn gives off two backward prolongations, which, fusing throughout a greater or lesser part of their extent, form the mesosternum and xiphisternum. It is hardly necessary to again point out the feeble relation of ribs and sternum in these last three figures.
 +
 +
Chirotes (fig. 30) gives the completion of the series; it is a sternum of the utmost importance in the consideration of the problem. Parker's description is so trenchant that a part is quoted :
 +
 +
In the whole range of vertebrate morphology there is nothing more beautiful or more instructive than the relatively large sternum of Chi
 +
 +
 +
ONTOGENY AND PHYLOGENY OF THE STERNUM 73
 +
 +
rotes; for if the sternum of the human embryo were to be demonstrated apart from the costal girdles, one diagram would serve to explain both that and what we find in this little snake-like lizard .... and if ribs had not been arrested we might have seen the counterpart of the ribs of the mammalian embryo.
 +
 +
The figure is of an adult, and, as Parker says, it might also be described as that of the mammalian embryo, except that no ribs are present and the sides of the presternum are closely applied to the coracoids. Now, if Paterson and the author be correct in their contention that the presternum and coracoids in the rat and mouse are continuous at an early stage, and this stage precedes the fusion of ribs and sternum, then the adult Chirotes sternum is a structure that far more closely approximates the early embryonic sternum of mammals than Parker suspected when he made the comparison above quoted between the two. This is a more striking parallel than is usually met between the adult structure in a lower group and an embryonic stage of the same structure in a higher group.
 +
 +
While probably all will admit the above argument, since both are Amniotes, the question may be raised as to whether there is any evidence for relating such a sternum as occurs in Chirotes with the amphibian sternum, for the crux of the whole matter of sternal homology lies between these two groups. It would seem that a direct comparison might be made with the Calamites sternum, and this structure, as has already been indicated, by a fusion of the sternal bars, and a retention of the sutural relation to the epicoracoids might be metamorphosed directly into the adult sternum of Chirotes, and this is especially strong evidence when we consider that in neither the reptile nor amphibian compared does the question of ribs enter at all, since there are none in either form in the region of the sternum.
 +
 +
Using the crocodile (fig. 31) as a contrast to Chirotes in showing the extreme of fusion of ribs and sternum in reptiles, it may be remarked at once that aside from the presence of ribs this sternum is directly comparable with the last one considered. The difference is due to the ventral growth and attachment of ribs to the sternum, otherwise it is essentially the same as more primitive
 +
 +
 +
 +
74 FRANK BLAIR HANSON
 +
 +
reptilian sterna having a presternum in close conjunction with the coracoids; a middle piece composed of the union of two longitudinal bars, with the line of fusion clearly evident, and the xiphisternal horns wide spread. A close comparison may be made between this and the later stages of the mammalian sternum, except that in them all connection with coracoids is early lost.
 +
 +
A Chamaeleo vulgaris adult sternum (fig. 32) is the last reptile considered here. It is mammalian-like and well ossified for a lizard. The presternum is a large, lobate structure, bearing two strong notches on either side on its posterior end, "this constriction answering to the transverse cleft so constant in the mammalian sternum" (Parker, '68). In the mesosternum the line of fusion of the two halves is well marked and extending also into the presternum. The ribs articulate by synovial joints with a series of enlargements on either side of the mesosternum. In describing the xiphisternum, Parker ('68) says, "The xiphisternum has a bilobate extremity that is quite mammalian in character .and no ribs ever reach this part .... the horns being free from ribs, grew not only towards each other and fused, but also grew backwards, so as to form a free, single xiphisternum, exactly like that of an ordinary mammal. That there is no real difference between these two classes in the formation of the xiphisternum, I feel certain "
 +
 +
The interesting fact about Chamaeleo is the statement of Parker that behind the xiphisternum there are seven pairs of floating ribs which later become fixed by growing toward each other and unite by suture at the midline. This is significant in the light of our contention of the non-relationship between pleural ribs and sternum. There is a distinct tendency in all vertebrates with ribs for these to grow ventrally. Now, if a sternum be present, it is likely that they would form an articulation with it; if none is present, that they should either remain free (floating) or unite (true ribs) with each other in the midline. In the chamaeleon both conditions are present in one animal ; those anterior thoracic ribs which, growing ventrally, met the sternum and articulated with it, while those ribs immediately behind the
 +
 +
 +
 +
ONTOGENY AND PHYLOGENY OF THE STERNUM 75
 +
 +
sternum kept on growing until the respective pairs met and fused in the midventral line. Chamaeleo is considered to present strong evidence of the secondary character of the relation between ribs and sternum, and it is an important intermediate stage in the development between the typical reptilian sternum and the same structure in the mammals.
 +
 +
The preceding account of the development and anatomy of the sternum in the Reptilia and a comparison with the same structure in the Amphibia must inevitably lead to the conclusion that, if the presternum be coracoidal in origin in the Amphibia, it is equally so in the Reptilia. For, beginning with that most primitive reptilian sternum in Anguis, and comparing with Pipa and other Amphibia, the gulf was bridged between these two phyla, and then by a series of successively more highly developed sterna in the reptiles a stage is reached (Chirotes) which spans the divide between the reptiles and mammals. We have also seen how in one amphibian (fig. 24) the beginning of sternal bands arises, and in the reptiles these are developed in the same way, and in higher reptiles fuse to form the mesosternum and, xiphisternum, preparing the way for the typically mammalian sternum, soon to be considered.
 +
 +
The last fact concerning the Reptilia is in regard to the extremely variable relation of ribs and sternum, both as to number and position. Rathke ('53) had an interesting paragraph on this which is quoted in part :
 +
 +
In typical scaly lizards several ribs are always in relation with the sternum; still .... it may be either only the anterior division (manubrium) which is connected with ribs, or it may be exclusively the posterior part. But, generally speaking, the number of ribs which are intimately connected with the sternum, and to which the name of true ribs can be applied, not only varies with the genus, but is also very variable in different species.
 +
 +
Both Rathke and Parker give long lists of species of reptiles showing this variability in the number and position of ribs reaching the sternum. Their lists comprise some fifty species, but only a few are mentioned here as indicating the range of variation found by them. Their figures show that in some species
 +
 +
 +
 +
— • , •
 +
 +
 +
 +
FRANK BLAIR HANSON
 +
 +
 +
 +
with the largest number and most intimate union of ribs to breast bone the sternum is but feebly developed; while, on the other hand, some of the largest sterna have the fewest number of ribs and feeblest connection of the two; or, as in Chirotes, a fully developed sternum showing the three typical divisions is present, but no ribs reach the sternum — conditions hardly to be expected if the ribs contribute the sternal materials.
 +
 +
The following table is a composite one from several authors and shows but a few of the numerous forms they lisl .
 +
 +
 +
 +
Chirotes canaliculatus
 +
 +
Anguis fragilis
 +
 +
Chamaeleo pumilis
 +
 +
Monitor dracaena
 +
 +
Draco viridis
 +
 +
Stellio vulgaris
 +
 +
Calates pictus
 +
 +
Iguana tuberculata
 +
 +
Crocodilus acutus
 +
 +
Gavialis schlegelii
 +
 +
 +
 +
RIBS TO
 +
 +
 +
RIBS TO
 +
 +
 +
MANUBRIUM
 +
 +
 +
MESOSTERNUM
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
1
 +
 +
 +
 +
 +
 +
2
 +
 +
 +
1
 +
 +
 +
3
 +
 +
 +
 +
 +
 +
3
 +
 +
 +
1
 +
 +
 +
3
 +
 +
 +
3
 +
 +
 +
4
 +
 +
 +
2
 +
 +
 +
2
 +
 +
 +
5
 +
 +
 +
2
 +
 +
 +
7
 +
 +
 +
 +
4. Birds
 +
 +
Birds, although not in the line of descent of the mammals and also having a sternal apparatus highly modified for purposes of flight, are still not difficult to bring into line in this argument. However, it is hardly necessary to do this as we are following successively more complex and highly differentiated groups in their phylogenetic course. Nevertheless, to show that there is nothing contradictory to our thesis among the birds, one figure of a bird is introduced. This is Vanellus cristatus (fig. 33) and is of a stage at the end of the first third of the incubation period. Parker's remarks upon this sternum give us the pertinent facts: "The longitudinal bands are long and wide, and in great contrast to the very slender pairs of ribs attached to them. On the other hand, a transverse cleft between the epicoracoids and the anterolateral margins of the longitudinal bands would give us the con
 +
 +
 +
ONTOGENY AND PHYLOGENY OF THE STERNUM 77
 +
 +
dition as here found. The sternum of Vanellus is not a highly complex structure, as such structures go in the higher birds, but shows many affinities to the reptilian stock."
 +
 +
It would be absurd to think that these large, heavy sternal bands were originally derived from the feeble and loosely attached pairs of ribs, while it is entirely plausible to suppose that in an earlier stage a continuous sheet of cartilage on either side was subsequently differentiated into sternal bands, coracoid, and scapula. That this is actually the state of affairs in early embryos of several mammals will soon be shown.
 +
 +
That the development of the sternum is largely independent of ribs in the birds, as in reptiles, is further shown by Parker's observations on Apteryx:
 +
 +
In the earliest stage in which the sternum is present it extends backwards to the level of the third thoracic rib; the first two ribs are united to it by joints, the third loosely attached by connective tissue. In the next stage, the first three ribs are attached by joints, and the fourth by connective tissue, that is, as it appears to me, the portion of the sternum corresponding to the third and fourth ribs is formed by a backward growth of the anterior region and quite independently of the last two ribs (italics mine), the union of which with it is a secondary process.
 +
 +
This is just about what Paterson says concerning the development of the sternum in the rat, except that it leads him to conclude that the sternum is not of costal origin, while Parker, giving all the evidence necessary to substantiate Paterson's view, is nevertheless himself oblivious of the logic of his own work, and makes the surprising statement that "the relation of the shoulder girdle to the sternum is altogether secondary, and forms no part of the axial skeleton, as the Transcendentalists vainly teach."
 +
 +
The above account of conditions in the birds seems to be sufficient to relate genetically the sternum in birds to that of reptiles and mammals, and while of little or no philogenetic value in this connection, yet the evidence shows the avian sternum to be homologous with the sterna throughout the entire vertebrate series, one of the theses for which this paper contends.
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 +
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78 FRANK BLAIR HANSON
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5. Monotremes
 +
 +
The monotremes (fig. 4) have long been held to have a sternum closely reptilian in character. In addition to the coracoids, which are firmly attached to the sternum, there is also an anterior paired element, usually called the epicoracoid. In these there is an overlapping in the midline much as in certain amphibians, and in a recent paper Watson ('17) holds that the epicoracoids of the monotremes are nothing other than the precoracoids of the lower forms. That typically reptilian structure, the interclavicle, is also present. As seen in the figure, there is noticeable a compactness of the elements of the shoulder-girdle and sternum, as if these might have been in the early embryo or in the ancient progenitor a single shield-like plate, such as occurs in Pipa (fig. 20) or in many of the reptiles.
 +
 +
6. Marsupials
 +
 +
In 1897 Broom discovered in the marsupial Trichosurus vulpecula, measuring 17 mm., a well-developed coracoid, which was at birth "structurally continuous with the sternum. " Figure 35 is an anterior view of the entire scapular arch which in general outline is strikingly like that of the shark. Its ventral middle portion is a part of the sternum, yet the parts are, as Broom says, not jointed, but constitute a single bar of cartilage.
 +
 +
In a smaller specimen (fig. 36) of 8.5 mm. of the same animal both cartilaginous and mesenchymatous elements are present in the coracoid; the cartilaginous part being that nearest the glenoid cavity, and the mesenchymatous spreading out in a fan-shape portion, which is continued without any interruption into the mesenchymatous sternum. It is apparent that this marsupial has a complete scapular arch crossing the middle line ; and that, from the history of the later stages, the sternum as well as the girdles are known to be its derivatives. Furthermore, just in front of the coracoid and posterior to the clavicle, there was "a thin, feebly developed continuous sheet of mesenchymatous cells;" lying therefore in the exact position of that anterior coracoidal element of the monotremes generally called the epicoracoid,
 +
 +
 +
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ONTOGENY AND PHYLOGENY OF THE STERNUM 79
 +
 +
but by some the precoracoid. That the two structures, i.e., the epicoracoid of the monotremes and this " sheet of mesenchymatous cells" are homologous is the belief of Broom. In the larger specimens (37 mm. and over) the coracoid becomes detached from the sternum by a process of degeneration, and this continues until the well-known adult condition is reached (fig. 37) where coracoids and sternum are far apart.
 +
 +
However, in a mammary fetus 23 mm. long an intermediate condition was found. As Broom describes it:
 +
 +
The coracoid process is similar to that in the large foetus, but from it there is produced backwards and inwards a small cartilaginous process, which nearly meets the outer process of the presternum. It may thus be concluded that during the later intra-uterine development of Trichosurus, and probably of other marsupials (later verified in other marsupials), there is a well developed coracoid, which, as in the adult Monotremes, most reptiles, birds, and amphibians, articulates with the sternum, and that shortly after birth, the coracoid loses its attachment with the sternum, and becomes rapidly absorbed, only the anterior part remaining as the coracoid process.
 +
 +
From this description it appears that the degeneration of the coracoid begins in its middle part and absorption progresses toward each end. In the marsupials that part of the coracoid attached to the sternum is completely absorbed and no trace of it is found in the adult, while the half connecting with the scapula is represented in the adult by the coracoid process. In this connection mention may be made of a peculiar structure I find in the mouse and rat, and an almost constant structure in rodents, as figured by Parker ('68) . In most of his figures of the Rodentia, there is a small bony process on either side of the presternum between the juncture of the clavicle and the first rib with the sternum (fig. 38). Parker calls this process the epicoracoid and says it was left by the retreating coracoids of the lower forms. It would seem from my observations on the mouse, described above, that Parker is correct to the extent that this is the median end of the coracoid, but wrong in making it simply an hereditary rudiment. It is, rather, the end of a complete embryonic coracoid, which in the rat, mouse and man, as in the marsupial, extends across to the sternum in an early stage. Whereas in the
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«
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THE AMERICAN JOURNAL OF ANATOMY, VOL. 26, NO. 1
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80 FRANK BLAIR HANSON
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marsupial and most other mammals, all trace of this sternocoracoidal piece is lacking in the adult, it persists in the rodents, and also in the narwhal (Monodon monocerus), and the blackfish (Globicephalus melas), and possibly other animals. In other words, in the rodents there is at first a complete coracoid reaching from the scapula to the sternum ; later, degeneration of the coracoid sets in at its middle portion, and working toward each end, stops just in time to leave the coracoid process attached to the scapula and the epicoracoid to the sternum.
 +
 +
From the foregoing it is apparent that Trichosurus passes through a stage in its development, as regards girdles and sternum, which is directly comparable with that of the adult condition in the monotremes.
 +
 +
In a series of later papers ('98, '99 '02, '08) Broom studied a number of marsupials, both Polyprotodonts and Diprotodonts, and found that conditions throughout the Marsupialia were as described for Trichosurus. In a mammary fetus of the common phalanger, 14 mm. long, the well-developed coracoid articulates with the sternum almost exactly as in the adult monotremes. In the earliest stage of Dasyurus (fig. 39) studied by Broom, the coracoid is still large and reaches nearly to the sternum. It would seem that here absorption had commenced at the medial end rather than in the middle of the cartilage. Figure 48 (petrogale) shows a complete, unjointed arch similar to that of Trichosurus. Pseudochiurus and permales were studied in various stages and agree in general with other marsupials, so that this is obviously a normal and constant phenomenon of the marsupial embryonic girdle, as it is of the adult girdle in the monotremes.
 +
 +
Prior to Broom's work, it was difficult, practically impossible in fact, to pass from the monotreme shoulder-girdle to that of the marsupial, albeit the relationship of monotreme girdle to that of the reptile was apparent. Since Broom's discovery, however, of the complete girdle in the early marsupial, and also in the rat by Paterson and the mouse and man by the present author, it is clearly seen that the girdles and sternum of the higher mammals are directly comparable to that of the marsupial fetus, and this in turn to the girdle of monotremes, which are unquestioned in
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ONTOGENY AND PHYLOGENY OF THE STERNUM 81
 +
 +
their reptilian affinities. It is thought that this makes a strong case for the homology of the sternum in the Amniota and its origin in connection with the coracoids ; and since this same structure is undoubtedly coracoidal in the Ichthyopsida, the two, Amniote and Ichthyopsidan sternum, are homologous.
 +
 +
As this work of Broom's is of considerable importance to our argument, it may be stated that Broom in his later papers verified his first discoveries in representatives of all groups of marsupials and made graphical reconstructions of the parts. His work has been checked up by Watson ('17), who made wax models of the girdles and sternum, and completely confirmed Broom's results.
 +
 +
7. Aquatic mammals
 +
 +
Among the aquatic mammals there are several interesting sterna. In the adult Manatus americanus (fig. 40) the sternum is moderately large and is typically divided into the usual three parts. This animal has seventeen pairs of ribs, but only three come anywhere near the sternum, and Parker ('68) says that only the second pair of ribs reaches it. The first and third pairs are connected with it by ligament only. Here in this adult mammal is a stage comparable to the embryo of the pig, man, etc., where ribs either do not reach the sternum or are connected with it by fibrous tissue. It is difficult to believe that this entire sternum was derived from the costal ends of three pairs of ribs, of which only one pair even approaches it.
