Difference between revisions of "American Journal of Anatomy 3 (1904)"

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=THE AMERICAN JOURNAL OF ANATOMY=
 
=THE AMERICAN JOURNAL OF ANATOMY=
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THE AMERICAN JOURNAL
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OF
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ANATOMY
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EDITORIAL BOARD
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LEWELLYS F. BARKEK,
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University of Chicago.
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THOMAS DWIGHT,
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Harvard University. JOSEPH MARSHALL FLINT,
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University of California. SIMON H. GAGE,
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Cornell University. 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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FRANKLIN P. MALL,
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Johns Hopkins University. J. PLAYFAIR McMURRICH,
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University of Michigan.
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CHARLES S. MINOT,
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Harvard University.
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GEORGE A. PIERSOL,
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University of Pennsylvania.
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HENRY Mc E. KNOWER, Secretary, Johns Hopkins University.
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VOLUME III J904
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THE AMERICAN JOURNAL OF ANATOMY BALTIMORE, MD., U. S. A.
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Baltimore, Md., U. S. A.
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I ^ I "1
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CONTENTS OF VOL 111
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No. 1. March 31, 1904.
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I. George L. Streeter. The Structure of the Spinal Cord
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of the Ostrich 1
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With 6 text figures.
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II. William J. Moenkhaus. The Development of the Hybrids between Fundulus Heteroclitus and Menidia Notata with Especial Eeference to the Behavior of the
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Maternal and Paternal Chromatin 29
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With 4 plates.
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III. John Lewis Bremer. On the Lung of the Opossum . 67 With 11 text figures.
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lY. A. M. Spurgin. Enamel in the Teeth of an Embryo
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Edentate (Dasypus Novemcinctus Linn) 75
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With 3 plates.
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No. 2. June 15, 1904.
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V. Bennet Mills Allen. The Embryonic Development of
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the Ovary and Testis of the Mammals 89
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With 7 plates and 5 text figures.
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VI. Arthur W. Meyer. On the Structure of the Human
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Umbilical Vesicle 155
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With 5 text figures.
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VII. E. H. Whitehead. The Embryonic Development of the
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Interstitial Cells of Leydig 167
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With 10 text figures.
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VIII. Florence E. Sabin. On the Development of the Superficial Lymphatics in the Skin of the Pig . . . . .183 With 7 text figures.
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iv Contents
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IX. Eoss Granville Harrison. An Experimental Study of the Relation of the ISTervous System to the Developing Musculature in the Embryo of the Frog . . . .197 With 18 text figures.
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No. 3. July 1, 1904.
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X. Thomas Dwight. A Bony Supracondyloid Foramen in Man. With Remarks about Supracondyloid and other Processes from the Lower End of the Humerus . . .221 With 1 plate.
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XI. Irving Hardesty. On the Development and ITature of
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the Neuroglia 229
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With 5 plates.
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XII. W. S. Miller. Three Cases of a Pancreatic Bladder
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Occurring in the Domestic Cat 269
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With 3 text figures.
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XIII. Leo Loeb and R. M. Strong. On Regeneration in the ' Pigmented Skin of the Frog, and on the Character of the Chromatophores 275
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XIV. Albert C. Eycleshymer. The Cytoplasmic and Nuclear Changes in the Striated Muscle Cell of Necturus . 285 With 4 plates.
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. XY. John P. Munson. Researches on the Oogenesis of the
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Tortoise, Clemmys Marmorata 311
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With 7 plates.
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No. 4. September 20, 1904.
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XVI. Eugene Howard Harper. The Fertilization and Early
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Development of the Pigeon's Egg 349
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With 4 double plates and 6 diagrams in the text.
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XVII. Harris Hawthorne Wilder. Duplicate Twins and
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Double Monsters 387
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With 2 plates and 11 figures in the text.
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Contents v
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XVIII. Lilian Y. Sampson. A Contribution to the Embryology
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of Hylodes Martinicensis 473
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With 3 plates and 17 figures in the text.
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XIX. Warren Harmon Lewis. Experimental Studies on the
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Development of the Eye in Amphibia 505
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With 42 text figures.
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XX. PEOCEEDINGS OF THE ASSOCIATION OF AMERICAN ANATOMISTS.* SEVENTEENTH SESSION I-XXVIII
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The Proceedings of the Assoc, of American Anatomists bare been removed to the
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end of the volume, from No. 1, March 31, 1904.
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THE STEUCTUEE OF THE SPINAL COED OF THE OSTEICH.
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BY
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GEORGE L. STREETER, M. D.
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Assistant in Anatomy, The Johns Hopkins University, Baltimore.
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From the Dr. Senckenberg Anatomie, Frankfort-on-Main.
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With 6 Text Figures.
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It is related by Herodian how the Kaiser Commodus beheaded ostriches and then watched them with delight and wonder as they continued running about the amphitheater, apparently to no great extent inconvenienced by the loss of their heads. That which served Kaiser Commodus as barbarous amusement frames itself for us into an interesting anatomical problem, and calls to mind a similar phenomenon so often observed among the domestic fowls. What is, then, this arrangement of the nervous elements of the spinal cord of a bird that enables it to functionate so completely after separation from the higher centers ?
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Our present knowledge and methods do not suffice for a complete explanation of this problem, but we can lead the way toward a future solution if we study out what can be learned at present concerning the histology of the bird spinal cord. In this sense, under the suggestion and guidance of Professor Edinger, I have undertaken the investigation of the structure of the spinal cord of the ostrich (Struthio camelus) . This, beyond all other birds, distinguishes itself by the great length of its spinal cord, and, in comparison with the brain, its great size.
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TtI flip lltpT"Clfm>Q ■fr'nn-itnr,-!- >.^-P«-««*, - - -* _ _ T i ii
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EEEATA TO VOL. III.
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■ MAKE THE FOLLOWING CORRECTIONS IN
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DE. E. G. HAEEISON'S PAPEE ON: "AN EXPEEIMENTAL STUDY OF THE EELATION OF THE NEEVOUS SYSTEM TO THE DEVELOPING MUSCULATUEE IN THE EMBEYO OF THE FEOG;"
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Page 209, line 14. Instead of "substances" read "substance"; instead of " nerve " read " muscle."
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Page 218, line 1. After " on " insert " the development of."
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THE STEUCTUEE OF THE SPINAL COED OF THE OSTEICH.
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BY
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GEORGE L. STREETER, M. D.
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Assistant in Anatomy, The Johns Hopkins University, Baltimore.
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From the Dr. Senckenberg Anatomie, Frankfort-on-Main.
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With 6 Text Figures.
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It is related by Herodian how the Kaiser Commodiis beheaded ostriches and then watched them with delight and wonder as they continued running about the amphitheater, apparently to no great extent inconvenienced by the loss of their heads. That which served Kaiser Commodus as barbarous amusement frames itself for us into an interesting anatomical problem, and calls to mind a similar phenomenon so often observed among the domestic fowls. What is, then, this arrangement of the nervous elements of the spinal cord of a bird that enables it to functionate so completely after separation from the higher centers?
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Our present knowledge and methods do not suffice for a complete explanation of this problem, but we can lead the way toward a future solution if we study out what can be learned at present concerning the histolog)^ of the bird spinal cord. In this sense, under the suggestion and guidance of Professor Edinger, I have undertaken the investigation of the structure of the spinal cord of the ostrich (Struthio camelus). This, beyond all other birds, distinguishes itself by the great length of its spinal cord, and, in comparison with the brain, its great size.
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In the literature, frequent reference is made to the spinal cord of birds. As early as 1868 Stieda* presents what could be seen in unstained preparations. He gives a review of the previous literature reaching back to Steno, 1667, and Perrault, 1699. All of the older investigators of the spinal cord, such as Stilling and Clarice, have also studied more or less that of the bird, but it is the above mentioned work of Stieda that gave us first a clear and complete description. Of the more recent anatomists, mention is to be made of the works of Gadotv ' and Eolliker." A number of investigations have been made which were limi
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Stieda, Studien iiber das centrale Nervensystem der Vogel und Saugethiere.
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' Gadow, Bronn's Klassen und Ordnungen des Thierreiches. Bd. 6, p. 406. = Kolliker, Gewebelehre, 1896.
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American Journal of Anatomy. — Vol. III. 1
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2 The Structure of the Spinal Cord of the Ostrich
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ted to various parts of the cord, as, for example, the study of DuvaV concerning the Sinus rhomboidalis, and an experimental work of Friedlander* on the fibre tracts. To these may be added also the works of Singer, MiXnzer, and others who devoted themselves more particularly to the brain. It is, further, not to be forgotten that the studies of Retzius, Ramon-y-Cajal, van Geliuchien, and v. LenliosseJc concerning the nervecells and fibres of the spinal cord in Golgi preparations were carried out largely on the chick. No attention, however, seems to have been directed toward the spinal cord of the ostrich. A cross-section, apparently of the thoracic region, is pictured by Edinger^ but is not otherwise described. The material on which this study is based consisted of three ostrich spinal cords taken from the neurological collection of the Anatomic. Two were practically intact; the third had been cut into segments. All three had been hardened in formol. After the macroscopic examination was completed, series of transverse sections were made in all segments. Unbroken sagittal and fronto-longitudinal series were prepared through three segments of the lumbar enlargement, and a fronto-longitudinal series of one segment in the cervical region. Sections were also prepared of a decalcified vertebra showing the cord in situ with its membranes, the nerve roots, and spinal ganglia. Where other than the usual stains were used they are specified in the text.
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The Meninges.
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The cord is supported in the vertebral canal by a connective tissue sheath which, like that in mammals, may be described as consisting of three separate membranes or envelopes. In order, from within outwards, they are the pia, arachnoidea, and dura. These structures are represented in Fig. 1.
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Of the three envelopes the dura is by far the strongest. It is this that forms the tough fibrous sheath surrounding the cord, which one sees on the removal of the latter from the vertebral canal. It consists of a membrane .011 to .012 mm. thick, made of thickly-lying coarse fibres, a.nd contains no blood-vessels. Outside the dura is a connective tissue layer which lines the vertebral canal, and forms the periosteum of the vertebrse. This, having the same histological character, may be described
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^ Duval, Recherches sur le Sinus Rhomboidal des Oiseaux, Journ. de I'Anat. et de la Phys., 1877.
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Friedlander, Untersuch. iiber das Riickeninark und das Klelnhirn der
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V6gel, Neurolog. Centrabl., 1898.
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"Edinger, Nervose Centralorgane, 1900, p. 76.
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Georo-e L. Streeter
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3
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as belonging to the dura, and as forming its outer layer. The cleft between the two, the epidural cavity, is bridged over by loose strands of tissue supporting a plexus of blood-vessels.
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More or less adherent to the inner surface of the dura is the arachnoidea. Whether or not this, in the fresh state, is completely adherent to and possibly a part of the dura, could not be decided, as all the material used in this study had been through a prolonged hardening in formol. In the preparations at irregular intervals, they were still adherent, but in the greater part there was a separation of the two membranes, having more the appearance of an artificial tearing apart, or slirinkage forma
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Arachnoidea
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Epidural plexus
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Epidural space
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'achnoid space
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Mucl. marg' minor Dorsal rool Venlral rool
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Fig. 1. Cross-section through the 4th cervical vertebra of the ostrich, showing the spinal cord and its membranes. One side is drawn at a point somewhat higher than the other. Enlargement x 6.
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tion, than a natural cleft. In the space thus formed, there was no trace of serum, blood-cells, or other tissue. In cross-sections the arachnoidea shows itself as a delicate, thickly nucleated membrane, connected from its inner surface with the pia by a network of fine strands which form a meshwork of lymph spaces for the cerebro-spinal fluid, siibaraclinoideal cavity.
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The rootlets forming the ventral and dorsal nerve roots take their course through this loose tissue caudad or cephalad to the nearest intervertebral foramen, where they pass outward, piercing the dural sheath. In their course they carry along with them a connective tissue contri
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4 The Structure of the Spinal Cord of tlie Ostrich
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bution from the pia and dura, which, through the intervertebral foramina, is directly continuous with the peripheral nerve-sheaths; this tissue furnishes the capsule and framework for the spinal ganglia, which are found just external to the foramina and attached to the fibres of the dorsal roots.
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The pia, in contrast to the two more external membranes, forms, as we may say, an integral part of the structure of the cord, and serves to some extent as a framework, inasmuch as it is closely adherent to the peripheral layer of neuroglia and follows the outline of the cord entering all clefts and depressions. In the anterior median fissure it sinks to the bottom as a thick, strong lamella, septum ventrale, supporting, just ventral to the anterior commissure, the arteria meduUaris ventralis.
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The pia throughout is richly supplied with blood-vessels. Brandies from these supply the cord, penetrating from the periphery inward and from the arteria med. ventr. outward. The vessels carry with them a connective tissue adventitia derived from the pia. In no case, however, were processes of pia seen entering the substance of the cord except as accompanying blood-vessels. This is easily demonstrated in specimens over-stained with iron hgematoxylin and differentiated with picrofuchsin. In such preparations the vessels, together with their connective tissue support, are stained brilliant red in contrast to the yellow-brown neuroglia septa which might otherwise be mistaken for pia.
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-At three places in its circumference, the pia receives an accession of thick dura like connective tissue fibres, producing ligamentous formations which extend as three longitudinal bands, lens-shaped in crosssection. Two of these are situated laterally, Ugamenta longitudinalia lateralia, and one is situated at the attachment of the septum ventrale. Ugamentum longitudinale ventrale. The former corresponds to what Berger^ has described as the Ugamentum dentatum in reptiles. Between the 37th and 38th segments in the region of the lumbo-sacral enlargement, these bands reach a special development; they become much stronger and are modified in form. From the ligamentum long, ventr. pointed, tooth-like processes extend laterally to join the ligamenta long, lat. These processes fit closely in the intersegmental grooves, the sulci transversi, of the ventral surface of the cord. This is represented in Fig. 3, a.
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A resemblance between this structure and the diiral tissue is at once noticed, but it is identified as modified pia from the fact that the arach " Berger, Ueber ein eigenthumliches Riickenmarksband einigen Reptilien und Amphibien, Sitzb. Wiener Akad. Wiss., Bd. Ixxvii, 3 Abth.
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George L. Streeter 5
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noitl lies external to it, and separates it from the dura proper. In the intervening spaces the pia becomes thinner and web-like; here the eminentise ventrales bulge forward and along their lateral border give oft' the ventral nerve roots which pierce the pia jnst ventral to the liga
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VENTRAL
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DORSAL
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Lig. long, lac. Lig. long, venti
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Eminenlia ventr.
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Sulc. Irans
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Radix venlr.
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X.
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Fiss. long, venlr — (Comissura ant)
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Lig. Irans. ventr f area relic. PIA
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[ area ligam, -■
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Fiss. long venlr.