 +
 +
According to W. K. Parker ('68) the sternum of the dolphin embryo (fig. 41) has reached its highest development in aquatic mammals. In the stage figured the sternal bands have fused, leaving a prominent longitudinal groove to mark the line of fusion, several centers of ossification are present, and in the presternum is an oval fontanelle such as is common in the lizards. In fact, this entire structure is very reptilian in character, as is seen by a comparison with Trachydosaurus (fig. 28) , even to the number of ribs.
 +
 +
In orders of mammals higher than those already mentioned the literature is scanty. Practically all papers based upon the
 +
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 +
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82 FRANK BLAIR* HANSON
 +
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human embryo accept Ruge's view, and the reason is apparent when we consider that in working exclusively with the highest mammals, one is at the disadvantage of not knowing what stages may have been suppressed in evolution. It would seem that, beginning with the lowest forms and working up through each successive group, as is attempted in this present paper, a foundation is laid upon which to interpret the greatly reduced history in the higher mammals.
 +
 +
Does the complete girdle, sternum, coracoid, and scapula, cross the midline in forms higher than the marsupial? Since the permanent adult coracoid articulating with the sternum in the monotreme is reduced to only a fetal stage in the marsupial, we might expect this stage to be very transitory or entirely suppressed higher in the scale. Since the marsupials, edentates, rodents, and insectivores are all ancient orders and probably lie not far from the monotreme stem, among them such a stage might be found, if present. Paterson was the first to discover this in an early embryo of the rat, and I found the same thing (section on ontogeny) in the mouse of 7.75-mm. and 17.2-mm. human embryo. This is shown in figures 5 and 6, where the girdles are very similar indeed to that of a shark (fig. 2). The next stages indicate that scapulae, coracoids, and sternum are all derivatives of this single mesenchymatous element. This stage is exceedingly brief in the mouse, as compared with the marsupial, for it cannot be detected with certainty in a 6-mm. mouse, and is hardly recognizable in the 8.75-mm. mouse. This rapid suppression would lead us to suppose that possibly no such stage is present in the human embryo, yet figure 6 shows man to have retained it identically as in the rodents.
 +
 +
Huntington ('18) identifies the costocoracoid ligament of man as indicating "'the original path of the sternal extension of the coracoid." It is interesting in this connection that the costocoracoid ligament often contains fibrocartilaginous nodules. Figure 42 shows this ligament in relation to the other parts in man on the left side of the figure, while on the right side, the fundamental plan of the vertebrate girdle as illustrated in the Anura is shown. Huntington does not say whether he considers the
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^ONTOGENY AND PHYLOGENY OF THE STERNUM 83
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costocoracoid ligament to be the remains of an embryonic coracoid occurring in man or only the rudimentary indication of man's phylogenesis. If this interpretation of the costocoracoid ligament as the evidence of a coracoidal connection with the sternum be correct, then from the elasmobranch to man the closest relation between coracoids and sternum is maintained. On the other hand, if Huntington's hypothesis is not sustained by later investigation, no harm is done to our attempt to overthrow Ruge's theory, for Rathke, Paterson, Whitehead and Waddell, and myself have already shown that in man and other mammals the sternum is an established structure prior to the union of ribs with it.
 +
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8. The adult human sternum
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 +
In the literature of the adult sternum in man there is evidence of a secondary, but interesting and corroborative sort. It may be related back to the tendency of the ribs to grow toward the ventral median line and fuse or articulate with each other, as was noted in Chamaeleo. This has been shown by several workers, Cunningham ('90), Dwight ('90), Tredgold ('97), Paterson ('09), and Lickley ('04), to be especially prominent in the human subject. Lickley, whose work is typical, studied a series of fifty-one human sterna with special reference to the relations of the seventh and eighth ribs to the sternum.
 +
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The number of ribs reaching the sternum in Lickley's material is shown in the following table :
 +
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BIGHT
 +
 +
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LEFT
 +
 +
 +
Sixth ribs
 +
 +
 +
2
 +
 +
43
 +
 +
6
 +
 +
 +
1
 +
 +
 +
Seventh ribs
 +
 +
 +
43
 +
 +
 +
Eighth ribs
 +
 +
 +
7
 +
 +
 +
 +
 +
 +
 +
Total
 +
 +
 +
51
 +
 +
 +
51
 +
 +
 +
 +
 +
 +
 +
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This constitutes a considerable percentage of variation at the posterior end of the thorax, yet whether six or eight ribs enter into union with the sternum, that structure is apparently not
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84 FRANK BLAIR HANSON #
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affected in length or shape. Advocates of a costal origin for the sternum derive the xiphisternum from the ends of the seventh or eighth ribs or both. However, when these are absent, the formation of the xiphisternum does not seem to be affected in the least. As in the reptiles, it is a constant feature regardless of the absence or presence of ribs.
 +
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In the work of Paterson ('09) and Lickley ('04) an even more suggestive fact is clearly apparent. This relates to the tendency of ribs to grow to the ventral side and fuse as was also seen in Chamaeleo and noted in the discussion of reptiles. In man the seventh rib normally reaches the sternum, but in its mode of attachment we have the remarkable statement of Lickley that in over 50 per cent the seventh costal cartilages are either fused or articulate with each other in a plane anterior (ventral) to the xiphisternum. That is to say, these ribs which are anterior to the posterior end of the sternum reach the median line and fuse just as did the ribs posterior to the posterior end of the sternum in Chamaeleo. In all the accounts here under review the ribs act as if entirely independent of and irresponsible for the being or well-being of the sternum.
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Paterson's results are not so striking in large percentages as Lickley's, but sufficiently so to give us pause, even if considered alone. He examined 236 human fetal sterna and found that the seventh costal cartilages articulated in front of the sternum in 14.4 per cent.
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In •this connection three special cases may be cited, the first two reported by Lickley and the last by Dwight :
 +
 +
1. The sixth costal cartilages articulated dorsally with the lower end of the mesosternum and ventrally with one another. The seventh cartilages articulated with the lower borders of the sixth cartilages and by their extremities with one another.
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2. Girl, eighteen years. Mesosternum terminated at level of insertion of fifth ribs. The extremities of the sixth and seventh cartilages on the left side were fused together, those on the right were closely united by fibrous tissue. The two bars formed in this way articulated with the mesosternum above, and with one another in front.
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ONTOGENY AND PHYLOGENY OF THE STERNUM 85
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3. The body of the sternum ends at 'the level of the fourth ribs. The fifth pair is attached to its lower end. The fifth, sixth, and seventh pairs meet one another and fuse.
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Kirchner ('98) and Adolphi ('05) also describe sterna in large numbers and find many cases of variation in the posterior thoracic ribs much in accord with the above. The results of these papers on the adult sterna of the human subject, together with similar observations on the lower forms, which are pertinent to our thesis are four :
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 +
1. Ribs posterior to the level of the sternum may grow ventrally and meet by fusion or articulation in the midline (Chamaeleo) .
 +
 +
2. Ribs at the level of the sternum may articulate firmly with it, as is the usual case in man.
 +
 +
3. Ribs may pass on the ventral side of the posterior end of the sternum and fuse in the midline much as they do when farther back where no sternum is present (Chamaeleo, man).
 +
 +
4. All ribs may fail to meet the sternum at any point without affecting the full development of that structure (Amphibia, Chirotes, Manatus, human embryo).
 +
 +
Plate 12, figures 43 to 49, inclusive, is designed to give at a glance a series of shoulder-girdles representing the main groups of vertebrates, in an effort to show how from the elasmobranch to the rodent, either in the embryo or throughout life, there is an intimate relation in all between shoulder-girdle and sternum, and in some it is demonstrated that they are for a time one structurally continuous element. In some (shark, lissotriton, lizard, and monotreme) this relation is maintained throughout life; in others (marsupial, mouse, rat, and man) it is of short duration in embryonic life. In all, however, it indicates a phylogenetic relationship between the shoulder-girdle and sternum in all stages of vertebrate evolution that cannot possibly be duplicated or even remotely approximated by a similar comparison of sterna and costal cartilages.
 +
 +
In most adult Amniotes the sternum is in close relation with both ribs and shoulder-girdle; in some, however, the connection with either ribs or shoulder-girdle may be lost (Chirotes, fig. 30,
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 +
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86 FRANK BLAIR HANSON
 +
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being an example of the first, and the pig of the second) ; but none is known in which the relation to both ribs and shoulder-girdle is lacking. Assuming that the sternum must be either a derivative of the shoulder-girdle or of the ribs, it is clearly evident from a phylogenetic viewpoint that since the costae verae extending to the ventral side of the body were not acquired by the vertebrates uutil the rise of the Reptilia, whereas the sternum and shoulder-girdle, in an ever-increasing closeness of relation and association, may be traced back to the very beginning of the Ichthyopsida in the elasmobranchs, that we cannot hope to find in the ribs any clue to sternal origin. If the sternum be homologous throughout, as the conclusions of this present investigation seem to warrant, then its origin may be sought in a structure which is coexistent with it and also in the closest possible relation to it in the lowest forms. In the shoulder-girdle of the Ichthyopsida we seem to meet with both of these requirements, while in each of them the ribs fail us.
 +
 +
In Huntington's ('18) paper, which appeared after this work was practically completed, are certain fundamental conceptions of the shoulder-girdle and its phylogenetic relationships which indirectly corroborate my conclusions. In the first place, Huntington recognizes the elasmobranch pectoral girdle as "the primordial fundament upon which all other vertebrate modifications are built." By a dual process of segmentation and replacement by bone, all structures of the complicated girdles of the higher classes of vertebrates are derived from this simple continuous unsegmented bar of cartilage found in the dogfish, sharks or rays. Huntington was not, however, primarily interested in the sternum and did not see in the midventral portion of the elasmobranch girdle the fundament of the presternum. Like other investigators, he finds the "first appearance of the sternal apparatus" in the amphibian girdle, but notes its intimate association with the epicoracoids, which latter cartilages are beginning to loosen by sutures on either side of the ventral midline. However, it must be pointed out that these structural relations of the Amphibia (clavicle excepted) are also present in Hexanchus, where a suture on either side of the ventral midline gives the
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ONTOGENY AND PHYLOGENY OF THE STERNUM 87
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'intercepted cartilage' interpreted by Howes ('91), Parker ('91), and myself as the rudiment of the presternum. If two dotted lines be added to the Hexanchus girdle (fig. 1), a sternum comparable to that of Rana is produced, with epicoracoids meeting in the midline and the fundaments of the omosternum and sternum present.
 +
 +
9. Conclusions
 +
 +
This somewhat lengthy review and discussion of the phylogeny of the sternum in the different classes of vertebrates, together with the accompanying figures, which have been adapted and modified from various sources, has revealed very clearly several facts of outstanding importance in relation to the problem of sternal origin:
 +
 +
1. That there is present a median ventral rudiment, derived from the coracoids, which may be identified as a presternum as far back in the vertebrate series as the shark, and can be followed up through a ganoid, a teleost, a dipnoan, and from there on through the Tetrapoda.
 +
 +
2. That in all cases of vertebrates, and as high as man in the Mammalia, there is in the embryo or throughout life a continuous girdle across the ventral side and connecting the two scapulae above.
 +
 +
3. That this girdle in its ventral aspect is in the most intimate relation to the anterior part of the sternum; sometimes the mesenchymatous material passes over insensibly from one structure into the other without any line of demarkation; or at most, adults of Amphibia and Reptilia, there being but a suture between the two parts.
 +
 +
4. That in all these forms from the lowest to the highest, the relation of sternum and ribs is purely secondary and the result of a comparatively late fusion of the two structures in the embryo. The presence or absence of ribs does not seem to affect the development or size of the sternum in any degree.
 +
 +
5. That plural ribs extending to the ventral side of the body are a recent acquisition of the vertebrates, while the shouldergirdle and sternum are coexistent and intimately related from the earliest appearance of the Gnathosomes.
 +
 +
 +
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88 FRANK BLAIR HANSON
 +
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6. That the number of ribs reaching the sternum varies from zero to a large number; sometimes the ribs are attached to the anterior part of the sternum, again exclusively to the posterior part, but apparently whatever the number or relation of ribs, the sternum remains unaffected, indicating strongly its independence of the costal cartilage.
 +
 +
7. That the evidence presented seems to bear out the homology of the sterna throughout the vertebrates; therefore, to classify them as coracoidal for the lower groups, and costal for the higher is unnecessary and artificial, for in the Amniota the sternum is as truly coracoidal in origin as it is in the Ichthyopsida.
 +
 +
8. That the mesosternum and xiphisternum are two backward prolongations of the coracoidal presternum, sometimes uniting in the midline (some reptiles, birds, and mammals), again remaining distinct as horns or bars (some Amphibia, some reptiles) .
 +
 +
V. SUMMARY
 +
 +
At the close of the section on the ontogeny of the sternum a number of conclusions were listed, and likewise at the close of the section on phylo'geny. These results may now be gathered up in the three main theses of this paper, which are stated thus :
 +
 +
1 . That the sternum is an homologous structure throughout all groups of vertebrates, and occurs in forms ranging from Hexanchus up to the highest mammals.
 +
 +
2. That the anterior element of the sternum has its origin in common with the shoulder-girdle, and in the embryo or throughout life is in intimate relation to the coracoids.
 +
 +
3. That the sternal bands are derivatives of the anterior median rudiment, and may be secondarily, but never genetically, associated with ribs.
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ONTOGENY AND PHYLOGENY OF THE STERNUM 89
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LITERATURE CITED
 +
 +
Adolphi, H. 1905 liber die Variation des Brustknorbes und der Wirbelsaule
 +
 +
des Menschen. Morph. Jahrb., Bd. 33. Anthony, R. 1901 Notes sur la Morphogenie du sternum chez Mammiferes a
 +
 +
propos de l'etude de Paterson sur le developpement de cet os. Bull.
 +
 +
Soc. Anthrop. Paris (5), T. 2, pp. 19-43. Broom, R. 1897 On the existence of a sternocorocoidal articulation in a fetal
 +
 +
marsupial. Jour, of Anat. and Physiol., vol. 31.
 +
 +
1898 Description of the shoulder-girdle in an 8.5-mm. embryo of trichosurus (not exact title). Proc. Linn. Soc. N. S. W., 1898.
 +
 +
1899 On the development and morphology of the marsupial shouldergirdle. Trans. Roy. Soc. Edinb., vol. 39, pt. 3, pp. 749-770.
 +
 +
1902 On the early condition of the shoulder-girdle in the Polyprotodont Marsupials Dasyurus and Permales. Linn. Soc. Jour. Zool., vol. 28.
 +
 +
1908 On the nomenclature of the elements of the amphibian shouldergirdle. Report S. Afr. Assoc. Adv. Sc, 6th. Meet., pp. 162-166.
 +
 +
Bruch, Carl 1852 Beitrage zur Entwicklungeschichte des Knochensystems von Dr. Carl Bruch. Neue Denkschriften der allgemeinen Schweizerischen Gesellschaft fur die gesammten Naturwissenschaften. Bd. 12, Zurich (quoted by Ruge).
 +
 +
Credner 1881-1893 Stegocephalen und Saurier. Zeitschr. deutsch. Geolog. Gesellsch., 1881-1893.
 +
 +
Cunningham, D. J. 1890 The occasional eighth, true rib in man and its relation to right-handedness. Jour. Anat. and Physiol., vol. 24, p. 127.
 +
 +
Dwight, Thomas 1890 Irregular union of the first and second pieces of the sternum in man and the apes. Jour. Anat. and Physiol., vol. 24, pp. 536-542.
 +
 +
Goette, A. 1877 Morphologie des Skelettsystems der Wirbeltiere: Brustbein und Schultergiirtel. Arch. f. Mikr. Anat., Bd. 14.
 +
 +
Goodrich 1909 Vertebrate Craniata (first fasicle) : Cyclostomes and fishes. A treatise on zoology, pt. 9, by E. Ray Lankester. London.
 +
 +
Gregory, W. K. 1915 Present status of the problem of the origin of the Tetrapoda. Ann. N. Y. Acad. Sc, vol. 26.
 +
 +
Gunther 1867 Anatomy of Hatteria (Sphenodon). Phil. Trans., 1867.
 +
 +
Haswell, W. A. 1884 Studies on the elasmobranch skeleton. Proc. Linn. Soc. N. S. W., vol. 9.
 +
 +
Hoffmann, C. K. 1879 Zur Morphologie des Schultergurtels und des Brustbeins bei Reptilien, Vogeln, Saugetieren, und dem Menschen. Niederland. Archiv. f. Zool., Bd. 5.
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 +
Howes, G. B. 1891 Morphology of the sternum. Nature, vol. 43.
 +
 +
Howes and Swinnerton 1901 Development of the skeleton of Sphenodon. Trans. Zool. Soc. Lon., vol. 16.
 +
 +
Huntington, G. S. 1918 Modern problems of evolution, variation, and inheritance in the anatomical part of the medical curriculum. Anat. Rec, vol. 14, no. 6.
 +
 +
Keibel and Mall 1910-1912 A manual of human embryology. J. B. Lippincott Co., Phila.
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 +
 +
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90 FRANK BLAIR HANSON
 +
 +
 +
 +
Kingsley, J. S. 1917 Comparative anatomy of vertebrates. 2nd ed. P.