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0'
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I!!
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sulc. dors, med
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-^^ — Radix dors.
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- Funic, dors 5inus rhomb.
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5ulc, lal, Sulc. dors.lat.
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■ Sulc. dors, med.
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Fig. 2. Ventral and dorsal surfaces of the lumbo-sacral enlargement of the ostrich spinal cord, enlarged to IVs natural size. The pial sheath has in part been stripped off in order to show the eminentise ventrales. X indicates the situation of one of the nuclei marginales majores.
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.menta long. lat. Strong fibrous processes, Ugamenta denticulata, extend also lateral from the ligamenta long. lat. to the dural sheath and thus render further support to this region of the cord. See Fig. 5.
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6 The Structure of the Spinal Cord of the Ostrich
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Caudal to the lumbo-sacral enlargement, the pia returns to the more simple sheath-like form as seen in the thoracic and cervical regions.
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A work on the comparative and embryological anatomy of the spinal cord meninges has recently been published by Sterzi.^
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In the mammalian embryo (Ovis aries), 15 mm. long, the author describes a mesenchyma perimeningeale which first produces a definite spinal cord membrane in the embryo of 20 mm. This he calls meninx primitiva. In the 80 mm. long embryo this membrane is differentiated into an outer layer or dura mater, and an inner layer or meninx secondaria. The two are separated by an intradural space. The dural layer is separated externally by the epidural space from an endorhachide which Sterzi finds always distinct from the dura. In the 157 mm. embryo the meninx secondaria is further differentiated into an outer or arachnoideal layer and an inner or pial layer. In his comparative series the author finds the Petromyzon as representing the 20 mm. embryonal stage. The Eana esculenta and Lacerta viridis represent the 80 mm. stage. The development shown by the 157 mm. embryo with a differentiated arachnoid he finds only in the mammals. Our findings in the ostrich do not correspond with this. In Sterzi's series the birds are represented by Gallus domestica in which he describes a meninx secondaria not yet differentiated into pia and arachnoid. In the ostrich we find, as is above described, an arachnoidal layer which presents all the distinguishing features of that of the mammalian cord.
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GENERAL MACROSCOPIC DESCRIPTION.
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The abrupt change from the slender cervical spinal cord of the ostrich to the thick medulla oblongata gives a rather definite level at which the cephalic end of the cord may be said to be located. From this point extending caudal ly it stretches throughout the entire length of the spinal canal, its slender tapering end extending to the last coccygeal vertebra. It measures 81 cm. long in a small ostrich, the middle of whose back stands about 60 cm. above ground, and whose head in the ordinary upright position is 45 cm. higher, or 105 cm. above ground.^
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From each side of the cord throughout its length is given off a series of fine rootlets which unite, within the dural sheath in segmental bundles, to form the dorsal and ventral nerve roots. These, together, pierce the
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'Sterzi, Anatomia comparata ed all'ontogenesi delle Meningi midollari: Atti del Reale Institute Venento di Scienze, Lettere ed Arti, Tomo LX, 19001901.
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All the measurements hereafter stated are taken from this same specimen.
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Fig. 3. Topography of the spinal cord of the ostrich. The transverse sections are all made on the same scale of enlargement and their proper levels are indicated on the drawing.
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8 . The Structure of the Spinal Cord of the Ostrich
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dural sheath, leave the spinal canal, and form the spinal nerves, as is described under the heading Meninges. Owing to the fact that the roots have a short intravertebral course, leaving the canal directly, a bundle of them forming a cauda equina is not here present, and the nerves thus correspond in position to the segments of the cord and to the vertebrae. There are in our specimens 51 pairs of nerves. We may classify the nerves and segments after the morphology of the vertebrae as follows :
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Region. Cervical Thoracic
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No. of Nerve 15 8
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Pairs.
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Segments.
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1st to 15th.
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16th to 23rd.
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Lumbo-sacral
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19
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24th to 42nd.
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Coccygeal
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9
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43rd to 51st.
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The topography of the cord is represented in Fig. 3. It will be observed that corresponding to the wings and legs the cord is in two places increased in size, the brachial and Iwtibo-sacral enlargements. The former is so barely visible that one notices at first only the enormously developed lumbo-sacral enlargement. A more careful observation however discloses a slight increase in size in the region lying betAveen the 16th and 19th pairs of nerves. The difference in size is much more apparent in cross-sections.
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Fiirbringer * describes the plexus brachialis of the ostrich as made up of the spinal nerves arising from the 17th to 21st segments, and this corresponds to our cervical enlargement. It is this region, therefore, that we must think of as the sensory and motor center for the wing musculature.
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The remainder of the cervical and thoracic portion of the cord is nearly uniform in size, and on section shows a rounded circumference. A fissura ventralis longitudinalis is to be seen, but dorsally in this region no fissure is present. The segments in a way are marked off at the attachment of the spinal nerves by a slight dorso-ventral compression and a corresponding increase in size laterally.
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In the lumbo-sacral region an entirely different appearance is presented. It is in this region of the cord that the crural and sacral plexuses are attached which supply nerve-fibres to the leg. The system of reflexes which is necessary for the control of the massive musculature of this member demands a large accumulation of nerve-cells and comiecting nerve-fibres, and this accumulation forms the lumbo-sacral enlargement, the so-called " Lumbar Brain." As is stated above, the lumbo-sacral
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" Ftirbringer, Untersuchungen zur Morphologie und Systematik der Vogel. Theil I., Amsterdam, 1888.
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George L. Streeter 9
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region of the cord extends from the •24th to the 4:-2nd segment. About one-half of this space, from the 26th to 3Tth, is occupied by the lumbosacral enlargement.
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A feature which contributes largely to the peculiar appearance of this part of the cord is the change occurring in the posterior longitudinal sulcus. What was a barely-perceptible furrow in the cervical and thoracic cord becomes, at the beginning of the lumbo-sacral region, more distinct, and, where the 31st pair of nerves are given off, it rather abruptly widens out into a broad boat-shaped groove, the sinus rhomhoideus sacralis. This reaches ventrally to the commissura anterior, and spreads apart the posterior funiculi from the 31st tp 36th segment, at which point the sides again come together and are continued as the posterior longitudinal furrow. This sinus is filled with a delicate gelatinous tissue, the structure of which will be discussed later.
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A drawing of the dorsal surface is reproduced in Fig. 2, b; lateral to the sinus can be seen the sharply-defined dorsal funiculi increasing in size from below upward. Each dorsal funiculns is bounded laterally by a dorso-lateral groove, at a point corresponding to the tip of the dorsal horn. Entering this groove are the enormous dorsal nerve roots, grouped into segmental fibre bundles.
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Fig. 2, a shows the ventral surface of the enlargement. At two places the pial sheath has been left intact. In this part of the cord the pia is considerably modified from the form which is present in other regions. Beginning at the 26th segment there is a marked increase in the size of the thickened strips of the pial sheath, or ligamentous bands. The pia in the intervening spaces becomes thinner and more web-like. Between the 30th and 37th segments the ligamentum long, ventr. sends out tooth-like intersegmental processes which join the ligamenta long, lat., and the ligamentous structure thus formed affords a strong support where, owing to its specialized character, the cord demands more than ordinary protection.
 +
 +
On removing the pia, there is seen an enlargement of the fissura longitudinalis ventralis, which forms a sinus resembling, to some extent, the sinus rhomhoideus of the dorsal surface, though it is shorter and narrower. Moreover it is not filled with the gelatinous semi-transparent tissue as seen in that sinus, and at the bottom one can see the cross-fibres of the commissura anterior. The space where the fissura long, ventr. may be called a sinus', extends from the 31st to the 35th segment, and is 1.3 mm. wide.