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 +
Blakiston's Sons & Co., Phila. Ivirchner, A. 1898 Das obere Brustbeinende und das ligamentum interclavi culare nebst zusammenstellungen uber das Verhaltnis des oberen
 +
 +
sagittalen Brustdurchmessers und der Brustbeinliinge zur Korper lange. Anat. Hefte., Bd. 10, S. 127-149. Kravetz, L. P. 1905 Entwicklungsgeschichte des Sternum und des Epister nalapparatus der Saugetiere. Bull. Soc. Imper. Natur. Moscow,
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 +
1905, 1906. Lickley, J. D. 1904 On the relations of the seventh and eighth ribs to the
 +
 +
sternum. Anat. Anz., Bd. 24. Markowski, J. 1905 Sollte der Verknocherungsprozess des Brustbeins von
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 +
keiner morphologischen Bedeutung sein? Aus anlass einer Publika tion von Paterson. Anat. Anz., Bd. 26, S. 248-269. Mueller, Charlotte 1906 Zur Entwicklung des menschlichen Brustkorbes.
 +
 +
Morph. Jahrb., Bd. 35. Parker, W. J. 1868 Structure and development of the shoulder-girdle and
 +
 +
sternum in the vertebrates. Ray Soc. Pub., London. Parker, T. J. 1891 On the presence of a sternum in Notidanus indicus.
 +
 +
Xature, vol. 43, p. 142.
 +
 +
1891 On the origin of the sternum. Trans. New Zealand Instit.,
 +
 +
vol. 23. Paterson, A. M. 1900 The sternum; its early development and ossification
 +
 +
in man and mammals. Jour. Anat. and Physiol., vol. 35, N. S., vol.
 +
 +
15, pt. 1.
 +
 +
1902 Development of the sternum and shoulder-girdle in mammals.
 +
 +
Brit. Med. Jour., vol. 2.
 +
 +
1904 The human sternum. Liverpool.
 +
 +
1909 Two abnormal sterna in living subjects. Jour. Anat. and
 +
 +
Physiol., vol. 43, pp. 322-323. Rathke, H. 1838 Sur le development du sternum. Arch. Anat. et Physiol.
 +
 +
1848 Uber die Entwicklung der Schildkroten. Brunswick.
 +
 +
1853 tJber den Bau und die Entwicklung des Brustbeins der Saurier.
 +
 +
Konigsberg. Huge, G. 1879 Uber die Entwickelung des Sternum. Morph. Jahrb., Bd. 5.
 +
 +
1880 Untersuchungen uber Entwickelungsvorgange am Brustbein
 +
 +
und der Sterno-clavicularverbindung des Menschen. Morph. Jahrb.,
 +
 +
Bd. 6. Sabatier, A. 1897 Morphologie du sternum et des clavicules. C. R. Acad.
 +
 +
Sc. Paris, T. 124, no. 15.
 +
 +
1902 Du systeme sternal des vertebres. C. R. Ass. Anat., 4me, pp.
 +
 +
99-102. Schatjinsland 1900 Entwicklungsgeschichte der Hatteria (Sphenodon). Arch.
 +
 +
f. Mikr. Anat., Bd. 56. Seeley, H. G. 1892 Researches on the structure, organization and classification of the fossil Reptilia. Phil. Trans., vol. 183-B, pp. 334-338.
 +
 +
1894 Researches on the structure, organization, and classification of
 +
 +
the fossil Reptilia. Phil. Trans., vol. 185-B.
 +
 +
 +
 +
ONTOGENY AND PHYLOGENY OF THE STERNUM 91
 +
 +
Tredgold 1897 Variations of ribs in the Primates, with especial reference to
 +
 +
the number of ribs in man. Jour. Anat. and Physiol., vol. 31. Watson, D. M. S. 1917 The evolution of the tetrapod shoulder-girdle and
 +
 +
forelimb. Jour, of Anat., vol. 52, pt. 1. Whitehead and Waddell 1911 Development of the human sternum. Am.
 +
 +
Jour. Anat., vol. 12, pp. 89-106. Wilder, H. H. 1903 The skeletal system of Necturus maculatus Rafinesque.
 +
 +
Mem. Bost. Soc. Nat. Hist., vol. 5, no. 9. Williston, S. W. 1903 North American Plesiosaurs. Field Col. Mus. Pub.
 +
 +
73. Geol. Ser., vol. 2, no. 1.
 +
 +
1903 On the osteology of Nyctasaurus (Nyctodactylus). Field Col.
 +
 +
Mus. Pub. 78. Geol. Ser., vo.. 2, no. 3.
 +
 +
1911 American Permian vertebrates. Chicago.
 +
 +
1914 Water reptiles of the past and present. Chicago. Woodward 1898 Vertebrate paleontology. Cambridge.
 +
 +
 +
 +
PLATE 1
 +
 +
EXPLANATION OF FIGURES
 +
 +
1 Median portion of pectoral girdle of Hexanchus. Note the medial 'intercepted cartilage' which bears all the relations of a presternum in higher forms. The dotted lines indicate how this girdle may be transformed into that of the Amphibia with pre- and post-omosternum. Drawing from a dissection made by the author in the U. S. National Museum.
 +
 +
2 Pectoral girdle of Acanthias vulgaris. Note that all the parts of the girdle of higher forms are present, including a sternum. Compare with figure 35 of the fetal marsupial.
 +
 +
Cr, coracoid Sc, scapula
 +
 +
POSt, pre-omosternum SSc, suprascapula
 +
 +
PSt, presternum St, sternum PtOSt, post-omosternum
 +
 +
 +
 +
92
 +
 +
 +
 +
ONTOGENY AND PHYLOGENY OF THE STERNUM
 +
 +
FRANK BLAIR HANSON
 +
 +
 +
 +
PLATE 1
 +
 +
 +
 +
 +
post
 +
 +
 +
 +
.PS t
 +
 +
 +
 +
 +
93
 +
 +
 +
 +
PLATE 2
 +
 +
EXPLANATION OF FIGURES
 +
 +
3 Cat embryo, 12 mm. Sternal bands are far apart. No presternum present at this stage. The first three pairs of ribs fail to reach the sternal bands. From the Princeton Embryological Collection, series no. 401. Graphic reconstruction.
 +
 +
4 Cat embryo, 14 mm. Sternal bands nearer to each other. Presternum has arisen and connects the anterior ends of sternal bands. All ribs reach the sternum. From the Princeton Embryological Collection, no. 37. Graphic reconstruction.
 +
 +
PSt, presternum R,* fourth rib
 +
 +
R\ first rib R, 7 seventh rib
 +
 +
R 3 , third rib StB, sternal bands
 +
 +
 +
 +
94
 +
 +
 +
 +
ONTOGENY AND PHYLOGENY OF THE STERNUM
 +
 +
FKANK BLAIR HANSON
 +
 +
 +
 +
PLATE 2
 +
 +
 +
 +
 +
S t B
 +
 +
 +
 +
 +
- — R
 +
 +
 +
 +
-R
 +
 +
 +
 +
---R
 +
 +
 +
 +
P St
 +
 +
 +
 +
 +
R
 +
 +
 +
 +
95
 +
 +
 +
 +
THE AMERICAN JOURNAL OF ANATOMY, VOL. 26, NO 1
 +
 +
 +
 +
PLATE 3
 +
 +
EXPLANATION OP FIGURES
 +
 +
5 Mouse girdle. Scapulae, coracoids, clavicles, and presternum, all in one continuous mesenchymatous girdle, which is similar to that of the dogfish (fig. 1) and the marsupial fetus (fig. 35). From the Washington University School of Medicine Embryological Collection, series no. 102, slide 4, section 16.
 +
 +
6 Human embryo girdle. Mesenchymatous stage. First appearance of clavicle; presternum and coracoids present. From The Johns Hopkins Embryological Collection, series no. 424, slide 5, section 16.
 +
 +
CI, clavicle PSt, presternum
 +
 +
Cr, coracoid Sc, scapula
 +
 +
 +
 +
96
 +
 +
 +
 +
ONTOGENY AND PHYLOGENY OF THE STERNUM
 +
 +
FRANK BLAIR HANSON
 +
 +
 +
 +
PLATE 3
 +
 +
 +
 +
 +
 +
 +
 +
 +
«^
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
^^«1
 +
 +
 +
 +
s^giS
 +
 +
 +
 +
 +
97
 +
 +
 +
 +
PLATE 4
 +
 +
EXPLANATION OF FIGURES
 +
 +
7 Sternum and ends of costal cartilages in pig two weeks old. Note in this and later figures that centers of ossification are intercostal.
 +
 +
8 Fetal sternum of Bradypus. After Hoffmann.
 +
 +
9 The ape sternum. After Anthony.
 +
 +
10 Sternum of Dasypus. After Hoffman. From the lowest to the highest mammals the sternebrae and centers of ossification are between the ribs, and not at their ends as would be expected according to Paige's theory.
 +
 +
Oc, ossific center
 +
 +
 +
 +
98
 +
 +
 +
 +
ONTOGENY AND PHYLOGENY OF THE STERNUM
 +
 +
FRANK BLAIR HANSON
 +
 +
 +
 +
PLATE 4
 +
 +
 +
 +
 +
99
 +
 +
 +
 +
PLATE 5
 +
 +
EXPLANATION OF FIGURES
 +
 +
11 Ventral portion of shoulder-girdle of the shark, Notidanus indicus, showing the presternum hi the midline. After Parker.
 +
 +
12 Ventral part of the shoulder-girdle of Polypterus. Note epicoracoid at junction of the clavicles. From Gregory, after Goodrich.
 +
 +
13 Clavicles and epicoracoid of the tropical fish, Gobius niger Linn., after Parker.
 +
 +
14 to 15 Upper and lower views of the girdle of the Dipnoid, lepidosiren. After Parker.
 +
 +
16 to 19 A series of stages in the development of the sternum of the frog, Rana temporaria, indicating that the sternum is coracoidal in origin. After Parker.
 +
 +
CI, clavicle PtOmSt, post-omosternum
 +
 +
Cr, coracoid OSt, omosternum
 +
 +
ECr, epicoracoid Sc, scapula
 +
 +
PrOmSt, pre-omosternum St, sternum
 +
 +
 +
 +
100
 +
 +
 +
 +
ONTOGENY AND PHYLOGENY OF THE STERNUM
 +
 +
FRANK BLAIR HANSON
 +
 +
 +
 +
PLATE 5
 +
 +
 +
 +
1 1
 +
 +
 +
 +
' PrQmS t
 +
 +
 +
 +
PtOmSt
 +
 +
 +
 +
 +
CI
 +
 +
 +
 +
 +
ECr
 +
 +
 +
 +
ECr
 +
 +
 +
 +
12
 +
 +
 +
 +
 +
ECr
 +
 +
 +
 +
ECr
 +
 +
 +
 +
 +
 +
/ECr
 +
 +
 +
 +
16
 +
 +
 +
 +
 +
OSt
 +
 +
 +
 +
•*— -ECr
 +
 +
 +
ECr
 +
 +
 +
 +
18
 +
 +
 +
 +
 +
-St
 +
 +
 +
 +
101
 +
 +
 +
 +
EC r
 +
 +
 +
 +
• 19
 +
 +
 +
 +
 +
—St
 +
 +
 +
 +
PLATE 6
 +
 +
 +
 +
EXPLANATION OF FIGURES
 +
 +
 +
 +
20 Shield-like plate on ventral side of Pipa dorsigera. Adult female, upper view. Would seem to indicate that sternum and coracoids were at one time structurally one; omosternum but feebly separated from coracoidal portion. After Parker.
 +
 +
21 Bufo vulgaris. First summer. Lower view. After Parker.
 +
 +
22 Large specimen of Siredon pisciformis. Sternum cut off from coracoids in adult, but retain evidences of having come from the overlapping epicoracoids. After Parker.
 +
 +
23 Dactylethra capensis. Adult female. After Parker.
 +
 +
24 Note the beginning of sternal bands in Calamites cyaneus. Compare with same stage in low-type reptile. After Parker.
 +
 +
CI, clavicle OSt, omosternum
 +
 +
Cr, coracoid PCr, precoracoid
 +
 +
Crf, coracoid fossa Sc, scapula
 +
 +
ECr, epicoracoid SSc, suprascapula
 +
 +
Gl, glenoid St, sternum
 +
 +
 +
 +
102
 +
 +
 +
 +
ONTOGENY AND PHYLOGENY OF THE CTERNUM
 +
 +
FRANK BLAIR HANSON
 +
 +
 +
 +
PLATE 6
 +
 +
 +
 +
OSt
 +
 +
 +
 +
CI
 +
 +
 +
 +
 +
of
 +
 +
 +
 +
— Cr
 +
 +
 +
 +
 +
ECr
 +
 +
 +
 +
21
 +
 +
 +
 +
"-ECr
 +
 +
 +
 +
PC
 +
 +
 +
 +
 +
 +
f— Cr
 +
 +
 +
 +
— St
 +
 +
 +
 +
ECr
 +
 +
 +
 +
--CrF
 +
 +
 +
 +
103
 +
 +
 +
 +
PLATE 7
 +
 +
EXPLANATION OP FIGURES
 +
 +
25 An old specimen of Anguis fragilis. Very similar to amphibian girdle. No ribs attached. Same intimate relation of sternum and coracoidal part as in the Amphibia. Modified after Parker.
 +
 +
26 In Stellio cordylinus long sternal bars make their appearance. No ribs are attached to them. Adapted from Parker.
 +
 +
27 Laemanctus longipes. After Parker.
 +
 +
28 Trachydosaurus rigosus adult. Sternal bars are fused and ribs are approaching. Adapted from Parker.
 +
 +
ECr, epicoracoid R 1 , first rib
 +
 +
Gl, glenoid Sc, scapula
 +
 +
ICl, interclavicle St, sternum
 +
 +
PCr, precoracoid Slf, sternal fossa
 +
 +
PSl, presternum XSt, xiphisternum
 +
 +
 +
 +
104
 +
 +
 +
 +
ONTOGENY AND PHYLOGENY OF THE STERNUM
 +
 +
FRANK BLAIR HANSON
 +
 +
 +
 +
PLATE 7
 +
 +
 +
 +
PCr
 +
 +
 +
 +
EC
 +
 +
 +
 +
 +
10.5
 +
 +
 +
 +
PLATE 8
 +
 +
 +
 +
EXPLANATION OF FIGURES
 +
 +
 +
 +
29 Cyclodus nigroluteus. Adult, lower view. After Parker.
 +
 +
30 Chirotes canaliculates. Adult male. Upper view. Mammalian-like sternum. No ribs ever reach sternum. After Parker.
 +
 +
31 Crocodilus acutus. Ripe embryo. Lower view. After Parker.
 +
 +
32 Chamaeleo vulgaris. Adult, lower view. After Parker. Cr, coracoid R 1 , first rib
 +
 +
ECr, epicoracoid R 2 , second rib
 +
 +
Gl, glenoid Sc, scapula
 +
 +
ICl, interclavicle Stf, sternal fossa
 +
 +
MSt, mesosternum XSt, xiphisternum
 +
 +
PSt, presternum
 +
 +
 +
 +
106
 +
 +
 +
 +
ONTOGENY AND PHYLOGENY OF THE STERNUM
 +
 +
FRANK BLAIR HANSON
 +
 +
 +
 +
PLATE 8
 +
 +
 +
 +
ECr
 +
 +
 +
 +
ICI
 +
 +
 +
 +
 +
— Sc — PSt
 +
 +
 +
 +
StF
 +
 +
 +
 +
29
 +
 +
 +
 +
StF
 +
 +
 +
 +
PSt
 +
 +
 +
 +
ICI
 +
 +
 +
 +
PSt
 +
 +
 +
'-EC
 +
 +
 +
 +
R2---
 +
 +
 +
 +
MSt
 +
 +
 +
 +
 +
— PSt
 +
 +
 +
 +
— MSt
 +
 +
 +
 +
X St
 +
 +
 +
 +
107
 +
 +
 +
 +
I
 +
 +
 +
 +
PLATE 9
 +
 +
 +
 +
EXPLANATION OF FIGURES
 +
 +
 +
 +
33 Vanellus custatus. One-third of incubation period. Lower view. Note slight attachment of ribs, but only sutural separation of sternum and coracoid. After Parker.
 +
 +
34 Echnidna histrix. Upper view of adult specimen. Drawn from a specimen in Washington University, Department of Zoology, and in part after Parker.
 +
 +
35 Shoulder-girdle of a marsupial embryo, Trichosurus. Scapulae, coracoids, and sternum are continuous parts as in the shark embryo. After Broom.
 +
 +
36 Anterior view of girdle in an 8.5-mm. Trichosurus embryo. Dotted portions are mesenchymatous. After Broom.
 +
 +
Ac, acromian Pro, pre-omosternum
 +
 +
CI, clavicle R 1 , first rib
 +
 +
Cr, coracoid R 2 , second rib
 +
 +
ECr, epicoracoid Sc, scapula
 +
 +
Gl, glenoid Sp, spine
 +
 +
ICl, interclavicle SSc, suprascapula
 +
 +
OSt, omosternum St, sternum
 +
 +
PCr, precoracoid XSt, xiphisternum
 +
 +
 +
 +
108
 +
 +
 +
 +
ONTOGENY AND PHYLOGENY OF THE STERNUM
 +
 +
FRANK BLAIR HANSON
 +
 +
 +
 +
PLATE 9
 +
 +
 +
 +
 +
CI
 +
 +
 +
 +
^ ICI
 +
 +
 +
 +
 +
 +
 +
109
 +
 +
 +
 +
PLATE 10
 +
 +
EXPLANATION OF FIGURES
 +
 +
37 Petrogale xanthopus, 3 inches long. Right scapula, outer view; sternum, inner view. After Parker.
 +
 +
38 Mus musculus, adult, inner view. Anterior end of sternum and medial end of clavicles, showing omosterna, and epicoracoids. After Parker.
 +
 +
39 Shoulder-girdle seen in a reconstruction of Dasyurus viverrinus. Front view of mammary fetus.
 +
 +
40 Sternum of adult Manatus americanus. Left half, inner view. After Parker.
 +
 +
41 Sternum of the embryo of the Dolphin. Inner view. After Parker. Ac, acromian PST, presternum
 +
 +
CI, clavicle R 1 , first rib
 +
 +
Cr, coracoid R 2 , second rib
 +
 +
ECr, epicoracoid R 3 , third rib
 +
 +
F, fontanelle R s , eighth rib
 +
 +
Gl, glenoid Sc, scapula
 +
 +
MSt, mesosternum SSc, suprascapula
 +
 +
OSt, omosternum XSt, xiphisternum
 +
 +
 +
 +
110
 +
 +
 +
 +
ONTOGENY AND PHYLOGENY OF THE STERNUM
 +
 +
FRANK BLAIR HANSON
 +
 +
 +
 +
PLATE 10
 +
 +
 +
 +
MSt "
 +
 +
 +
 +
 +
111
 +
 +
 +
 +
THE AMERICAN JOURNAL OF ANATOMY, VOL. 26, NO. 1
 +
 +
 +
 +
m
 +
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«  O
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 +
 +
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c/J
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o
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4
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113
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PLATE 12
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EXPLANATION OF FIGURES
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43 Girdle of Hexanchus. Pre- and post-omosterna. After Parker.
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44 Diagrammatic transverse section through shoulder-girdle of adult frog.
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45 Section through edges of shoulder-girdle and sternum of adult Lissotriton punctatus. After Parker.
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46 Diagrammatic transverse section of arch in the Australian lizard, Trachydosaurus rugosus.
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47 Diagrammatic transverse section of Echnidna histrix, from a mounted specimen in Washington University.
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48 Anterior view of cartilaginous girdle of petrogale, 21 mm. in length. The different parts of the girdle are outlined, but the whole is a continuous piece of cartilage. After Broom.
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49 Transverse section of mouse embryo, 7.75 mm. long. Washington University School of Medicine series no. 102, slide 4, section 16. X 38.
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Ac, acromian PrOmSt, pre-omosternum
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Cr, coracoid PtOmSt, post-omosternum
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ECr, epicoracoid Sc, scapula
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Gl, glenoid SSc, suprascapula
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Hu, humerus St, sternum
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114
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ONTOGENY AND PHYLOGENY OF THE STERNUM
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FRANK BLAIR HANSON
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PLATE 12
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4 5
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46
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ECr
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115
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Resumen por el autor, George W. Corner Universidad de California.
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Sobre el origen del cuerpo amarillo de la cerda a expensas de la
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granulosa y teca interna.
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El ovario de la cerda presenta algunas ventajas para la solucion de este problema. El presente trabajo esta basado en la inspecci6n de una larga serie en la cual se determin6 el estado del ciclo reproductor por la observaci6n de los animales vivos y sus 6vulos. Los resultados obtenidos pueden resumirse del siguiente modo: En la cerda la membrana granulosa persiste intacta despues de la ruptura del foliculo de Graaf . Sus celulas aumentan de tamano sin dividirse; su citoplasma se carga de substancias lipoides y finalmente se transforman en los grandes elementos del cuerpo amarillo completamente formado, llamados comunmente "celulas luteinicas." Los capilares sanguineos procedentes de la teca interna invaden la membrana granulosa ramificandose para formar un extenso plexo vascular en la nueva estructura. Las grandes celulas cargadas de lipoides, presentes en la teca interna, aumentan en numero a consecuencia de divisiones mitdsicas perdiendo muchas o la mayor parte de sus inclusiones grasas y pasando al cuerpo amarillo, alojandose entre las celulas de la granulosa, en toda la extensi6n de esta ultima. No hay pruebas de la transformacidn de la celulas de la teca interna en fibroblastos de tipo fusiforme corriente ni tampoco de su participaci6n en la formacidn de las fibrillas del reticulo de mallas angostas que existe en el cuerpo amarillo. El autor ha encontrado pruebas sobre la persistencia de algunas de las celulas de la teca interna durante la prefiez, las cuales forman elementos bien patentes en el cuerpo amarillo; pero su destino no puede reconocerse por los metodos actuales, a causa de la semejanza entre algunos de los derivados de la teca y la granulosa.
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Translation by ,Jos6 F. Nonidez Carnegie Institution of Washington
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AUTHOR S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, AUGUST 11,
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ON THE ORIGIN OF THE CORPUS LUTEUM OF THE
 +
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SOW FROM BOTH GRANULOSA AND
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THECA INTERNA
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GEORGE W. CORNER
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From the Anatomical Laboratory, University of California
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CONTENTS
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Introduction 117
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Recent investigations 122
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Previous work on the corpus luteum of the sow 127
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Material and methods 131
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Special cytology of the lutein cells of the sow 137
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The mature follicle 140
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The freshly ruptured follicle 148
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Invasion of the granulosa 157
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The fully formed corpus luteum, until the termination of pregnancy 168
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Retrogression of the corpus luteum 177
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Discussion 178
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Conclusions 180
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INTRODUCTION
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The history of the discussion, now of more than seventy years' standing, as to the origin of the corpus luteum, has been repeated so many times that it has become traditional, and the names of von Baer and Bischoff have been passed down to us as the original proponents of the two chief doctrines in question. It is said that the former, in his monograph "De ovi mammalium genesi" ('27) first stated that the corpus luteum is derived from the theca interna of the Graafian follicle, and that Bischoff first discarded this view in favor of the membrana granulosa as the site of origin. I have not been able to see von Baer's work, but judging at least from Bischoff's account of the early embryology of the rabbit ('42), there was no such clear-cut opposition of view as tradition declares, for Bischoff considered himself, rather, as an upholder of von Baer (and was so quoted by contemporary investigators).
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117
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118 GEORGE W. CORNER
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It must be remembered that the first of these monographs appeared a decade before Schleiden and Schwann's enunciation of the cell theory, and the other not five years after; histology was studied with pincettes and the needle rather than by sections, and the first nuclear stain was not discovered. The layers of the follicle were as yet imperfectly differentiated, and the early descriptions are so vague that it is difficult to interpret them in present-day terms. New steps toward the solution of this problem have always followed fast upon the development of histological technique, and thus it is in the writings of Wilhelm His ('65) and Waldeyer (70) that we first find opinions and descriptions approaching those of recent years.
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The studies of His led to the complete formulation of the view that the corpus luteum is derived from the theca interna of the Graafian follicle, which in the next two decades was supported by a number of investigators and still holds a place in the field against strong opposition. The chief arguments in favor of this view are that, first, the membrana granulosa of large follicles is often degenerated, and is believed to be cast off at the time of rupture; second, as the Graafian follicle ripens, the cells of the theca interna show marked changes — they swell in volume, become rounded, in some species they acquire granules of a yellowish pigment, and in short come to present a striking resemblance to the large cells of the corpus luteum; third, this resemblance is enhanced by the fact that not only are the large cells of the theca interna folliculi and the corpus luteum similar, but the presence of many blood-capillaries and connective-tissue cells causes a resemblance as well in the general structure of the two tissues; and, fourth, such follicles as do not rupture lose their granulosa by degeneration, become obliterated by proliferation of the theca interna, and in this process of atresia attain also a resemblance to the corpus luteum. .
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None of the contributions disagreeing with this view in favor of the granulosa origin of the lutein cells were at all convincing, until the appearance in 1895 and 1896 of Sobotta's first researches, which mark the beginning of modern work upon the question. Here again the chief contribution was one of method. Sobotta
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ORIGIN OF THE CORPUS LUTEUM 119
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pointed out that the arguments quoted above are based merely upon analogies between the layers of the follicle and the fully formed corpus luteum, and that from the writings of his predecessors it is apparent that few had actually seen corpora lutea in process of formation. Even when descriptions are given of mature follicles or supposed early corpora lutea, there is usually no proof that the structures in question actually represent the results of normal follicular development or recent ovulation. The problem should be worked out from a series of specimens gathered at known periods after rupture of the follicle; and in order to avoid confusion with atresia or other irrelevant processes, each follicle or corpus luteum studied should be certified as to its normal condition and stage of development by comparison with the fertilized ovum or embryos proceeding therefrom. To fulfill these high requirements calls for long and tedious labors — the investigator must spend hours and days in observation of his animals ; the reproductive cycle of the species used must be known well enough to acquaint him with the time of ovulation, the animals must be killed at definite times thereafter, and the ova must then be sought in the ovary, the oviducts, or the uterus. Sobotta himself chose the mouse, in which he had found that an ovulation takes place about twenty-one days after the birth of a litter, and in which the small size of the animal permits serial sectioning of the entire ovaries and Fallopian tubes. It must be admitted that his own postulates could not be followed to the full; the individual corpus luteum corresponding to a given ovum cannot be identified, because many ova are extruded at one ovulation in this species; the exact time of ovulation may vary by hours, and again there is so much variation of the interval between ovulation and the entrance of the spermatozoon into the egg that the condition of the ovum cannot be used as an exact measure of the age of the corpus luteum. Study of the ova merely provides assurance that the corpora lutea are normal and gives a rough means of determining their ages. The judgment of the investigator must finally be used to rank the corpora lutea in an orderly series. It is beyond denial, however, that Sobotta possessed such a series, collected from about 200 mice and based upon the
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120 GEORGE W. CORNER
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study of nearly 1500 ova during the stages of maturation, fertilization, and the segmentation of the blastomeres. He found no degeneration of the membrana granulosa; instead the cells of this layer remain and undergo hypertrophy (without division), finally becoming the characteristic large cells of the corpus luteum. Meanwhile the cells of the theca interna undergo mitotic division, are converted into spindle-cells and invade the granulosa to form the connective- tissue reticulum of the corpus. In this process all the theca interna cells are used up, and the layer therefore disappears. Capillary blood-vessels grow in from the vessels of the theca interna, ultimately providing the corpus luteum with a rich circulation.
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There will be no need to enter here upon a detailed account of the debate which began immediately upon the publication of these epoch-making studies. A full analysis of the literature upon the origin of the corpus luteum up to 1901 will be found in the papers just quoted and in the two reviews of Sobotta in the Ergebnisse der Anatomie ('99a, '02). In 1897 Sobotta himself studied corpus luteum formation in the rabbit, and found the process in all important points exactly as in the mouse. A year later, Stratz ('98) printed a research upon which he had been engaged before the publication of Sobotta's work, in which he had followed the early stages of the corpus luteum on ovaries of an insectivore, Tupaia javanica, the lemuroid ape Tarsius spectrum (the specimens being those of Hubrecht's well-known Javanese collections,) and of the 'Spitzmaus' or shrew, Sorex vulgaris. Although he did not have large numbers of cases, all were checked by the study of the ova or embryos. As far as the persistence of the granulosa cells and their direct conversion into the lutein cells was concerned, Stratz agreed fully with Sobotta, but with regard to the fate of the theca interna there is a minor difference. If I understand Stratz correctly, he considers the theca interna of the mature follicle merely as a zone of blood-vessels, in which all the cells are either constituents of the vascular wall or of the adventitia. Considered in this light, it is easy to see how this angioma-like thecal tissue would enter into the growing corpus luteum to form its blood-vessels and its connective tissue without
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ORIGIN OF THE CORPUS LUTEUM 121
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the necessity of transformation or regression of the specialized theca cells into fibroblasts, as described by Sobotta. So far as is known to me, no subsequent investigator has confirmed the view of Stratz, all others being agreed that the theca interna consists of a distinct layer of highly specialized cells derived from the mesenchymatous elements of the ovarian stroma, and containing a network of blood-vessels, supported by cells and fibrils of connective tissue.
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A third theory as to the fate of the theca interna is proposed in the important papers of 0. Van der Stricht, of which the first appeared in 1901. The study was carried out upon the ovaries of large numbers of European bats, chiefly Vesperugo noctula. As with the two previously cited investigations, the animals were collected primarily for the study of the ova and the early embryos of the species used, and the series is therefore accurately controlled by the condition of the ova. Van der Stricht demonstrates beyond doubt that in these species the granulosa layer persists in situ after rupture of the follicle, and that its cells enlarge, acquire granules of lipoids staining black with osmium tetroxide, and finally become the typical lutein cells. Contrary to Sobotta, he thinks that mitotic division may occasionally occur in these cells, so that the filling of the follicular cavity is brought about by a slight increase in their number as well as by the vast increase in their individual bulk. The connective tissue of the corpus luteum arises chiefly as Sobotta described it in the mouse. After rupture of the follicle the membrana propria disappears, and fibroblasts invade the metamorphosing granulosa layer. Van der Stricht thinks that these are the spindle-cells of the theca interna or their descendants. Of the distinctive cells of the theca, some seem to disappear, but others remain, chiefly about the periphery of the new corpus luteum, or enter a short distance into the granulosa, and here they remain almost in their original condition. After a few days, however, when the deposition of fatty droplets in the granulosa cells has progressed, the two types of cells so closely resemble each other that Van der Stricht could no longer distinguish them in his Flemming-fixed tissue. He believes, in fact, that they have become identical, and therefore
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122 GEORGE W. CORNER
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that in the fully formed corpus luteum most of the lutein cells are of granulosa origin; a few of them, however, are from the theca interna.
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A nearly identical theory is that proposed by Rabl ('98), who studied human corpora lutea (the youngest estimated at ten days), and found in them about the periphery a layer of cells differing from the rest of the lutein tissue; this, he suggested, might be the theca interna, which he supposed to persist in its original position until its cells were lost to view, some by becoming converted into lutein cells, others by degenerating.
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During these five years from 1896 to 1901 there was no lack of publications restating the total loss of the granulosa before rupture, in opposition to the descriptions of Stratz, Sobotta, and Van der Stricht. A few of these investigations were carried out upon the ovaries of swine, and will therefore be discussed more fully later in this paper. As Sobotta pointed out in his resume of 1902, not one writer among those who taught the non-participation of the granulosa in corpus luteum formation had been able to prove that his specimens were normal mature follicles and corpora lutea by presenting the ova which had proceeded therefrom.
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RECENT INVESTIGATIONS
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From 1901 to 1917 there have been about thirty-five more publications upon the question, of which some twenty-five represent actual original investigations. As the subject has not been brought up to date in any publication in English, it may be as well to take up in some detail the work of the past sixteen years, especially as the old differences of opinion still persist.
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 +
Jankowski ('04) reports studies upon a series of ovaries of sows and guinea-pigs, collected without an attempt to learn the reproductive cycle of the animals or to test the normal conditions of the specimens according to the postulates of Sobotta (which he says he had found impracticable to apply and whose value he questions). He believes the granulosa to be intact until the rupture of the follicle and even afterward, but to degenerate before the ingrowth of the theca interna, to which he ascribes the
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ORIGIN OF THE CORPUS LUTEUM 123
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origin of the lutein tissue. The value of his results with the pig will be discussed below; Sobotta has presented directly opposite evidence and a vigorous criticism with regard to his work on the guinea-pig ('07).
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The contributions of Pottet ('10) on the human corpus luteum and Delestre ('10) on that of the cow are based on evidence which can hardly be considered conclusive. Delestre had no bovine corpora lutea of pregnancy at an earlier stage than two and a half months. He had twelve corpora lutea from non-pregnant animals, four of which he thought to be in the first stages of formation, but there was no effort, by observing the animals alive or by searching for the ova, to determine that ovulation had actually been recent. Pottet studied twenty-two human corpora lutea of pregnancy, the youngest already six weeks old. Both of these authors speak for the degeneration of the granulosa before rupture.
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We cannot judge the work upon the human ovary very critically until the relation of ovulation to menstruation is better known or some other method of estimating the age of young human corpora lutea and of obtaining really young specimens is at hand. Buhler ('00) collected ovaries of rabbits according to Sobotta's methods, but found the distinction between theca and granulosa so difficult that he turned to the human corpus luteum. The only specimen of importance described by him is one from an operative case, without menstrual history or other means of estimating its age, except that it showed a point of rupture in process of healing (see below, p. 179, as to the possibility of error on this point) . In this supposedly early corpus luteum the granulosa is degenerating and a 'typical lutein tissue' appearing in the place of the theca interna. Cristalli ('03), a pupil of Paladino, whose peculiar views will be quoted below, believes also in the total degeneration of the granulosa layer before rupture, but gives no data as to his specimens. Teacher, in discussing the TeacherBryce-Kerr case of early ovarian pregnancy ('08), states that he had been studying corpus luteum formation in the human, and interprets his preparations to indicate quite clearly that "whatever the source of the cells (of the corpus luteum) may be in the
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124 GEORGE W. CORNER
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lower animals, they do not in man arise from the membrana granulosa," which latter membrane he thinks is probably shed with the ovum. Hegar, in 1910, reported an examination of six human ovaries removed four and two days before the onset of menstruation, all containing corpora lutea which he supposes to be fairly young. While admitting the epithelial origin of the structure in mammals lower than man, he is inclined to view his preparations as indicating a thecal origin of the lutein cells in the human species. J. Whitridge Williams, whose text-book of obstetrics ('03-' 17) is based to so great an extent upon original study that it is regularly quoted in scientific literature as authoritative, retains in his last edition the views of the preceding writers, of whose correctness he feels convinced by the study of several hundred corpora lutea.