 +
 +
The great increase in the anterior horn elements, which occurs in the enlargement, is segmental in character, and forms segmentally projecting
 +
 +
 +
 +
10 The Structure of the Spinal Cord of the Ostrich
 +
 +
masses of grey substance whose outline can be seen on the ventral surface of the cord as rounded elevations, eminentiae ventrales, which bulge forward through the ligamentous framework. There is thus formed a series of hill-like prominences separated by intersegmental grooves, sulci iransversi. In the grooves lie the lateral prongs of the ligamentum long, ventr. From the lateral border of the segmental elevations arise the motor nerve roots as a row of fine rootlets which pass through the web part of the pial sheath just ventral to the ligamenta long. lat.
 +
 +
On examining the lateral surface of the cord in the region from the 81st to 36th segment, one sees, just dorsal to the ligamenta long, lat., at the level of each sulcus transversus, a small oval greyish projection measuring 1.4 mm. long and 0.4 mm. wide. These projections are the nuclei marginales majores, or the Large Hofmann Nuclei. They are easily seen with the naked eye, but better with a lens and under water. A description of them will be included under the heading Nerve-Cell Groups.
 +
 +
Caudal to the lumbo-sacral enlargement the cord decreases abruptly in size and extends, gradually tapering, to the end of the spinal canal. There is no cauda equina. A section of the most caudal pieces of our specimen shows a central canal and a similar general arrangement of grey and white matter as present in other parts of the cord.
 +
 +
From Gadow's^" work we can localize the peripheral parts that are controlled by this region of the cord. Gadow describes the sacral plexus as consisting of three individual groups: plexus cruralis; plexus ischiadicus; plexus pudendus. The nervus sacralis, which, by means of its bifurcated root, joins the latter two plexuses, he locates in the ostrich at the 37th segment. The nervus furcalis, which separates the plexus iscliiadicus from the plexus cruralis, he places at the 31st segment. Thus the plexus cruralis is attached to the cord from the 27th to the 31st segment, or the cephalic half of the enlargement. We may, therefore, locate here the nerve-cell groups belonging to the trochanter muscles and the muscles situated on the medial and anterior side of the femur, to which area the plexus cruralis is distributed. The plexus ischiadicus arises by 7 roots from the caudal half of the enlargement, the 31st to 37th segment. The roots of this plexus unite to form nervus ischiadicus which supplies the massive group of muscles on the lateral and posterior sides of the femur and the muscles of the lower leg. Caudal to the 37th segment is situated the pudendal plexus which innervates the anal and genital musculature. Beyond the 43rd segment arise the delicate caudal nerves which supply the coccygeal muscles.
 +
 +
'" L. c, p. 406
 +
 +
 +
 +
George L. Streeter 11
 +
 +
Arrangement of White and Grey Substance.
 +
 +
A cross-section of the cord shows, in a general way, a central fourhorned area of grey matter surrounded by a much larger area of white matter. The two dorsal horns of grey matter separate off a portion of the latter forming the dorsal funiculi, so called in distinction to the remainder of the white matter, or ventro-lateral funiculi. The entline and relative size of these individual areas in different levels of the cord are shown in Fig. 3.
 +
 +
A great variation exists in the size of the dorsal and ventral horns, as well as the anterior commissure. These structures are apparently closely interrelated, as they undergo the size-variation in unison. All of them reach their greatest development in the lumbo-sacral enlargement. Of the ventral and dorsal horns, the latter show less increase in size in the two enlargements. In the cervical region the dorsal horns are reduced to a narrow strand of grey matter and fail to reach the border of the cord. The white commissure, commissura ventralis, connecting the two halves of the cord is present at all levels, and will be described more in detail in connection with the fibre tracts. The grey commissure from the 31st to the 36th segments entirely fails. Its place is filled by the tissue of the sinus rhomboideus.
 +
 +
A more exact knowledge of the total area of transverse sections made at different levels, and the relative area of the antero-lateral funiculi, the dorsal funiculi, and the grey substance was obtained by a method which allows the calculation of the areas in square mms.
 +
 +
In this method one makes a series of outline drawings (in our case the Edinger drawing apparatus was used) of the various segments on a sheet of evenly-rolled lead or tin foil. Thick cardboard can also be used when the drawings are large. The drawings of the individual segments thus outlined on the sheet of lead are all magnified on the same scale. A drawing is also made in a similar way and with the same enlargement of a square cm. which has been outlined in ink on a glass slide. The drawings of the different segments and of the square cm. are then cut out from the lead sheet, and the segments further cut apart into the different areas. These pieces are all separately weighed. The ratio then, between the weight of each individual part and the weight of the piece representing the square cm., is equivalent to the area of this part.
 +
 +
Sections were taken from each segment of the ostrich cord, and the area of the various fields was thus calculated. The sections were taken uniformly near the departure of the nerve to avoid the discrepancy that might occur from differences in the same segment. This variation in the upper part of the cord is hardly appreciable. In the lumbo-sacral enlargement, however, it is more marked, and we have a distinct segmental character given to the cord by the increase in the size of the ventral horns, which occurs in the middle of the segment. Taking the sections at the level of the roots has the
 +
 +
 +
 +
12
 +
 +
 +
 +
The Structure of the Spinal Cord of the Ostrich
 +
 +
 +
 +
further advantage that here the boundary of the dorsal funiculi is more sharply defined, owing to the larger number of entering dorsal root-fibres.
 +
 +
The results of the method in our case are represented in the adjoining table :
 +
 +
TABLE
 +
 +
SHOWING SIZE OF VARIOUS AREAS OF CROSS-SECTIONS OF CORD AT DIFFERENT LEVELS. THE NUMBERS GIVEN INDICATE SQUARE MMS.
 +
 +
 +
 +
Segment.
 +
 +
 +
Grey Matter.
 +
 +
 +
Veiitro-lateral Funiculi.
 +
 +
 +
Dorsal Funiculi.
 +
 +
 +
Total Area.