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This completes the list of recent authors who see in the corpus luteum a structure of connective-tissue origin alone. All other investigators of the past sixteen years uphold in general the epithelial origin of the lutein cells, but among themselves they vary according to their views as to the fate of the theca interna. The rabbit has been studied in 1897 by Sobotta, who found the process exactly as in the mouse, but Honore, three years later, in the same animal, found that not all the theca interna cells are converted into fibroblasts, but that some of them linger about the periphery of even the fully formed corpus luteum. Cohn, in 1903, repeated the work, apparently without study of the ova, but dating his specimens from an observed copulation, (in the rabbit ovulation occurs only after coitus), and confirmed the results of Sobotta. Marshall, in the next year, described the corpus luteum of the sheep, dating his specimens from observed copulation. He did not seek the ova, but as in this species ovulation and coitus can occur at no time except during a short oestral period, so that coitus dates the time of ovulation within a very few hours, the presumption is great that Marshall possessed normal corpora lutea of ages accurately known. He found the granulosa to persist and to be vascularized by sprouts from the theca interna, all the cells of which were finally used up, having been converted into spindle-cells of connective tissue. A part of
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ORIGIN OF THE CORPUS LUTEUM 125
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the connective tissue of the corpus is contributed also by the theca externa, which is drawn inward in places by the folding of the follicular walls. O'Donoghue ('12, '14, '16) has given a confirmation of Sobotta's views for the marsupials (which I believe were first studied by Sandes ('03), whose paper was not accessible to me). Strakosch ('15) is the last to repeat the Sobotta theory in its original purity, basing his statements upon the human ovaries which were used by Robert Schroeder in his study of the time relation between ovulation and menstruation ('14).
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Van der Stricht's belief that the theca interna cells are not converted into fibroblasts, but remain in the corpus luteum, no longer distinguishable from other lutein cells, found support in the study of L. Loeb ('06) upon the guinea-pig. The specimens were collected in a series dated from copulation without study of the ova. The theca interna cells, after a few hours, could no longer be distinguished from the granulosa lutein cells. This work is open to the criticism that haematoxylin and eosin (the only staining combination used) do not accentuate differences between cells of the types met with in this problem. In 1908 Van der Stricht himself repeated his ideas as the result of researches upon the ovaries of dogs, carefully checked up by examination of the ova, and in 1912 he repeated his findings in the bat.
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A somewhat different view has found exposition in the very careful work of Volker ('05) upon Spermophilus citellus, a European marmot allied to the gophers of the western United States. The ova and embryos were recovered and examined in all cases, and there appears to have been a sufficient number of stages, though the author does not state the number of specimens studied. The theca interna cells were found to persist unchanged between the granulosa and the theca externa, even until the end of pregnancy. The few spindle-cells found in the fully formed corpus luteum are said to proceed from the theca externa. Practically the same view is presented in the thesis of Niskoubina ('09), who worked on the rabbit, but does not give an account of the methods used. Cohn ('09) studied the human ovary, but appears to have seen no really young stages. His descriptions agree with
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126 GEORGE W. CORNER
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those of Rabl, and he thinks the layer of theca cells is not destined to persist, but is a 'matrix' or source of origin for the newly forming connective tissue of the corpus luteum.
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With this group should be placed one of the most ambitious of the recent attempts to work out the origin of the human corpus luteum, that of R. Meyer ('11 a). The paper describes five corpora lutea in process of formation, of which one is claimed by the author to be the youngest ever obtained in the human. The appearance of the structures and the menstrual histories were the only guides to their age. The specimens show first a proliferative stage, during which the granulosa cells swell and acquire granules of a fatty substance, and, second, a stage of 'glandular metamorphosis' through vascularization of the granulosa layer. The first spindle-cells seen in the lutein layer arise from the bloodvessels, which are sprouting inward. The wall is thrown into folds, in which the larger fat-infiltrated theca interna cells are crowded. Here they remain until the pressure of the swelling lutein tissue crushes them out of existence, an event which may be early or late according to the internal conditions of pressure. Groups of them, serving as sources of nutrition for the growing organ, may be seen about the periphery of the corpus luteum and in the folds of its wall, until fairly late in the life of the corpus luteum. The name theca-lutein cells has been given them.
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As an example of the difficulty of proving anything about the origin of the corpus luteum by specimens 'whose age can only be guessed, it may be mentioned that the genuineness of Meyer's first and supposedly youngest corpus luteum has been sharply attacked. Ricker and Dahlmann ('12) have hinted that it is not even a naturally ruptured follicle, and J. W. Miller ('11) believed it was an atretic follicle, because Meyer had stated the granulosa cells to contain 'Fett,' while, according to Miller, neutral fat is never found in the normal fresh corpus luteum. It must be admitted that this criticism was rescinded when Meyer ('lib) stated that 'Fett' meant merely lipoids in general, and that Miller is himself no opponent of Meyer's views. But after all we shall never be certain of the early human corpus luteum until skill and good fortune enable someone to obtain the tubal ovum with the ovary.
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ORIGIN OF THE CORPUS LUTEUM 127
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Four recent writers upon the human ovary have repeated the same views as Meyer with but slight modifications. Elizabeth Wolz ('12), from the study of a few specimens, believes that none of the theca interna cells suffer change into connective tissue. Some degenerate by atrophy, others remain in situ a long time. Timofeiev ('13) l and Wallart ('14) appear to have given as accurate and modern a description as is possible in the face of the particular difficulties of the human material. Careful menstrual histories are given, and both used varied and interesting histological methods. According to both, the theca interna cells remain in groups about the periphery, of the corpus luteum for a long time, as described by Meyer, and they slowly atrophy. None of them are converted into spindle-shaped connective-tissue cells. Timofeiev describes also the deposition of lipoid bodies in the granulosa lutein cells during the first days of the new corpus luteum. Lastly, Novak ('16) reports five early corpora similar to those of Meyer, whose conclusions he follows.
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PREVIOUS WORK ON THE CORPUS LUTEUM OF THE SOW
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It is said that von Baer's celebrated monograph ('27) announcing the discovery of the mammalian ovum, contains a description of the early corpus luteum of the sow. The first account of the histological development in swine which has come into my hands, however, is that of Zwicky ('44), entitled "De corporum luteorum origine." Zwicky was a medical student who was set to work by the distinguished Henle to study the formation of connective tissue in fibrin clots, Henle being under the mistaken impression that the corpus luteum represented the conversion of the clotted follicular haemorrhage into scar tissue. The student was acute enough to correct his master's error, even though he found haemorrhage into the follicle in two-thirds of the early corpora lutea of swine. As a result of his studies, he announced himself as on the side of von Baer and Bischoff in favor of the granulosa
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1 This Russian dissertation seems to me the best contribution to the origin of the human corpus luteum yet presented. As it was abstracted for me by a Russian-speaking scientific colleague, not especially acquainted with histological methods, I quote it with some slight hesitation, but I believe our interpretation is correct.
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THE AMERICAN JOURNAL OF ANATOMY, VOL. 26, NO. 1
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128 GEORGE W. CORNER
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origin of the corpus luteum, but his description shows that he Mas probably including the theca interna as part of the granulosa. The little dissertation must remain as a not uninteresting example of the effect produced upon the work of an active student by the recently announced cellular theories, rather than as an important contribution to its subject.
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The next to use the sow for study was Paladino (79, '80, '81, '87), who collected about 500 corpora lutea of 100 sows. His extensive papers propose a peculiar theory of his own, namely, that the entire granulosa is lost before rupture of the follicle, and that the theca externa, carrying blood-vessels, proliferates inward, to form the corpus luteum tissue, pushing the theca interna before it to form a central connective-tissue core. In 1900 and 1905 he repeated his views of twenty years before in criticism of Sobotta, who pointed out in return that Paladino 's writings contain no evidence at all, in text or plates, that he had ever seen developing stages of the corpus luteum; a just criticism, as study of the originals has convinced me.
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Far different is the work of Benckiser ('84), who has given a very careful description of a small series of stages. He had, without doubt, normal and mature Graafian follicles just prior to rupture. These contained the membrana granulosa intact. In his recently collapsed follicles, containing large clots, the granulosa had been torn off in places; when there was no hemorrhage, this layer remained very largely in situ. His next stage is much further developed, its wall showing only one homogeneous layer, and the gap was bridged by the assumption that the granulosa had degenerated during the interval. The account is clearly written, its author was describing what we can now state to be normal specimens, and it was only lack of sufficient intermediate stages that led him into an error of interpretation.
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One of the most frequently quoted works is that of Clark ('98), whose material consists of ovaries collected at random in the slaughterhouse. It is said that among the sows used by the butchers there were many undergoing oestrus, but it is not stated that any of those ovaries used in the research were known to be from the animals in heat. Clark gives a good description of the
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ORIGIN OF THE CORPUS LUTEUM 129
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immature follicle at growing stages. For study of the mature follicle he selected large follicles, without discovering whether or not they contained normal maturing ova. From these large follicles all the granulosa cells had disappeared. The author was willing to consider the possibility that they might be atretic, but inclined to rate them as normal because they seemed logical predecessors of his next stage. Much is made of the fact that the theca interna cells of ripening follicles contain granules of yellowish fat, which are taken to be the 'lutein' already present in the future lutein cells before rupture. This assumption rather overreaches itself, as one glance at fresh corpus luteum tissue of the sow will show that there is no microscopic yellow pigment present in the so-called lutein cells. In the one pair of ovaries next described, some follicles were ruptured, others were not. In the latter, the granulosa was no longer visible, except for a few cells lying in the cavity. The theca interna was thickened and closely resembled lutein tissue. In a later stage there was a central cavity rimmed by connective tissue, supposed to represent the membrana propria pushed before the thickening theca interna. Sobotta ('99 b) has given a vigorous criticism of Clark's specimens, explaining his so-called mature and just-ruptured follicles as cases of atresia. Volker ('05) has also pointed out what he considered errors of interpretation of the specimens.
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However, Doering ('99) came to the defense of Clark, also using material collected at random. He states that the wall of a recently ruptured follicle shows no granulosa. His principal evidence, however, is from one corpus luteum of the sow, which shows, near the center of the section he figures, a flattened circle of granulosa cells. This he interprets as the granulosa of the same follicle in which the corpus luteum formed, which had been pushed inward by the proliferating theca interna, and which for some reason had not degenerated. I have seen very much the same appearance when a young growing follicle had crowded itself into the side of an old corpus luteum, so that a tangential section appeared to show a follicle within a corpus luteum.
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Jankowski ('04), using the same method of collection, would seem to have had normal mature follicles of swine; at any rate,
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130 GEORGE W. CORNER
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the granulosa was intact. The ova were not seen. His veryearly corpora lutea, showing stigmata at the point of rupture, also contain the granulosa in situ and completely preserved, except that the cells are swollen, irregular in form, and contain vacuoles. The theca interna cells are large, contain lipoid granules, and resemble lutein cells. No specimens between this and the solid corpus luteum are presented. Upon such evidence he confirms Clark's account.
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Kopsch ('01) demonstrated at a meeting of the Anatomische Gesellschaft certain preparations by Menzer of the corpora lutea of swine three, six, and ten days after copulation. This contribution appeared by title only, and our sole information as to its nature is the statement of Sobotta that Menzer's specimens are in general agreement with his own views.
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It is fair to say that the theory of the origin of the corpus luteum from the theca alone, though it still holds a place in current literature, has no good evidence in its favor. Every investigator whose methods assure us that accurately dated specimens of a sufficient number of stages were in his hands has declared the persistence of the granulosa cells and their transformation directly, with little or no mitotic division, into the characteristic large 'lutein cells' of the corpus luteum. The problem has shifted during the sixteen years whose progress I have reviewed; the present point of interest is as to the fate of the theca interna. Are its cells all converted into connective tissue; do they persist as a special peripheral layer of the corpus luteum; do they assume an impenetrably close resemblance to the granulosa lutein cells; or can we find some other and clearer explanation of the problem of their disappearance?
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The following pages contain the results of an attempt to answer these questions. Choice of the species to be studied was influenced by several reasons. There is a frequently expressed idea that perhaps the larger and smaller animals differ in the formation of their corpora lutea, as they do in many other features of their reproductive cycles; the sow is large and has an ovulation cycle not unlike the human species. Other considerations in
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ORIGIN OF THE CORPUS LUTEUM 13]
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elude previous experience of the author with the ovaries of swine; certain presumed histological advantages of the species, to be explained later, and, above all, the fact that the previous work on swine has been much quoted by writers in confirmation of the thecal-origin theory. Here, if anywhere, the application of modern methods of research should settle the old difference once for all.
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MATERIAL AND METHODS
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As it has been pointed out that the only hope of trustworthy results in this problem depends upon the possession of an unbroken series of normal specimens of known ages, a description of the material in the author's hands and the methods of obtaining it will be given in detail. The first step was a preliminary investigation to obtain exact knowledge of the period in the reproductive cycle at which the ova are shed from the ovary in order that mature follicles and very early corpora lutea might be obtained. The results of this study have already been published (Corner and Amsbaugh, '17) and will not be repeated in detail here. We were able to confirm the current supposition that in swine ovulation is coincident with the oestral period, and by this fact we are at once provided with the means of obtaining the desired stages of corpus luteum formation.
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The females of the wild swine of Europe are monoestrous, according to Kaeppeli ('08), having but one period of heat in the year; but under domestication the sow becomes polyoestrous, coming in heat at intervals of two to four weeks, usually about every twenty-one days, as all breeders agree. The period of heat commonly lasts three days and is characterized by sexual excitement and in some individuals by swelling, reddening, and slight eversion of the vulva, or even at times by a serous, mucous, or partially sanguineous discharge from the genital orifice. If a boar be present, the sexual excitement is made apparent by ready acceptance of coitus (commonly on the second or third day of oestrus) ; if none but females are in the pen, the sow in heat will be seen to sniff at the genitals of her neighbors and 'ride' them in imitation of coitus. Frequently the sow is the recipient
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132 GEORGE W. CORNER
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rather than the donor of these attentions. The period is not terminated by coitus, but continues until the end of three days.
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For the purpose of the present investigation, the condition of oestrus was observed while the animals were alive in the yards of the packinghouse. The sows were marked, and on the day of killing they were traced through the processes of the abattoir and the internal genitalia received from the hands of the eviscerator. The Fallopian tubes were then removed by cutting across the upper portion of the uterine horns, were carried to the laboratory in 0.7 per cent saline solution, and there washed out by inflating them with salt solution through a slit in the wall near the fimbriated extremity. After inflation with the fluid, the tubes were gently 'milked' into a Syracuse dish, and the washings examined with the dissecting microscope. This simple and almost infallible method of finding the ova was suggested to us by Professor Evans as an improvement upon Martin Barry's practice of milking the tube without injected fluid ('39). As we have subsequently found, it had been used by Sobotta (in the rabbit) and no doubt by others as well.
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We found that ovulation occurs on the first or second day of oestrus, and that the stimulus of copulation is not necessary to cause rupture of the follicles. The ovaries of all sows killed during heat contain mature Graafian follicles ready to rupture or just ruptured, in which latter case the ova are in the tubes and may be recovered therefrom for study by the method described below. Little or nothing has been known of the mature ovum of the sow, and we have found no record of any previous observation of the unsegmented ovum from the tube. We measured fourteen fresh tubal ova from nine sows and found the diameter, including the zona pellucida, to vary from 155^ to 165m, the zone being about 10^ in thickness. The ova are plainly visible to the naked eye if placed against a strong light. We have not noticed a radial striation of the zona pellucida either in fresh or fixed ova. The ovum is filled with yolk granules of varying sizes, usually about 3m to 5m in diameter, which are so numerous and so retractile that they quite conceal the nucleus.
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ORIGIN OF THE CORPUS LUTEUM 133
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The author has presented ('17 c) a brief study of the maturation of the pig's ovum, based upon some of these specimens, which indicate that the sequence of events is the same as in other mammals. The first polar body and the second polar spindle are formed in the ovary just before rupture. After the entrance of the spermatozoon, which occurs in the tube, the second spindle completes its division, and the presence of two polar bodies is therefore a sign of fertilization. If the ova are not fertilized, they degenerate in the tube with the second spindle undivided. Just how long they survive is not known, but by analogy with the smaller and better-known mammals, we may assume that after three or four days they are no longer capable of segmentation; the degenerating ova may be found in the tubes a few days longer. It is said that pregnancy is more likely to result when the sow is served on the second day of oestrus. The number of ova extruded at one ovulation, and consequently the number of fresh corpora lutea in one animal, may be quite large. One prolific sow is known to have given birth to twenty-three pigs in one litter. However, in the mixed stock, not especially adapted for breeding, which is found in the abattoirs, small litters are the rule. Records of 128 sows raised in Maryland, presented in my paper of 1915, show that the corpora lutea of pregnancy in both ovaries numbered one to sixteen, averaging eight, and that the number of foetuses in the uteri of the same sows varied from one to ten, averaging six. Failure of fertilization, abortion, and resorption of embryos dying in utero account for the fact that not all the eggs of one ovulation proceed to full development.
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About 133 embryos of the sow younger than two weeks, taken from twenty-three sows, have been observed and described in the literature. The youngest of all are the three ova found in three different sows by the present writer and Amsbaugh ('17), in which conjugation of the pronuclei had not occurred. It was not possible to know the exact time of insemination in these animals, but in one case it is believed that the animal had not been in heat, and consequently had not copulated, more than forty hours before killing. R. Assheton ('99) studied about 100 specimens during the first ten days, the youngest stage being that of two blastomeres. It
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134 GEORGE W. CORNER
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would seem that fertilization may occur about the end of the first day or may be postponed until two or three days after copulation — a conclusion which he draws from finding embryos of the same stage in two sows killed on the fourth, fifth, and sixth day post coitum. Likewise, embryos in the same uterus may vary rather markedly as to their state of development, for instance, one uterus contained ova of two segments, of nine segments, and completed morulae. For this reason it is possible to give only an approximate time schedule of early development. The ova pass down the tube rapidly and enter the uterus about the fourth day post coitum. Assheton did not find any stage further advanced than four blastomeres in the Fallopian tubes. (A specimen found by the present writer and Mr. Felix H. Hurni contained ova of two, four, and six blastomeres, all in the tube.) Assheton found that various sows killed on the sixth day presented uterine embyros from the stage of six blastomeres to fairly well-developed blastodermic vesicles. By the seventh day the zona pellucida has usually disappeared and the inner cell mass of the early vesicle has differentiated into two layers, the epiblast and the hypoblast. By the twelfth day the great elongation of the blastodermic vesicle which is so characteristic of the pig, is well under way and the vesicle is already 10 to 12 mm. long. By the fourteenth day each vesicle may measure 20 cm. ; in the embryonic area the primitive streak is well developed and there are from one to three somites. In addition to Assheton's studies, thirty embryos of the ninth, tenth and eleventh days have been described by Weysse ('94), and from the fourteenth day to about the twenty-fifth we have the accurate tables of Keibel ('97). For older (foetal) stages, no good age-length ratios have been determined. The period of gestation is usually 116 to 120 days. It is stated that sows undergo oestrus and may become pregnant again five weeks after littering.
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During the progress of this investigation the ovaries and uteri of several thousand sows have been examined macroscopically, and the corpora lutea of about 300 have been studied under the microscope. The permanent preparations upon which the following description is based comprise sections from the Graafian
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ORIGIN OF THE CORPUS LUTEUM 135
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follicles and corpora lutea of 171 sows of which there are records sufficient to determine the stage of the reproductive cycle. In 162 of them the ova, developing embryos, or foetuses were examined and recorded.
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Twenty-four were killed during the oestral period or within the first week after the onset of heat. Some of the tubal ova found were unfertilized, others were fertilized and were in stages from the one-celled to the six-celled embryo. Five of the twentyfour sows mentioned were obtained before a method of discovering the ova had been acquired, and the ova were therefore not sought, but as the dates of copulation were noted at the University of California Farm by Professor Thompson, it seems proper to include them, since their corpora lutea agree with the others in structure.
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Six sows were taken in the second week after ovulation. As the ova were unfertilized, they had degenerated, and were not found, except shriveled eggs in two of the sows. It chanced that none of those sows which had copulated were killed during this period, and thus the opportunity to obtain embryos of the second week did not fall to my lot.
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Fifteen contained embryos of the third week, from five to thirty-nine somites. The ovaries of the eleven youngest of these were given me by Prof. F. R. Sabin; some of the embryos to which they were related are described and pictured in her recent contribution to the early vasculogenesis of the pig (Carnegie Institution of Washington, Contributions to Embryology, No. 18, 1917).
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One hundred and twenty-four compose a complete series from animals containing embryos of the fourth week to the end of pregnancy, the embryos or foetuses being measured in each case. Two were obtained from sows which had littered seven and ten days before killing, respectively. Most of the older corpora lutea of pregnancy were prepared in the Anatomical Laboratory of Johns Hopkins University, and formed part of the material for my previous monograph ('15). They have been restudied in the light of the results gained from the specimens of the first fourteen days after ovulation, all of which were obtained in California.
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136 GEORGE W. CORNER
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The collection of this material would have been impossible without the special and unusual cooperation which has been extended to this laboratory by the Western Meat Company of San Francisco. I refer to their donation of permanent laboratory quarters in their West Berkeley plant (Oakland Meat and Packing Company) to the Anatomical Laboratory of this institution. I owe especial thanks to Mr. J. 0. Snyder, general superintendent of the Western Meat Company, and to Mr. Ralston B. Brown, superintendent of the Oakland Meat and Packing Company, and to many other members of the staffs and employes of both these establishments; to Dr. H. H. Hicks, U. S. Supervising Inspector, Dr. G. R. Ward, and other members of the U. S. Inspection Service at the South San Francisco plant, and to Dr. Thomas Presst, of the California State Inspection Service. To Mr. R. B. Brown in particular I owe the opportunity of observing living animals and of obtaining their pelvic organs, often at the cost, I fear, of some inconvenience to the routine of his plant. The permanent laboratory space provided by him at the packinghouse has been invaluable during the prosecution of this work. I am further indebted to Professors Evans and Sabin for the contribution of ovaries with the corresponding early embryos; to Prof. J. I. Thompson, of the Department of Agriculture of the University of California, for observing and marking five animals, and to Messrs. A. E. Amsbaugh and Felix H. Hurni for assistance in the collection and preparation.
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In general the younger specimens were fixed in Bouin's fluid, the older in 10 per cent formol, these fluids being selected to secure the advantage of fixation in slow aqueous coagulants, as will be explained in the next section; small pieces of many ovaries were placed in osmium tetroxide for study of the lipoids. Blocks were imbedded in paraffin and celloidin. The chief stains used with the specimens herein described comprised haematoxylin and eosin, Heidenhain's iron haematoxylin, Mallory's triple connective-tissue stain, Van Gieson's mixture, and several lipoid-soluble dyes (Nile-blue sulphate, Sudan III, Scharlach R), besides many special procedures applied to fresh and fixed tissues.
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ORIGIN OF THE CORPUS LUTEUM 137
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SPECIAL CYTOLOGY OF THE LUTEIN CELLS OF THE SOW
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Four years ago the writer undertook, at the suggestion of Professor Mall, to study the corpus luteum at different stages of pregnancy, with the aim of learning through the varying appearances to standardize the stages as a means of determining the ages of embryos and foetuses (Corner, '15). It was very good fortune that led to the choice of the pig for the first studies, for a useful peculiarity of cytoplasmic structure was found to occur in this species. If we take a section of the corpus luteum of a pregnant sow whose foetuses are perhaps 100 mm. long, fixed in formol, and stain it with any strong cytoplasmic stain, study of the lutein cells shows that the cytoplasm contains unstained areas which are roughly concentric to the nucleus, and which appear to form canal-like paths in the cell (fig. 1) . In the younger corpora the canals grow more and more complex, assuming the form of wide V-shaped spaces, long clefts, and circles in the cytoplasm, so extensive that the nucleus is surrounded only by a narrow zone of endoplasm. But it is in the corpora lutea of pregnancies under 30 mm. that the highest development of the exoplasmic zone is found. Here the entire outer part of the cell is occupied by a curiously elaborate system of vacuoles, almost every one of them in turn containing a spherule of substance which, although it takes the same stain as the cytoplasm, yet has a more hyaline appearance, and is seen in the section as a bright ring. Within many of the spherules is found another and tiny vacuole (fig. 2, r). Corpora lutea of pregnancies with foetuses more than 140 mm. long contain no trace of this system (figs. 23 and 24), and by careful attention to the degree of its development it is possible, therefore, to estimate the age of the corresponding embryo with some accuracy. Taking other histological features into consideration, I find myself able to detect the stage of pregnancy within close limits by examination of the corpus luteum alone. The same bodies are present in the corpora lutea of dogs, and were seen in the lutein cells of rabbits by Cohn ('03), who undertook certain microchemical studies upon their nature, which I have been able to extend. It was tentatively suggested, in my former paper, that they represent an elaborate
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IMS
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GEORGE W. CORNER
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modification of the Golgi-Holmgren intracellular apparatus. This view I have had to discard as a result of work which had led to the correct interpretation (Corner, '17 a, b). The spherules are due to the presence of a lipoid, probably of phosphatid nature, which is sufficiently oily to round up in the presence of water. The round droplets thus produced usually surround the preexisting globules of neutral fat present in consider
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Fig. 1 Cells of corpus luteum of pregnant sow (foetuses 100 mm. long), showing spaces in cytoplasm. Mallory's connective-tissue stain. Formol fixation. X 810.
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Fig. 2 Cells of corpus luteum of pregnant sow (embryos 20 mm. long). Formol fixation. Mallory's connective-tissue stain. X 810. r, vacuoles in lutein cell; th.l.c.l, theca lutein cell, type 1; th.l.c.2, theca lutein cell, type 2.
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Fig. 3 Cells of corpus luteum of pregnant sow (embryos 20 mm. long). Formol fixation followed by osmium tetroxide. X 810.
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ORIGIN OF THE CORPUS LUTEUM 139
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able numbers in the early corpus luteum cells of swine (fig. 3). After immersion in alcohol, xylol, ether, or other lipoid solvents, both the fatty center and the phosphatid substance of the spherical droplet are dissolved out, leaving only a hollow sphere (appearing as a ring in thin sections), probably composed of proteid constituents of the cytoplasm precipitated in the spherules during fixation. The bodies are not seen in fresh tissues nor in material fixed with very rapid coagulants like osmium tetroxide, which precipitate the proteids before the oil droplets round up. The microchemical evidence of these conclusions is given in the articles cited.
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The appearances in question, therefore, are simply the result of methods of fixation which do not preserve certain obscure lipoids in* their natural diffused state. But the artifact is a useful one. In the first place, it enables us to follow the changes in amount of the phosphatid substance during the advance of pregnancy. It also allows us to estimate the age of a corpus luteum of pregnancy from the histological appearance alone, and it gives us a constant (even though artificial) cytological characteristic of the cell which can be used in determining the early history of the lutein cells. Due no doubt to different physical state of the cell lipoids, the phenomenon does not occur in the human and bovine corpora lutea (a former statement of the author to the contrary).
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THE MATURE FOLLICLE
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As the reader has perceived, one of the crucial points in this debate has been as to the condition of the granulosa of the mature follicle. Some investigators think that this layer degenerates before rupture, others that it remains intact. It would seem, offhand, an easy matter to obtain mature follicles and settle the question at once. To be certain that a given follicle is really mature is very difficult, however, and particularly so in some species. Mere size is no criterion, for full-sized follicles are not infrequently in a state of advanced atresia. The presence of maturation processes in the ovum is no more certain
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140 GEORGE W. CORNER
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a sign, for the formation of the polar bodies, as was pointed out by Flemming ('85), is a frequent occurrence in early atresia. As atresia may set in at any time in the life of a follicle, even up to the last, it is obvious that we can never state with complete assurance whether a given Graafian follicle is doomed to degeneration or is about to rupture and give rise to a corpus luteum.
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We shall probably not be in error, however, in assuming that a follicle is normal and mature if it is taken from the animal at a time when ovulation is known to be imminent, and if it contains a normal ovum in which the process of maturation is under way.
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To satisfy these requirements is easy when the animal is small enough to be under observation in the laboratory, when an impending ovulation can be predicted (as, for instance, in the rat and mouse, which are now known to ovulate about eighteen hours after littering) and when the small size of the ovaries and tubes readily permits serial sectioning. In animals like the hog, however, it is more difficult to observe these two criteria of the mature follicle, and no previous investigators of this species have watched the animal during life in order to determine the imminence of ovulation, nor have any taken the pains to find and study the ova of the follicles which they described as ripe.
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In the author's material, of sixteen animals known to have been in heat when killed, only two were taken early enough in oestrus to contain unruptured follicles. In one, all the follicles were still unruptured. Three of them were successfully sectioned ; two of them contained ova with nuclei presenting 'germinal vesicles,' the third showed the first polar body and the second polar spindle. In the second sow, one of the follicles had ruptured; the tubal ovum could not be found; one of the remaining follicles, upon sectioning, showed its ovum to be in the matured state, with the second polar spindle formed. There can be little doubt, therefore, that the follicles in question were perfectly normal and would have immediately shed their ova and developed into corpora lutea had the sows not been slaughtered.
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These follicles possessed clear, translucent, almost spherical walls, protruding a great part of their bulk from the ovary, as is characteristic of the species. They all measured about 7 mm.
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ORIGIN OF THE CORPUS LUTEUM 141
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in diameter, the measurement ranging from 6 to 8 mm. in some which were distorted by crowding. 2 The surface presented no 'stigma' or other sign of impending rupture. On section, they were found to possess the usual three layers, and the membrana granulosa was present and intact, showing no sign of degeneration. The wall of that part of the follicle lying deepest in the ovary presents a slightly wavy contour toward the cavity.
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The cells of the granulosa (fig. 4, a) form a layer about six to nine cells deep, or about 0.13 to 0.17 mm. thick. Those cells nearest the membrana propria form an irregular columnar layer, but the upper cells show less semblance of order in their arrangement. The cells are round or polyhedral, from 8 x 8/* to 10 x 16^ in diameter (in celloidin sections), with round nuclei 5n or 6/x in diameter. The cytoplasm appears homogeneous after the usual fixing reagents, except for a few vacuoles due to the presence of lipoid substances, as will be explained later. Often the cells possess short processes which meet those of the neighboring cells so as to make the tissue resemble a syncytium.
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The theca interna is about 0.09 to 0.10 mm. thick, or a little more than half as thick as the granulosa. Its most striking characteristic is the presence of three to five layers of large 'epithelioid' cells, usually from 10 x 17/x to 12 x 17m in diameter, but occasionally reaching larger sizes, up to perhaps 16 x 24/x (fig. 4, b). On section, they are oval, spindle-shaped, or almost rectangular, with their long axes in the circumference of the follicle, so that they usually lie at right angles to the columnar layer of the membrana granulosa. Between the larger cells, and especially along the inner border of the layer formed by them, are others of small size (though still somewhat larger than the granulosa cells), which are similar in appearance to the large theca cells. In material fixed in Bouin's fluid the theca cells
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2 In a previous publication ('15) I fell more or less into the same error of method which I am now imputing to others, and attempted to determine the size of the 'ripe' follicle without knowing the state of the enclosed ovum. The larger follicles there mentioned, however, agree histologically with the accurately known specimens described in these pages, and it is therefore likely that my previous conclusions were correct, namely, that the normal mature follicle may attain a diameter of 10 mm.
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142
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iti. ext.--
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tti. int:
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>ran.- - .
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ap. cz.
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Fig. 4 a, Portion of wall of unruptured Graafian follicle (sow in heat, ova maturing). Iron haematoxylin. X 380. b, A few cells from theca interna of same specimen. X SOU. gran., membrana granulosa; th.int., theca interna; th.ext., theca externa; sp.cz., spindle-cell zone of theca interna; b.v., bloodvessels.
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ORIGIN OF THE CORPUS LUTEUM 143
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are notable for the presence in their cytoplasm of a number of vacuoles, giving them a striking honeycombed appearance (fig. 4, b). These vacuoles are due, at least in part, to the presence in the fresh tissue of granules of fat-like substance, packed closely into the theca cells, whose chemical nature has not been determined (fig. 5, 6). In some species they are quite yellow, since they hold in solution some of the lipochromes common in the ovaries of certain animals, but in the pig they are practically colorless. It is of course the appearance of these large fatty cells of the theca which has helped establish the belief that they are the precursors of the ' lutein cells' of the corpus luteum. The granules are soluble in alcohol; in osmium tetroxide they take a color varying from gray to deep black; they take a decided reddish color with Herxheimer's alkaline Scharlach Rot, but appear not to stain at all with Nile-blue sulphate; they are not anisotropic. From these reactions we may assume that the substance is of lipoid nature, but is perhaps not a neutral fat. The granules are variable in diameter, from 0.5/x to 1.5ju, a few even reaching 2.5/*. Many of the theca cells contain, instead of lipoid granules, vacuoles which are not stained even in osmium preparations, and which therefore must contain either a modified form of the lipoid or some other substance which is not rendered insoluble by combining with Os0 4 (fig. 5, b). The smaller cells of the theca interna mentioned above usually have finer granules, but there are all transitions between the large and small types. The cells of the granulosa contain a few very small granules, uniformly black after osmium fixation; these are usually more numerous in the basal layer of the follicular epithelium.
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Between the granulosa and the cellular layer of the theca interna just described is a narrow zone (0.03 to 0.04 mm.), which contains chiefly spindle cells without fatty inclusions or vacuoles (figs. 4, a, and 5, a). These cells appear to be of two types: first, the endothelium of the blood-vessels which lie in this zone and, second, the fibroblasts of the perivascular tissue, which are part of a light network of connective-tissue reticulum supporting the theca and forming a base for the granulosa, for I agree with J. G. Clark that the membrana propria is nothing more
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THE AMERICAN JOURNAL OF ANATOMY, VOL. 26, NO. 1
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Ri/f,., $i'<J. H(f.
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Fig. 5 a, Portion of wall of unruptured Graafian follicle (sow in heat, ova maturing). Osmium tetroxide fixation without further stain, showing distribution of i'ats. X 380. b, A few cells from theca interna of same specimen. X 800. gran., membrana granulosa; th.int., theca interna: th.ext., theca externa; sp.c.s.,
 +
 +
spindle-cell zone.