 +
 +
 +
3
 +
 +
 +
.7
 +
 +
 +
8.7
 +
 +
 +
.7
 +
 +
 +
10.1
 +
 +
 +
5
 +
 +
 +
.7
 +
 +
 +
9.1
 +
 +
 +
.7
 +
 +
 +
10.5
 +
 +
 +
7
 +
 +
 +
.7
 +
 +
 +
9.1
 +
 +
 +
.7
 +
 +
 +
10.5
 +
 +
 +
12
 +
 +
 +
.8
 +
 +
 +
9.2
 +
 +
 +
.7
 +
 +
 +
10.7
 +
 +
 +
13
 +
 +
 +
.9
 +
 +
 +
8.8
 +
 +
 +
.7
 +
 +
 +
10.4
 +
 +
 +
14
 +
 +
 +
.9
 +
 +
 +
8.9
 +
 +
 +
.7
 +
 +
 +
10.5
 +
 +
 +
16
 +
 +
 +
1.3
 +
 +
 +
10.9
 +
 +
 +
.9
 +
 +
 +
13.1
 +
 +
 +
17
 +
 +
 +
1.9
 +
 +
 +
12.2
 +
 +
 +
1.1
 +
 +
 +
15.2
 +
 +
 +
19
 +
 +
 +
1.7
 +
 +
 +
10.9
 +
 +
 +
.9
 +
 +
 +
13.5
 +
 +
 +
20
 +
 +
 +
1.3
 +
 +
 +
9.9
 +
 +
 +
.7
 +
 +
 +
11.9
 +
 +
 +
21
 +
 +
 +
1.2
 +
 +
 +
9.9
 +
 +
 +
.6
 +
 +
 +
11.7
 +
 +
 +
22
 +
 +
 +
1.2
 +
 +
 +
8.9
 +
 +
 +
.5
 +
 +
 +
10.6
 +
 +
 +
24
 +
 +
 +
1.5
 +
 +
 +
10.5
 +
 +
 +
.7
 +
 +
 +
12.7
 +
 +
 +
26
 +
 +
 +
2.1
 +
 +
 +
11.9
 +
 +
 +
1.1
 +
 +
 +
15.1
 +
 +
 +
27
 +
 +
 +
4.4
 +
 +
 +
14.9
 +
 +
 +
2.1
 +
 +
 +
21.4
 +
 +
 +
28
 +
 +
 +
6.3
 +
 +
 +
18.3
 +
 +
 +
3.1
 +
 +
 +
27.7
 +
 +
 +
29
 +
 +
 +
8.6
 +
 +
 +
21.9
 +
 +
 +
4.3
 +
 +
 +
34.8
 +
 +
 +
30
 +
 +
 +
9.6
 +
 +
 +
23.5
 +
 +
 +
5.i
 +
 +
 +
38.5
 +
 +
 +
31
 +
 +
 +
7.8
 +
 +
 +
18.9
 +
 +
 +
4.4
 +
 +
 +
31.1
 +
 +
 +
32
 +
 +
 +
7.2
 +
 +
 +
16.6
 +
 +
 +
3.5
 +
 +
 +
27.3
 +
 +
 +
33
 +
 +
 +
5.9
 +
 +
 +
14.3
 +
 +
 +
3.1
 +
 +
 +
23.2
 +
 +
 +
34
 +
 +
 +
4.8
 +
 +
 +
9.3
 +
 +
 +
2.1
 +
 +
 +
16.2
 +
 +
 +
35
 +
 +
 +
3.5
 +
 +
 +
6 9
 +
 +
 +
1.1
 +
 +
 +
11.5
 +
 +
 +
36
 +
 +
 +
1.9
 +
 +
 +
5.2
 +
 +
 +
.6
 +
 +
 +
7.7
 +
 +
 +
38
 +
 +
 +
1.0
 +
 +
 +
3.1
 +
 +
 +
.5
 +
 +
 +
4.6
 +
 +
 +
44
 +
 +
 +
.4
 +
 +
 +
.9
 +
 +
 +
.2
 +
 +
 +
1.5
 +
 +
 +
 +
These areas and their relative size are more graphically represented in the diagram given in Fig. 4. The size of the grey substance, the ventrolateral funiculi, and the dorsal funiculi in typical segments are represented by curves, the height of which signifies square mms. as shown by a scale on the left.
 +
 +
From this diagram it is apparent that the ventro-lateral funiculi form by far the greatest area at all levels. The proportion is much greater cibove than below the lumbo-sacral enlargement. This could be accounted for in part by the presence of tracts connecting the enlargement with the brain centers. In both the cervical and lumbo-sacral enlargements the increase in area of the ventro-lateral funiculi is greater than that of the grey matter and dorsal funiculi. This is doubtless due to the large number of association fibres which form a field of fine fibres surrounding the anterior horns.
 +
 +
 +
 +
14 The Structure of the Spinal Cord of the Ostrich
 +
 +
Between the curves which represent the grey matter and the dorsal funiculi there is a closer uniformity in size; although the former shows a greater increase in the regions corresponding to the wing and leg musculature.
 +
 +
Of all three curves on the diagram that of the dorsal funiculi indicates the smallest as well as the least variable area. It is smallest at the 44th segment, and presents practically no change as we proceed cephalad until the 36th segment. If we look at Fig. 2, b it is to be seen that the dorsal nerve roots from the 36th to 31st segment are enormously increased in size. Corresponding to the entrance of these large dorsal nerve roots, in the- same segments in the diagram there is an abrupt ascent of the dorsal funiculi curve. Attention is called to the fact that the increase in the size of the dorsal funiculi extends cephalad from the point of increased dorsal root fibres. Therefore we may assume that the collaterals in the dorsal funiculi extending caudalward from the dorsal roots are either very few in number or very small in diameter, and that the general course of the entering impulses is in the cephalic direction.
 +
 +
The descent of the curve of the dorsal funiculi from the 30th to the 26th segment is as abrupt as the previous ascent. While in a space of six segments the area of the dorsal funiculi was increased nine times in size, this area, four segments higher up, has lost already more than three-fourths of this increase, and so the area at the 26th segment is only one-fourth of that at the 30th. If we take for granted that all the fibres that leave the dorsal funiculi enter the grey substance, and that there is very little variation in the size of the fibres from the 30th to 26th segment (both of which facts are confirmed by microscopical study of the cross-sections) then we may say that three-fourths of the fibres present in the dorsal funiculi at the 30th segment have entered the grey substance before the 26th segment. In other words the course of the dorsal root fibres ivithin the dorsal funiculi is a short one, and not more than a small proportion of these fibres ever reach the medulla by this tract.
 +
 +
That which is apparent regarding the dorsal funiculi in the lumbosacral enlargement is seen again in the cervical enlargement, though in the latter it is less marked. Above the cervical enlargement the rate of accession and loss of fibres in the dorsal funiculi maintains a constant balance, and the curve of area runs as a horizontal line.
 +
 +
FINER STRUCTURE OF THE CORD.
 +
 +
By the usual methods of staining, the cord resolves itself into three elements: Neuroglia, which forms the general framework; Nerve-Cells
 +
 +
 +
 +
George L. Streeter 15
 +
 +
and Myelinated Axis-Cylinders, w^hich form the fibre tracts and make up the bnlk of the white substance. The histology of the cord will be discussed under these heads.
 +
 +
The neuroglia was studied in preparations stained by the iron haematoxylin picro-fuchsin method of Weigert. This method cannot be spoken of as a glial stain; on the contrary, the glia does not stain with fuchsia as in the original Van Grieson method, but remains a yellowish brown and is seen in sharp contrast to the brilliant red connective-tissue elements. By combining the original Van Gieson method and the Weigert modification we may study the glial distribution by a process of exclusion ; this permits the following general description :
 +
 +
The glia fibres are more numerous in the grey substance than in the white, and are more numerous in the ventral horns than in the dorsal horns. They form an especially thick mass in the region of the central canal. In the white matter on the periphery, adjoining the pia, is a rather uniform layer of closely-lying fi.bres which forms a glial sheath to the cord, the peripheral glia sheath. This layer, at a point corresponding to Lissauer'"s fasciculus, is thickened and extends into the substance of the cord as a broad strand to meet the tip of the dorsal horn, which fails to reach the border of the cord. This strand spreads laterally to the dorsal horn and forms the web-like formatio reticularis situated in the median part of the lateral funiculus.
 +
 +
In most sections another glial process is seen extending from the sulcus longitudinalis dorsalis toward the central canal, the septum, longiiudinale dorsale, supporting a blood-vessel with its connective tissue sheath. Aside from the peripheral sheath and the processes as mentioned, the glia of the white substance forms a more or less uniform framework, supporting the nervous elements proper. There remains to be mentioned a special modification of the glial arrangement associated with the formation of the sinus rhomboideus.
 +
 +
Sinus Ehomboideus.
 +
 +
A macroscopic description of this structure has already been given, and we have spoken of the delicate gelatinous tissue with which it is filled. From the study of a series of transverse sections through this region it is our conclusion that this tissue is not a new structure, but is identical with the peripheral glia sheath and the septum dorsale which have become modified in their histological character.
 +
 +
In sections through, the 29th segment there is a marked increase in the size of the sulcus longitudinalis dorsalis, which penetrates ventrally one-half the length of tlie septum dorsale and splits it in wedge-shape
 +
 +
 +
 +
16 The Structure of the Spinal Cord of the Ostrich
 +
 +
fashion. In the 30th segment the sulcus extends the entire distance to tlie grey commissure completely separating the dorsal funiculi and forming the cephalic end of the sinus rhomboideus. At this level a change in the character of the glia^hows itself in that part of the peripheral sheath between the ventral and dorsal nerve roots, as well as in the grey commissure and the adjoining divided septum dorsale. In these places instead of a compact mass of fibres the glia shows a looser and more sponge-like appearance. In the succeeding sections this glial modification rapidly increases in extent, coincident with the increase in the size of the sinus, and reaches its maximal development between the 30th and 36th segments. A drawing from this region is reproduced in Fig. 5, and a portion of the glial web is shown under higher magnification. It is thus seen that the peripheral glia sheath throughout the circumference of the cord, except at the attachment of the ligamenta denticulata, is changed into, or replaced by, a tissue consisting of enormous cells (.003 to .004 mm. in diameter), the body of each of which is filled with a transparent fluid of undetermined nature which crowds the small nucleus to one side, or the nucleus is suspended in the fluid supported by a slender stalk of cell tissue. It resembles fat tissue to some extent. It however fails in frozen section to stain with Herxheimer's solution of Fettponceau. In iron-hgematoxylin picro-fuchsin preparations there is no trace seen of connective tissue fibres. The cells remained unstained like the neuroglia cells of other parts of the section. By exclusion, then, we are led to consider them as modified neuroglia cells, though we unfortunately lack the definite evidence of a selective stain.
 +
 +
The sinus rhomboideus of birds has always been an object of interest to investigators, especially as to the character and significance of the gelatinous material with which it is filled. Of the earlier writers the work of Duval " may be referred to, the results of which were more or less confirmed recently by Kolliher.^' Both of these authors from embryological evidence agree as to the glial nature of the tissue filling the sinus. They, however, do not make mention of the presence of this weblike material around almost the entire circumference of the cord.
 +
 +
The grey commissure and the septum dorsale are entirely changed into this tissue, which thus fills the sinus as a broad network separating the blunt ends of grey substance and the dorsal funiculi and extending ventralward to the commissura ventralis. In the ventral part lies the
 +
 +
" Duval, L. c.
 +
 +
^-Kolliker, Ueber die oberflachlicheu Nervenkerne im Marke der Vogel und Reptilien. Zeitschrift f. wiss. Zool., LXXII, 1.
 +
 +
 +
 +
George L. Streeter
 +
 +
 +
 +
17
 +
 +
 +
 +
central canal held in suspension by a few coarser strands of glia fibres which lie among the cells and bridge over the space separating the grey matter. I'liis meshwork formation of these modified glia cells extends somewhat into the territory of the white substance along the borders of the dorsal, lateral and ventral funiculi. Under low power this ragged edge of white substance gives the deceptive appearance of an artifact.
 +
 +
From the 38th segment caudalward there is a gradual retrogression of neuroglia to the form as previously described.
 +
 +
 +
 +
 +
 +
Invasioaof v/hite substance.
 +
 +
 +
 +
Lal.Jroup gang ce.
 +
 +
 +
 +
Nucl. margindli major
 +
 +
 +
Ligamenlum denlic Psnph glia unmodified
 +
 +
Periph glia modified
 +
 +
Ligamenlum long'ventr
 +
 +
 +
 +
L gameiilum long lal, (Ligamentum dcntic)
 +
 +
Ventral root fibres.
 +
 +
 +
 +
Anterior commissure Central C=ip.il
 +
 +
Fig. 5. Cross-section of the lumbo-sacral enlargement of the ostrich spinal cord, ai the 36th segment, enlarged 12 X- A portion of the sinus rhomboideus tissue is shown above with an enlargement of 270 x
 +
 +
 +
Central Canal.
 +
 +
The cylindrical epitlielial cells lining the central canal form a layer .007 to .015 mm. thick which is supported by a thick mass of glial tissue, 2 •
 +
 +
 +
 +
18 The Structure of the Spinal Cord of the Ostrich
 +
 +
the substantia gelatinosa centralis, in the middle of the grey commissure. Where the grey commissure is lacking, in the region of the sinus rhomboideus, the central canal is supported just dorsal to the commissura ventralis by the loose strands of glial fibres which bridge over the space between the blunt ends of grey substance.
 +
 +
The lumen of the canal varies in irregular manner from round to oval, and Avhere it lacks the support of the grey commissure it is no more than a narrow slit. Where it is round or oval it has a diameter averaging from .035 to .04 mm. In both cross and longitudinal stained preparations there is seen within the lumen the so-called Rcissner'sche C entralfaden. Kolliker " in a recent contribution gives the opinion that it is a " natiirliche Bildung beim Vogel, Eeptilien, und Amphibia," and also finds in it " eine iiberraschende Aehnlichkeit mit einem Achsencylinder." This is contrary to Gadoiv " who considers it a product of shrunken cerebro-spinal fluid and lymph corpuscles. In favor of the view as held by Gadow may be stated the three following facts : The structure shows a marked and irregular variation in form and size in different sections; in some transverse sections it was seen as multiple " Centralfaden " ; in sections stained with toluidin blue it retains a deep blue stain while the axis-cylinders in all other parts of the section are unstained.
 +
 +
Xerve-Cell Groups.
 +
 +
The, majority of the nerve-cells of the spinal cord of the ostrich are situated in the grey matter of the ventral horn. There are, however, many cells in the grey commissure and the dorsal horn, and there are still other cells among the fibres of the white substance, especialty near the periphery. These cells vary at different levels in their form, size, and manner of grouping. For their descriptions the following classification has been found advantageous:
 +
 +
1. Lateral Group — a. Lateral cells.
 +
 +
b. Dorso-lateral cells.
 +
 +
c. Ventro-lateral cells.
 +
 +
2. Central Group — a. Small mixed cells.
 +
 +
b. Giant cells.
 +
 +
3. Commissural Group—
 +
 +
i 4. Dorsal Group — a. Clarke cells.
 +
 +
b. Dorsal horn-cells. 5. Peripheral Group — a. Nuclei marginales majores.
 +
 +
b. Nuclei marginales minores.
 +
 +
c. Scattered cells.
 +
 +
" Kolliker, L. c, p. 159. " Gadow, L. c, p. 338.
 +
 +
 +
 +
George L. Streeter 19
 +
 +
The lateral group consists of more or less imiformly large multipolar cells, which in finer histology closely resemble the motor cells of the ventral horns of the higher vertebrates. Their distribution in typical sections is shown in Fig. G. They are seen in every section, but vary in number, being most numerous in the lumbo-sacral region and least numerous in the cervical segments. Corresponding with the number there is some variation in the size; those in the cervical segments average .03 mm. in diameter, while in the lumlio-sacral region there are many cells over .04 mm. This group may be further subdivided into cells having lateral, dorso-lateral, and ventro-lateral positions. A particularly well-defined group of the ventro-lateral cells occurs in the region of the sinus rhomboideus (Fig. 6, segm. XXXYI).