 +
 +
m
 +
 +
 +
 +
ORIGIN OF THE CORPUS LUTEUM 145
 +
 +
nor less than a network of these fibrils applied closely to the base of the granulosa layer.
 +
 +
The theca externa consists of a layer of long spindle-shaped cells, shading off into the stroma of the ovary (or into the capsular connective tissue, over that part of the follicle which is jutting out from the ovary). It is composed chiefly of collagenous fibrils and their associated fibroblasts, but it is highly interesting to note in connection with the subsequent collapse of the follicle, that there are also a good many smooth muscle fibers, as is readily seen by the use of Van Gieson's stain. There are no elastic fibers, except in the walls of the larger bloodvessels.
 +
 +
Just before rupture there are many mitotic figures in the cells of the theca externa, but only occasional signs of cell division in the theca interna and the granulosa.
 +
 +
Injected specimens show the blood- vascular distribution to be as described by His and J. G. Clark (fig. 6). Large vessels form a network in the theca externa, sending twigs inward to form a generously anastomosing plexus which lies in the abovedescribed spindle-celled zone of the theca interna.
 +
 +
I have found a curious arrangement of the blood-vessels in the ovum-bearing area of the follicle. The discus proligerus is a cone-shaped or rounded projection of the granulosa bearing the ovum near its apex, which until shortly before maturation of the ovum is composed of densely packed granulosa cells. At its base, in this particular species, are found a number of little vascular loops sprouting up from the vessels of the theca interna well into the granulosa of the discus, and pushing before them the cells of the basal columnar layer (fig. 6, loops). The basal cells appear as if radiating from the loops, and like all the cells of the area occupied by the loops are enlarged and have a much less dense cytoplasm than the other granulosa cells. Those loops which are near the center of the discus are longer than those toward the periphery. Such vascular loops penetrating the granulosa have apparently not been mentioned previously. They are not to be found in the rat and mouse, the only other species which I have studied in this regard. It would seem that
 +
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 +
146
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GEORGE W. CORNER
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ORIGIN OF THE CORPUS LUTEUM
 +
 +
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 +
147
 +
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 +
the large size of the discus proligerus (as large as the entire follicle of the mouse) places the ovum at such a distance from the vascular bed that special vessels are needed for its nutriment.
 +
 +
Be this as it may, by the time maturation of the ovum is in progress, the character of the discus proligerus has become considerably modified (fig. 7). Its cells are very much swollen by a
 +
 +
 +
 +
 +
Fig. 7 Portion of wall of mature Graafian follicle (sow in heat), showing mature ovum in situ, and discus proligerus in process of dissociation. X 50.
 +
 +
vacuolization of the cytoplasm, so that they stand farther apart from each other, and in many places seem to be no longer in contact. About the ovum the cells of the corona radiata still hold together, but the rest of the discus has nearly crumbled away, and the slightest disturbance must complete the freeing of the ovum, bound as it now is to the parietal granulosa only
 +
 +
 +
 +
148 GEORGE W. CORNER
 +
 +
by a few strands of cytoplasm. This rearrangement of the discus proligerus was long ago shown by Bischoff ('78) to be a trustworthy sign of impending rupture of the normal follicle. Certain subsequent observers, wondering how the ovum could be freed from its apparently secure moorings, were led to conjecture that there is a total desquamation of the granulosa— an error in which they were confirmed by the fact that for lack of the proper stages they did not see the mechanism for cutting off the ovum described by Bischoff, but did see, on the other hand, the complete dissolution of the granulosa, in follicles which we now know to have been atretic.
 +
 +
THE FRESHLY RUPTURED FOLLICLE
 +
 +
Four animals of my series contained follicles which had ruptured very recently. One of these sows had shown the first signs of heat at some time between thirteen and twenty-two hours before killing, another between sixteen and thirty-nine hours before killing, and the other two were in the second or third day of oestrus (probably the second). As we have shown on page p. 132, rupture of the follicle occurs on the first or second day of oestrus. In all four of these cases, unfertilized ova were found in the tubes, there having been no copulation.
 +
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Apparently the act of rupture begins by the production of a small slit in the exposed part of the follicle, through which the ovum escapes to enter the tubal fimbria (figs. 8 and 9). A varying amount of the follicular fluid, usually a considerable portion, is extruded with the egg, and the follicle collapses as the volume of its contents suddenly lessens. It seems that an important part in this collapse must be taken by the fibers of involuntary muscle which lie in the theca externa; through their contraction the follicle is greatly diminished in all dimensions. At the point of rupture, the muscle fibers draw the theca externa away from the torn area, and the result is a slight eversion of the wound, through which the theca interna and granulosa protrude, forming a small reddish papule 1 mm. or less in diameter, the so-called stigma (fig. 12). The eversion of the inner layers
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ORIGIN OF THE CORPUS LUTEUM 149
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appears at times to close the orifice immediately, but in other cases the follicular walls do not come together about the opening at once; the tiny slit is first plugged by fibrin, and later closed permanently by proliferation of its edges, much as described by Strakosch ('15) in the human corpus luteum. From the minute blood-vessels whose torn ends lie in the stigma there is often a slight oozing of serum or of blood, so that the surface of the ovaries at this time may be roughened by tags of pale or bloody
 +
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Theca mUma,
 +
 +
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Thee a externa
 +
 +
 +
 +
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Giircmuloe.a
 +
 +
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Poirvt of fupt ure
 +
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Fig. 8 Diagram of ruptured Graafian follicle (sow in heat, ova in tubes), illustrating partial collapse without great infolding of walls. X 14. (Compare with figure 9.)
 +
 +
fibrin, sometimes forming temporary adhesions to the fimbriae of the tubes. The entire ovaries and the tubes are usually much congested during oestrus.
 +
 +
On section, the follicular cavity is collapsed to a mere slit in some cases, in others it is still partially distended, owing to the continued presence of more or less of the follicular fluid. For this reason, the size of the structure varies, but in general it is much smaller than the mature unruptured follicle, its diameters vary
 +
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150
 +
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 +
 +
GEORGE W. CORNER
 +
 +
 +
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ing from 3.5 to 6.5 mm. when not distended by hemorrhage. Most frequently the ruptured follicles are ovoid in form, about 4x4x5 to 5x5x6 mm. in diameter. Owing to the collapse of the follicle and to the contraction of the theca externa, the inner walls of the cavity are no longer smooth, but are thrown into folds whose complexity varies from that of low ridges in those
 +
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Point of rupture
 +
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Cavity of
 +
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corpus luteu
 +
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Unripe
 +
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?
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follicle _aR|
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Theca externa
 +
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~s Theca interna
 +
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Granulosa
 +
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Theca externa in N^
 +
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interior of cavity
 +
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0edematou6 spaces in theca mterr\a
 +
 +
 +
 +
Fig. 9 Diagram of ruptured Graafian follicle (sow in heat, ova in tubes), illustrating complete collapse with much infolding of walls. X 14. (Compare with figure 8.)
 +
 +
 +
 +
follicles where much follicular fluid still lingers (fig. 8) to elaborately interwoven folds such as those shown in figure 9.
 +
 +
Microscopically, the follicular wall is found to consist of the same layers as before rupture (fig. 10). There is no sign of any degeneration of the granulosa, which is now somewhat thicker,
 +
 +
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 +
ORIGIN OF THE CORPUS LUTEUM
 +
 +
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151
 +
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since it lines a smaller cavity than before. The individual cells are about the same size as formerly, but in many places are now elongated into oval or spindle forms by the stresses of the collapse, appearing to have slid upon each other as the granulosa thickened. The theca interna is the layer most affected, in a mechanical way, by the sudden collapse, for in some of the
 +
 +
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gran
 +
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th.
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int.
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mernb prop.
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 +
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 +
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■■•
 +
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 +
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Fig. 10 Portions of wall of recently ruptured Graafian follicle (sow in first clay of oestrus, ova in tubes). X 330. a, Mallory's connective-tissue stain, Bouin's fixation, b, Osmium tetroxide after formol fixation, showing distribution of fats, gran., membrana granulosa; th.int., theca interna; Ih.ext., theca externa; memb. prop., membrana propria.
 +
 +
folds it is violently torn apart, so that there are many wide spaces either within the theca interna or between the two thecae (fig. 11, tear). These spaces are occupied either by networks of fibrin, which may be altogether devoid of cells, or contain an occasional theca interna cell, or a leucocyte; or the space may
 +
 +
 +
 +
152
 +
 +
 +
 +
(JK()H(JK W. CORNER
 +
 +
 +
 +
not be an actual tear, but merely an oedematous area in which the cells, connective-tissue fibers, and blood-vessels of the thecae interna and externa are held apart by the tissue fluids. However, over many of the folds, and always in the depressions between the folds, the theca interna is neither torn nor oedema
 +
 +
 +
dep
 +
 +
 +
 +
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 +
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fear--
 +
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y
 +
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 +
 +
 +
 +
 +
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 +
"~ : .: : ' J : .'
 +
 +
 +
 +
Fig. 11 Portion of wall of recently ruptured Graafian follicle (sow in heat first day of oestrus, ova in tubes; same animal as in figures 9 and 10), showing torn area in theca interna in a fold of the wail. X 80. gran., membrana granulosa; th.int., theca interna; tear, torn area in theca interna; dep., depression or recess between folds of wall.
 +
 +
 +
 +
tous (fig. 11, dep.). The tears do not separate the theca interna from the granulosa; these two layers are everywhere in apposition, and the boundary between them is still marked at most points by the slight wall of condensed connective-tissue fibers, originating in the innermost layer of the theca interna, the so-called
 +
 +
 +
 +
ORIGIN OF THE CORPUS LUTEUM 153
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membrana propria (fig. 10). The theca externa is of course drawn into the folds of the wall, and where these folds are verydeep, long spindle-cells of the externa may thus penetrate almost to the center of the former follicular cavity — though of course they are walled out by the inner layers (fig. 9). Dividing cells are found not infrequently in the theca externa, but are quite rare in the inner layers. The distribution of lipoid substances, as indicated by the use of osmium tetroxide and Herxheimer's stain, is exactly as in the mature unruptured follicle (fig. 10, b). Leucocytes are found in the walls of all developing corpora lutea. The blood-vessels are exactly as in the unruptured follicle, the picture presented by them being modified only by the elaborate infolding of the walls (fig. 12). At the point of rupture the torn vessels of the thecal plexus present to the outside, and within a few days of rupture have sprouted into a little rosette of capillaries about the stigma, which helps to make this spot conspicuous by its redness. In the production of the curious torn spaces of the theca interna, described above, vessels of the theca interna are not infrequently ruptured, with resultant haemorrhage into the theca. If the loss of blood is very slight, the broken-down blood is taken up by the large cells of the theca interna, in which the phagocyted golden-brown pigment may remain for some days at least (fig. 13) . In one of my cases there was a single local haemorrhage into the theca externa, and the nearest cells of the theca interna were full of blood-pigment granules. However, when the thecal haemorrhages are large, the resultant haematomata may burst through the granulosa into the cavity. I am inclined to think that we have here the source of most of the bleeding into the early corpus luteum cavity. The now almost forgotten doctrine of Henle and Paterson, that the corpus luteum is formed from the blood clot of the newly ruptured follicle, naturally led to investigations into the importance and constancy of the haemorrhage in various species, which have been summed up by Sobotta in his paper of 1896. In the pig, Zwicky ('44) held that bleeding is frequent, Paladino ('80) that it occurs in two-thirds of the cases, Benckiser ('84) that it is inconstant, Spiegelberg ('65) that it is important, and Bonnet ('91) that it is constant and
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154
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GEORGE W. CORNER
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Fig. 12 Recently ruptured Graafian follicle (ova found in tubes), bloodvessels injected with India ink. X 15. gran., membrana granulosa; th.int., theca interna; th.ext., theca externa; stig., stigma (blood-vessels at point of rupture).
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ORIGIN OF THE CORPUS LUTEUM
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155
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marked in extent. Sobotta ('96), reviewing the evidence, is inclined to the last view. Pfluger ('63), in an experimental investigation, found that in cats and rabbits killed violently there was much more frequent bleeding into young corpora lutea than in animals killed without struggle and very carefully autopsied. In my own specimens, out of sixteen sows whose ovaries contained very early corpora lutea, dressed at a packinghouse using the relatively gentle method of scraping by hand, four showed more or less blood in the corpora lutea and twelve
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Fig. 13 Cells from theca interna of recently ruptured follicle (sow in heat, ova in tubes). Iron haematoxylin stain, showing pigment and broken-down erythrocytes in theca cells. X 1000.
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were entirely free of macroscopic haemorrhage. In another establishment, where the carcasses are conveyed 150 feet dangling from a chain and are scraped by engine-driven revolving vanes (so that in the bodies of pregnant sows, young foetuses frequently suffer an effusion of blood into the amniotic sac), there chanced to be a somewhat higher proportion of haemorrhagic follicles; but even there, in spite of such excessive violence, it is common enough to see delicate corpora lutea one or two days old come through with no blood at all in their cavi
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156 GEORGE W. CORNER
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ties. Again, trauma at the time of killing does not explain awayall the haemorrhages; for instance, in those cases in which among a number of solid, bloodless corpora lutea several days old, one or two others are found distended with dark clotted blood to a size exceeding the normal corpora. I feel that the present evidence indicates that haemorrhage into the corpus luteum of the sow, while not uncommon, is the exception rather than the rule, and is of no anatomical or physiological importance. Indeed, the arrangement of the follicle seems well adapted to prevent any considerable loss of blood into the cavity, for the tiny vessels at the place of rupture are promptly directed outward toward the peritoneal cavity, while the follicle is provided with smooth muscle, which keeps the walls tensely contracted, even after rupture. When small haemorrhages occur, undoubtedly they are readily resorbed, and the corpus luteum then goes on to develop normally. When great enough to distend the follicle and compress the growing wall, inhibition of corpus luteum formation presumably occurs, and we have here one of the causes of corpus luteum cysts, which are very common in swine.
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INVASION OF THE GRANULOSA
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The next stage is represented by seven animals in my collection, all of which were killed during oestrus, as normal ova were found in the tubes. Moreover, four of them were observed during life, and were actually seen to be in the second or third day of oestrus. In three, copulation had not occurred; in three others, fertilization had taken place, the ova showing the pronuclei approaching conjugation; and in the seventh, the ova were segmented into two, four and six blastomeres.
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The first sign of an advance upon the previous stage consists of the breaking-down of the membrana propria, at first at the apices of some of the folds, later over the entire follicle, so that the former sharp line of division between granulosa and theca interna is no longer present (fig. 14). Wherever the membrana propria is disappearing, slender spindle-cells are seen to be insinuating themselves between the still closely packed granu
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ORIGIN OF THE CORPUS LUTEUM
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157
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losa cells (fig. 14, sp.c). The nature of the inwandering cells is difficult to decide. In places there can be no doubt that they are endothelial in nature and represent the first sprouts from
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ih. ext.
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Fig. 14 Portion of wall of young corpus luteum (ova found in tubes), showing swollen cells of granulosa with inwandering spindle-shaped cells. Mallory's connective-tissue stain, formol fixation. X 810. gr.l.c, granulosa lutein cells; th.int., theca interna; th.ext., theca externa; sp.c, spindle-shaped cells.
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the walls of the thecal capillaries, growing inward to the granulosa. In the endothelial cells mitoses are not uncommon at this time. It seems quite likely that all the early invading cells are
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158 GEORGE W. CORNER
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of endothelial nature, but at some few points, however, it is impossible to convince oneself that the spindle-cells have any connection with the vessels, for they are not always arranged in tubular form and are sometimes well disseminated throughout the granulosa in advance of any circulation of blood. It cannot be denied absolutely, therefore, that some of them may be inwandering cells of the perivascular spindle-cell zone of the theca interna. During these early changes the large cells of the theca interna remain in their place, and I have never seen convincing evidence of their conversion into spindle cells.
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Practically all of those observers who have been convinced of the persistence of the granulosa have described such an early invasion of the innermost layer by spindle-cells, a stage which was called by Robert Meyer the stage of proliferation, but as in the pig there is doubt as to the interpretation of the observed facts. Sobotta ('96) holds that all the cells of the theca interna are converted into spindle-cells (fibroblasts), and wander into the granulosa, dividing frequently, to form the connective-tissue framework of the corpus luteum. In this view he is supported by Marshall ('04) in his work on the sheep, and by O'Donoghue ('16), who studied the marsupial ovary; but several authors, including Volker ('05), Loeb ('06), and R. Meyer ('11), working, respectively, with the corpora lutea of the marmot, the guineapig, and man, are inclined to consider the first inwandering cells as endothelial, and deny the conversion of the theca interna cells into fibroblasts.
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It has been mentioned that the breaking-down of the raembrana propria and the invasion of the granulosa by spindle-cells does not take place at once over the entire inner surface of the collapsed follicle, but begins first at the apices of the folds, where the structure has presumably been subjected to the greatest mechanical strain. Because of this very important fact, we are able to observe a definite stage at which, while in places there is an actual intermingling of the two layers going on in part of the structure (fig. 15 a, X), in other parts the two inner layers of the wall maintain their original relations. During the same period there is a marked and rather sudden change in the granu
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ORIGIN OF THE CORPUS LUTEUM
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159
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losa cells (fig. 15 b). Their cytoplasm increases in volume so that the cells are now much larger in size, varying from 9.5 x 11/x to 14 x 21 \i in diameter in sections, or half again as large in diameter as before rupture. The nuclei become rather more vesicular. Most important is the fact that many of the larger
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a.