 +
 +
If we compare this lateral group with the cells of the human cord as classified by Waldeyer it is apparent that it corresponds to his median and lateral groups, each of which he subdivides into anterior, middle and posterior subgroups. Tlie cells of the lateral group in segment XXXVI coidd have been separated in a similar manner into a median and a lateral group; the ventro-lateral cells would then Avell correspond to Waldeyer's median group, and the lateral group could l)e further sulidivided into anterior, middle and posterior groups. Such a classification in the ostrich however serves only irregularly and for isolated segments, and therefore this distinction between the cell groiips was not attempted ; but all the large multipolar cells of the ventral horns, the so-called motor cells, were put under the one general class, the lateral group, as described above.
 +
 +
]\Iost of the cells of the lateral group apparently send their axis-cylinders into the ventral nerve-roots. The axis-cylinders of the ventrolateral cells, however, seem to enter the commissura ventralis. Xo attempt to establish such relations could be made without Golgi preparations, and these unfortunately were not to be had from our material.
 +
 +
The central group occupies the area of junction of the ventral and dorsal horns, and invades the territory of the horns proper. It consists of loosely-scattered cells which vary greatly in size and average a third smaller than the cells of the lateral group. They also stain less intensely and have fewer processes, consequently having less tendency to a multipolar form.
 +
 +
In the lumbo-sacral enlargement there appear cells among this group which from their size we may speak of as giant cells. They are quadrilateral or rounded in shape, and vary from .03 to .09 mm. in diameter.
 +
 +
" Waldeyer, Das Gorillariickenmark, Abhandl. der kgl. preuss. Akad. der Wissensch. zu Berlin, von Jahre
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20
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The Structure of the Spinal Cord of the Ostrich
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They are distinguished from the Clarke cells and cells of the lateral group by having fewer processes, by their tendency to easy disintegration, staining less intensely and having finer granules in the cell body. These cells are present throughout the whole enlargement, but are more numerous in the upper part (37th to 31st segments). A
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C£NT(7AL'
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SeOm.V.
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DORS. L AT.
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Segm.XVII.
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DOPtSAl^
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5egm. X X.
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I-ATC««L<
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DORS. L/\T.>
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^^^mi.
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Segm.XXIX.
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Segm. XXXVI.
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i,«"r£;R/M_
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Segm.XLIV.
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C£"/w rffflu
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Fig. 6. Cell-groups in the grey substance of the spinal cord of the ostrich.
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few are also seen in the 13th to 16th segments, just above the cervical enlargement. As can l)e seen in Fig. 6, segm. XXIX, they are scattered over the entire area of the central group. Very often they are seen on the extreme ventral or dorsal border of the grey matter. Thelargest number seen in any one section (20 ^u, thick) was eight. These giant cells present a striking similarity to the large cells seen in the lateral group of the nucleus funiculi gracilis of the human medulla.
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George L. Streeter 21
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The (ominissnml group is made up of a compact group of small intensel3^-staining multipolar cells, which are found in the grey cominissure in the thoracic division of the cord, from the 20th to 27th segments. It suggests, by its position, a possible relation with the viscera.
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The dorsal group includes in sections through the 26th to 31st segments a small group of cells on the median border of the grey matter at the junction of the two dorsal horns. The cells of this group resemble tliose of the lateral group, though slightly smaller. From their similarity, in position and appearance, to the group in the mammalian cord these are classed as Clarke cells (see Fig. 6, segm. XXIX). Otherwise as. noteworthy are classed under the dorsal group the occasional small multipolar or spindle-shaped cells, which are seen on the periphery of the dorsal horn both median and lateral, and frequently on the tip of the horn near the entrance of the dorsal root.
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Peripheral Group. In 1889 Lachi^" described a peripheral group of nerve-cells forming a series of segmental projecting nuclei, occurring in the lumbo-sacral enlargement of the spinal cord of doves. This nucleus was seen later by Gaslell and Schafer but attracted little attention until KdUiker" originally unaware of Lachi's work, published the results of a most complete study of this structure, both in the embryo and the adult bird. Kolliker finds three varieties of peripheral cell-groups, namely: Hofmann'sche Grosskerne, so named after his Praparator P. Hofmann, who had called his attention to them; Hofmann'sche Kleinkerne; and a scattered group.
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In the embryonal cord, 41/2- to 5-day chick, Kolliker describes a group of cells separating itself from the superficial cells of the ventral horn. This group in the 10-day chick is completely separated and forms a definite peripheral nucleus, the Hofmann'sche Kerne. There are 28 of these nuclei on each side of the cord, segmentally arranged according to the 28 spinal nerves and ganglia. Of these nuclei the 5 or 6 pairs, corresponding to the level of the sinus rhomboideus, undergo a marked development, and in the 15-day chick can be seen bulging from the periphery of the cord just dorsal to the ventral nerve roots, Hofmann'sche Grosskerne. The nuclei in the other regions of the cord do not share this development, but remain more or less
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'" P. Lachi, Alcune particolarita anatomische del ringonfiamento sacrale nel midollo degli uccelli, Memorie della Societa Toscana di Scienze Naturali. Vol. X, Pisa, 1889.
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" Kolliker, a. — Ueber einen noch unbekannten Nervenzellenkern in Riickenmark der Vogel, Akad. Anzeiger (Wien), Nr. XXV, 1901. b.— Weitere Beobachtungen iiber die Hofmann'schen Kerne am Mark der Vogel, Anatom. Anzeiger, Bd. XXI, Nr. 3, 1902. c— L. c.
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22 Tlie Structure of the Spinal Cord of the Ostrich
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rudimentary, the Hofmann'sche Kleinkerne. The third or scattered group is made up of cells similar to the lateral group cells of the ventral horns, and occurring at irregular points on the periphery of the ventro-lateral funiculi, more especially near the exit of the ventral nerve roots and near the Hofmann'sche Grosskerne. Kolliker considers these cells to he detached elements from the ventral horns.
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In the ostrich the occurrence and arrangement of peripheral cells is similar to that found by Kolliker and Ijachi in the dove and hen. In describing them we follow Kolliker's classification, but would substitute more descriptive names.
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The nuclei 'marginal es majores (Lobi accessori. — Lachi : Hofmann'sche Grosskerne. — Kolliker) lie on each side of the cord Just dorsal to the ligamenta longitudinalia lateralia at levels marked off by the sulci transversi ventrales. Of these nuclei fi pairs could be readily seen with the naked eye. They appear as elongated oval greyish semi-translucent elevations measuring macroscopically 1.0 to 1.4 mm. long. The interval between successive nuclei averages 6.0 mm. Each segment of the lumbo-sacral region was cut in transverse or longitudinal series, mostly the former. In studying these sections this nucleus was identified in the 30th, 31st (32d injured in removing the cord), 33d, 34th, 35th, and 36th segments. Thus the nucleus occurs in the region of the sinus rhomboideus, extending a little cephalad as well as somewhat caudad to it. Microscopically in the preparations the nucleus is seen projecting from the lateral border of the cord Just dorsal to the attachment of the ligamentum denticulatum, as is represented in Fig. 5.
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The size of the nuclei averages among the larger ones .10 to .18 mm. antero-ventral diameter, and .08 to .13 mm. lateral diameter. In a continuous series of sections 20 /x thick the nucleus is present in 62 ; that is the nucleus is 1.24 mm. long. These dimensions are somewhat smaller than the macroscopic, as could be expected from shrinkage associated with the embedding process, and possibly partly due to greater accuracy in measuring, the l^oundaries of the nuclei being more definite in the prepared and stained specimen.