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Fig. 15 a, Part of wall of developing corpus luteum in stage of spindle-cell invasion (ova in tubes). Formol fixation, Mallory's connective-tissue stain. X 110. X, area of active invasion by spindle-cells (at apex of fold in wall).
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cells now have in their cytoplasm large spaces containing rounded bodies resembling rings in section, which we have already seen (pp. 137, 139) to be characteristic of the so-called 'lutein cells' of the young corpus luteum in swine and to be due to the presence in the cytoplasm of an oily lipoid substance.
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THE AMERICAN JOURNAL OF ANATOMY, VOL. 26, NO. 1
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160
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(;kok<;k w. corner
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Furthermore, these changes in the granulosa cells are found to occur in all parts of the wall, as well in those areas as yet uninvaded as in those where granulosa and spindle-cells are already intermingled. To sum up the evidence, there is a time in the development of the corpus luteum, about three days after the
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Fig. 15 a, Enlarged view of portion of same as shown by rectangle. X 1000. gr.l.c, granulosa lutein cells; th.c, theca cells; b.v., blood-vessel.
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rupture of the follicle, when changes of structure within the organ are already under way, and when many of the cells have begun to acquire adult characteristics, but in some parts of the organ the original relations of granulosa and theca interna are
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ORIGIN OF THE CORPUS LUTEUM 161
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still intact; in these areas we find that it is the granulosa cells alone which have assumed the appearance of the large cells of the corpus luteum, commonly called lutein cells.
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The breaking-down of the membrana is followed by a rapid sprouting and branching of the blood-capillaries throughout the entire granulosa (figs. 16 and 17). This stage is represented, in my material, by seven sows, of which one contained fertilized ova (some unsegmented and some with two blastomeres) ; one was killed about five days after the onset of heat, no ova being found, probably having degenerated; one was killed about six days after the onset of heat, one degenerate ovum being found; the other four were among those received from the University Farm School, in which the ova were not sought, but in which the dates of copulation were accurately known, in two on the third day and in two on the fifth day before killing.
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Coincidently with the spread of the blood-vessels in a network throughout the granulosa, there continues a marked swelling of the cells of this layer, which double or more than double in diameter, thus making an eightfold increase in volume; some of them reach dimensions of 30V to 35^- The nuclei are larger and more vesicular. I have never seen a mitotic figure in a cell of the granulosa at this or later stages, and feel sure that the generalization of Sobotta on this point is correct. In the formol- or Bouin-fixed specimens, the periphery of the cells is studded with the striking ringlike phosphatid artifacts of fixation (fig. 18, gr.l.c). The rest of the cytoplasm is thin and contains irregular vacuolar spaces, due partly perhaps to the shrinking away, during fixation, of the cell-substances which form the ring bodies, and partly to the solution in the alcohols, xylol, or ether, of other lipoid substances, which osmic preparations show as small black globules in the center of each ring and also scattered about the nucleus or throughout the cytoplasm. I have not been able to apply other microchemical tests to the tissues at this stage, but the globules are morphologically like the neutral fat of later stages (fig. 3), and I assume that they are indeed the neutral fat or its forerunner.
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102
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GEORGE W. CORNER
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Fig. 16 Young corpus luteum (segmenting ova with one and two blastomeres found in tubes); blood-vessels injected with India ink. Compare with figure 12. X 15.
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ORIGIN OF THE COHl'l'S LUTEUM
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163
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Meanwhile, changes have also been taking place in the large cells of the theca interna. Mitoses are more common, and the former definite internal limit of this layer has been blurred by the breaking-down of the membrana propria and the ingrowth of the capillaries at all points of the follicular wall. Within the
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ith int.
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th. extf'
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Fig. 17 Enlarged view of small part of figure 16 as indicated by rectangle, showing blood-vessels of theca interna branching throughout granulosa. X 4").
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cells there are changes in the lipoid inclusions which are their chief distinguishing characteristic. In some cells of osmic preparations the granules are larger, in others smaller than before; in some they do not form an insoluble black compound with
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h. int.
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Fig. 18
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ORIGIN OF THE CORPUS LUTEUM 165
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mium tetroxide, but leave vacuoles of varying sizes, between which a few stained granules may remain giving a characteristic foamy appearance to the cytoplasm (fig. 19) ; and in others practically all the fatty bodies and vacuoles have disappeared, leaving a smooth homogeneous cytoplasm. In ordinary stained sections, then, the theca interna cells are of about the same size as before rupture, their nuclei are perhaps slightly more vesicular, and the cytoplasm is either homogeneous or contains many densely packed vacuoles, usually uniform in size within any one cell, resulting in the foamy appearance.
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Fig. 19 Theca interna cells of corpus luteum in stage of invasion, osmium tetroxide fixation without further staining, showing varying degrees of fatty inclusion. X 1000.
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It will be obvious that the previous clean-cut distinction between the two layers is now lost. Heretofore we have been able to distinguish them by position, size, and content; there has been a wall of connective-tissue fibrils between the layers; the cells of the granulosa have been smaller than those of the theca interna ; and the former have contained but small numbers.
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Fig. 18 a, Part of wall of developing corpus luteum in stage of invasion (ova in tubes). Bouin fixation. Mallory's connective-tissue stain. X 110. b, Enlarged view of portion of same as indicated by rectangle. Mallory's connectivetissue stain. X 1000. gr.l.c, granulosa lutein cells; th.l.c, theca lutein cells; th.int., portion of original theca interna, near a fold in wall of corpus luteum; b.v., blood-vessel.
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166 GEORGE W. CORNER
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of lipoid granules, the latter considerable amounts. But now the membrana propria is done away with, the granulosa cells have increased in size and are becoming rich in lipoids, while the theca interna cells are losing their lipoids. It is not strange that investigators have become involved in uncertainty regarding the further fate of the theca cells. I have seen the abrupt ending, at stages similar to those now being described, of two careful attempts, by students in our histological courses, to follow the theca cells of the mouse and rat by means of their osmiumstaining inclusions, owing to failure to observe further distinctions between the two cell types. In the pig, however, we possess a peculiar advantage in the tendency of the phosphatid material to form the previously mentioned cytoplasmic rings when fixed with slow aqueous fixatives, giving the granulosa cells a distinctive appearance. There are other less regularly present criteria, which when added together afford the practiced observer means of partially distinguishing the two cell types; these are a tendency of the cytoplasm of the theca cells to take acid stains somewhat more deeply than the granulosa cells (perhaps this indicates merely a denser cytoplasm) and also the regularity in size and closely packed disposition of the lipoid granules or the vacuoles left when they disappear or are dissolved. Following these clues, we find that many of the theca interna cells remain in their original location about the periphery of the follicle, running into the interior of the folds produced by the collapse; but also that many of them, as the blood-vessels grow inward, are carried or wander with the vessels, and become disseminated among the cells of the membrana granulosa, where they are finally lodged, either singly or in small groups, frequently along the capillaries (fig. 18). It must be remembered that a general scattering of the theca cells among the granulosa in this way will not require a longer journey for any single cell than the thickness of the inner layer, which is not more than 0.2 mm. The cells thus immigrating resemble in every way their mates left behind at the periphery and in the folds, some of them containing large granules, staining black with osmium tetroxide, others showing almost no fatty inclusions (fig. 19).
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ORIGIN OF THE CORPUS LUTEUM
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167
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They are often bioadly spindle-shaped or irregular in form, sometimes compressed between the granulosa cells or applied demilune-fashion to one of them.
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Further changes are brorght about by the great swelling of the granulosa cells, which proceeds so far that the contents of the follicle begin to equal and finally to exceed the capacity of the contracted theca externa. The first effect of the internal pressure is to fill whatever remains of the original follicular cavity solidly with new tissue, then to compress the thecal cores of the folds of the walls so that all fibrin-containing cavities and
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Fig. 20 Diagram showing outline of section of a corpus luteum about four days after ovulation. X 5. pr., 'Pfropf or hernia-like bulging of contents through point of rupture.
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oedematous spaces are obliterated, and the folds become merely connective-tissue septa containing the remains of the theca interna in the shape of a diminished number of theca cells enmeshed by reticular fibrils; in the bases of the folds the bloodvessels of the young corpus luteum enter, usually accompanied by fibroblasts and fibrils proceeding from the theca externa. In many young corpora lutea the swelling of the granulosa cells finally causes a bulging of the contents through the outer pole of the wall, at the point previously weakened by the rupture; which in the prolific ovaries of the sow may be exaggerated by
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168 GEORGE W. CORNER
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the pressure of the many neighboring corpora lutea of the same crop, until there is produced a knoblike hernia of corpus luteum tissue sometimes containing a tenth or more of the whole corpus luteum (fig. 20). This appearance is sometimes called by the handy German name 'Pfropf.' In some species, as, for instance the cow, it seems to occur invariably and to persist throughout pregnancy, but in swine the hernia is not always produced, the whole wall of the corpus distending evenly instead; and later it seems to subside, as in most corpora lutea in more advanced pregnancy there are no 'Pfropf en.'
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THE FULLY FORMED CORPUS LUTEUM AND ITS MORPHOLOGICAL CHANGES UNTIL THE TERMINATION OF PREGNANCY
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Invasion of the granulosa by the thecal vessels and cells begins about the third day after the onset of oestrus (or about the second or third day after rupture of the follicle) and is completed about the sixth or seventh day. My series contains five sows killed during the second week after ovulation and a large number from all stages of pregnancy from fifteen days after ovulation on to full term and into the period of lactation, so that altogether there is an unbroken series representing almost every day of the entire reproductive cycle.
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By the seventh day the corpus luteum may be considered to have completed the first stage of its metamorphosis. It is solid (unless there has been a decided haemorrhage into the cavity), and it is already larger than the follicle in which it arose, reaching diameters of 8 and 9 mm., although a slow increase in size is yet to go on until the second or third week, by which time the full diameter, 10 to 11 mm., is reached. The blood-vessels have grown into a very narrow-meshed plexus, reaching every cell. The remains of the former great folds of the walls are seen as thin septa of connective-tissue fibrils running radially into the corpus luteum, carrying the larger blood-vessels of the organ. In some specimens, just inside the theca externa capsule and along the septa is a layer of theca interna cells or sometimes a few scattered clumps of them which have not chanced to invade the granulosa. These clumps may be found as late as the
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ORIGIN OF THE CORPUS LUTEUM
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169
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second month of pregnancy or may disappear long before (fig. 21). In appearance the cells of these clumps resemble the theca cells of the stage of invasion. In osmium-tetroxide preparations,
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5 ir>r~
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\K. c.
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Fig. 21 Portion of corpus luteum from second month of pregnancy (foetuses 35 mm. long), showing a clump of theca interna cells still in their original position. Formol fixation. Mallory's connective-tissue stain. X 450.
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some retain many lipoid globules, others have lost all of their fatty inclusions; but many present the previously noted foamy appearance of the cytoplasm, due to the presence of many
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170 GEORGE W. CORNER
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small evenly packed vacuoles with a few tiny black granules interspersed.
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In the substance of the corpus luteum are now found two types of cells whose differing characteristics become well marked after the beginning of the third week. One type is that whose identity with the granulosa is fully demonstrated by the presence of the peculiar lipoid spherules about the periphery of the cytoplasm (figs. 2 and 21). From now on these cells grow slowly in size, until just before delivery some of them have attained the
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'if 22
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immense size of 30 x 45^. During the first few weeks the neutral fat increases until it greatly exceeds the small amount found in the cells of the granulosa before rupture, and then grows progressively less, finally almost disappearing by the 110th day of pregnancy. The phosphatid substance which produces the artifacts of fixation, so frequently referred to, disappears also, and therefore during the last third of pregnancy the cells, in ordinary formol preparations, possess a homogeneous cytoplasm, strikingly different from the greatly vacuolated cell substance of the earlier stages (fig. 23). Just before delivery, however, great globules of an osmium-staining material, presumably a fat, appear about the periphery of some of the cells.
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ORIGIN OF THE CORPUS LUTEUM
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171
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Fig. 22 Cells of corpus luteum of pregnancy eighteen to twenty days old (embryos of twenty-seven somites). Osmium tetroxide after formol fixation, a, Diagram of a small portion of the periphery, showing a clump of theca cells still present at the periphery at the site of a fold in the follicular wall, and indicating by small rectangles the location of the cell groups in b and c. b, Cells from persisting theca interna. X 1000. c, Cells from interior of corpus luteum, showing two granulosa lutein cells and a third cell exactly resembling those of the theca. X 1000.
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172 GEORGE W. CORNER
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The cells of the other type are those described in my contribution of 1915 as "additional cells of the corpus luteum, type 1." They are found throughout the corpus scattered between the larger cells or in small clumps along blood-vessels and connective-tissue septa. They are smaller than the granulosa lutein cells, having diameters of 15^ to 20/*. In form they are adapted to their interstitial position, being rounded, almost rectangular, or at times compressed into polyangular shape (figs. 23 and 24). Their cytoplasm is either finely granular or contains regular vacuoles so closely packed as to give a foamy appearance. Indeed, in form, size, and in intracellular characteristics they present a most striking resemblance to those cells of the theca interna which in the same preparations are still belatedly situated at the periphery of the corpus luteum (fig. 22). Especially in osmium preparations is the similarity so great that one is forced to the hypothesis that we have scattered throughout the organ, among the granulosa lutein cells, the multiplied and immigrated cells of the theca interna. In the light of the apparent origin of these cells, it would seem well to give them the name, already established in the literature, 'theca lutein cells,' for though there are certain just reasons for criticism of this term and the name 'granulosa lutein cells' as applied to the other great class of corpus luteum elements, there would seem to be no better names at hand.
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While the cells derived from the granulosa lose their lipoids after the first few weeks, the smaller cells just described again gradually increase their content of osmium-staining lipoids during the span of gestation, and some of them come at last to be laden with these bodies, which, how r ever, do not altogether resemble the lipoid granules of their earlier days (fig. 24). At the end of pregnancy the cells of this type are still present among those derived from the granulosa, apparently having maintained separate identity during the entire term of gestation. Even those which remain for a while in clumps or a definite layer about the periphery are not found to degenerate, but seem to be drawn in among the neighboring granulosa cells as the corpus grows older.
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gr.l.c
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23
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24
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Fig. 23 Cells of corpus luteum of advanced pregnancy (foetuses 230 mm. long). Formol fixation. Mallory's connective-tissue stain. X 810. gr.l.c, granulosa lutein cells; th.l.c, theca lutein cells.
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Fig. 24 Same stage as in figure 22, osmium tetroxide fixation without further staining, showing distribution of fatty inclusions in cells. X 810. gr.l.c, granulosa lutein cells; th.l.c, theca lutein cells.
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173
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174 GEORGE W. CORNER
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However, when an attempt is made to classify all the elements of the fully formed corpus luteum, the picture is complicated by the fact that numerous cells are found which are intermediate in size between the two classes described above, and whose cytoplasmic vacuoles and fatty inclusions are of nature too indifferent to place them definitely with either granulosa or theca derivatives. The evidence, therefore, is not yet conclusive as to the exact fate of all the theca lutein cells. Either the intermediate forms represent genuine transitional stages in the formation of 'lutein cells' from theca interna cells or else they are merely cells, actually of one line or the other, in which the all too slight distinguishing features of the type (size, form, lipoid inclusions and vacuoles) have not been obvious. Toward the latter view 7 — the intermingling of the two cell lines without actual conversion of one into the other — the author is inclined to lean, without more positive evidence than has already been given.
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As a digression, it may be mentioned that some few of the theca lutein cells retain the primitive characteristics of the theca interna, even exaggerating them at times; they are variable in shape and size, they have a cytoplasm which stains deeply with acid stains, becoming dark blue, brown, or even orange with Mallory's triple stain and very dark with iron haematoxylin; the cytoplasm is usually somewhat shrunken, and contains clear vacuoles about 1^ to 2/x in diameter, which are quite uniform in size, are closely packed, and which either stain intensely black with osmium tetroxide or remain as vacuoles. The nuclei are often very dense, even pyknotic, and sometimes stain bright orange with Mallory's stain. The cells are often spindle-shaped, branched, or compressed in such a way that they give the appearance of amoeboid motion, as if they were active wanderingcells. These are the cells described in my paper of 1915 as "additional cells of the corpus luteum, type 2" (fig. 2). Although their origin is now explained, I have no more light upon their nature or possible function than before. They appear to be more common in the earlier half of pregnancy, but their number varies greatly from animal to animal.
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ORIGIN OF THE CORPUS LUTEUM
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175
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The cells of the young fully formed corpus luteum are supported by a reticulum of delicate connective-tissue fibrils with denser strands along the septa. In sections stained by Mallory's anilin-blue mixture and by the Bielschowsky technique as modi
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th.X.c.
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Fig. 25 Corpus luteum of pregnancy (embryos 20 mm. long). Formol fixation. Bielschowsky's silver-impregnation method, showing reticular fibrils. X 1000. gr.l.c, granulosa lutein cells; th.l.c, theca lutein cells; cap., capillary blood-vessel containing an erythrocyte.
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fied by Ferguson ('11), it is clear that neither the granulosa nor the theca lutein cells are intimately related to the fibrils, which form dense baskets about them, but are not found within the cytoplasm of the 'lutein cells' of either type (fig. 25). In