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The free border of the nucleus is overlapped by pia, and the inner border merges gradually into the wliite substance of the cord. It consists of a network of glia tissue, somewhat looser and more vascular than the adjoining cord. In this sponge-like framework lie a number of multipolar nerve-cells and myelinated axis-cylinders. The cells resemble those forming the lateral group of the ventral horn, but are not more than one-fourth to one-sixth as large. In one nucleus 10 of these were seen in which the cell nucleus was cut throug-h. In the
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George L. Streeter 33
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majority of sections there are not more than 5 such cells present. The myelinated axis-cylinders have mostly a longitudinal course, and are about the same size as those in the neighboring periphery of the cord. Throughout the greater part of the nucleus they are uniform in number, 112 were counted in one section, but such a count is subject to error as it is often difficult to say whether the fibres belong to the lateral funiculus or to the nucleus owing to the indistinct inner border of the latter. In studying a complete series of transverse sections through the nucleus, prepared after Weigert's myelin-sheath method, one gets the impression that these large axis-cylinders belong properly to the lateral funiculus. In such sections near its caudal and cephalic ends the nucleus appears as a small island of increased glia tissue lying in the midst of the axis-cylinders near the border of the cord. In the succeeding sections this glia tissue rapidly increases in amount and envelops and carries with it the surrounding nerve-fibres, until finally it bulges from the side of the cord as an exuberant overgrowth. The large size of the nerve-fibres compared to the cells of the nucleus, and their uniformity in number at difl'erent levels, would also lend support to the view that they are independent of the cells and not properly a part of the nucleus. There are, however, a certain number of fine axis-cylinders seen in the sections, both with longitudinal and oblique course, which may be related to the cells embedded in the nucleus.
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The nuclei marginnles minores (Hofmann'sche Kleinkerne) are seen in sections taken from the cervical region at levels where the nerve roots make their exit from the dural sheath and vertebral canal. Their size and general position are indicated in Fig. 1. They do not project from the periphery of the cord and have no appearance of activity. The cells are small and are not definitely multipolar. The glia in which they lie is only slightly increased over that present in other regions of the periphery of the cord.
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The scattered group includes multipolar cells similar to those of the ventral horn, both in shape and size. One or two of these are found in nearly all sections of the lumbo-sacral enlargement, lying among the fibres of the periphery of the ventro-lateral funiculi. They are found most often near the nuclei marginales majores, or among the fibres leaving the cord as the ventral root. It is this group that Kolliker regards as detached elements from the ventral boras.
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In regard to Kolliker s^^ suggestion of a relation between the Hofinann'sche Grosskerne and the enormous size of the commissura ven 1'^ Kolliker, L. c. (c), p. 176.
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24 The Structure of the Spinal Cord of the Ostrich
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tralis we may state the fact that a longitudinal ventro-dorsal section cut through the commissura ventralis, from the 32nd to 34th segment, shows that the commissure here is practically imiform in the ventrodorsal diameter. It presents no segmental increase in size at the levels of the Hofmann'sche Kerne which would be expected if the size of the commissure in the lumbo-sacral enlargement were due to the presence of these nuclei.
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Fibre Tracts.
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Myelinated fibres are present both in the grey and white substance of the cord. In the former they are seen in the preparations in cross and longitudinal section, and form a network which cannot be resolved into definite fibre tracts.
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The great bulk of the spinal cord fibres make up the white matter, and form a thick envelope surrounding the grey substance. This envelope may be separated into ventral, lateral and dorsal funiculi. The boundary between the first two is an artificial one, produced by the fibres of origin of the ventral nerve roots. At levels where those fibres are few or absent there is no point of division between the two funiculi.
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The dorsal funiculi are more sharply defined. They are separated from each other by the septum posterior, and separated from the lateral funiculi by the dorsal horns and the glial processes which extend from the tip of the horns to the peripheral sheath of the cord.
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The general variation in size and shape of the dorsal funiculi occurring at different levels of the cord can be seen in Fig. 3. The definite area is recorded by a table and by Fig. 4, in which a diagram gives the area in a curve indicating square mms. Thus a further mention of the shape and size of these funiculi is here not necessary.
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In their finer structure the dorsal funiculi consist of fibres of entrance and departure, and fibres having a longitudinal course. The bundles of fibres entering as the dorsal nerve roots vary greatly in size, as is seen macroscopically. Those in the lumbo-sacral enlargement are two or three times larger than those in the cervical enlargement, and about five times larger than those of the upper cervical region. These fibres enter obliquely as a compact bimdle at the dorso-lateral border of the funiculi. The bundle then breaks up into loose strands, disappearing among the longitudinal fibres. No fibres could be seen to enter the grey matter directly. In longitudinal sections most of the fibres could be seen to bend upwards, and could be traced a short distance in the longitudinal direction. A few fibres were seen which, on entering, turned caudalwards. In neither Van Gieson nor Weigert prej)arations, how
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George L. Streeter 25
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over, was a " Y " form seen, where the entering fibre had both a eephalad and caudad collateral. That so few fibres take a downward course accounts for the fact that in the 35th and 36th segments, where there is a pronounced increase in the size of the dorsal nerve roots, the corresponding increase in the size of the dorsal funiculi is in the cephalic direction. Furthermore, the course of these fibres in the dorsal funiculi cannot be a long one, and this is shown by the rapid decrease in the size of the funiculi coincident with the decrease in the number of entering fibres, a fact which we have already referred to in the consideration of the diagram, Fig. 4.
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The collaterals from the dorsal funiculi to the grey matter vary in number in correspondence to the number of fibres from the dorsal nerve roots. In the lumbo-sacral enlargement these fibres enter the dorsal horn as a large, strand of fibres which could be traced to the region at the base of the horn. In the cervical region fibres entering the grey matter are found only as single separate collaterals.
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No definite subdivision of these funiculi into separate fasciculi or tracts could be made. In general, however, the fibres of the ventral one-third are smaller and form a triangular field of fine fibres, averaging .2 fi. These are apparently association fibres. This field is not present in the lumbo-sacral enlargement; here the grey commissure is absent, and the dorsal funiculi are separated by the sinus rhomboideus and lie further dorsal. The size of the fibres of this enlargement is uniformly large, the myeline ring in Weigert preparations averaging 1.0 to 1.5 fjb. We have already seen that the majority of the fibres of this region do not remain in the dorsal funiculi for a course of more than 3 to 4 segments, and that a small proportion of them reach the medulla through this tract. It would seem, then, tliat large fibres do not necessarily indicate long fibres; because in the lumbo-sacral enlargement the fibres are uniformly large and it is right here that we have shown that at least three-fourths of the fibres have a course in the dorsal funiculi shorter than 4 segments.
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The lateral funiculi present an inner zone of fine fibres and an outer zone of coarser fibres, the latter fibres averaging 1.0 yu,. The inner zone, or formatio reticularis, makes up a third to one-half the area. It is connected with the grey substance by numerous radiating strands of fibres, and apparently consists of association bundles. The outer field is connected with the central grey substance by less numerous strands of fibres. It is in this outer zone that Friedldnder^^ found ascending
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^^ Friedlander, L. c.
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26 The Structure of the Spinal Cord of the Ostrich
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and descending cerehellar tracts by experimental secondary degeneration in doves.
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The ventral funiculi have an inner zone which is a ventral extension of the inner zone of the lateral fnnicnli. The outer zone, tractus cerebello-spinalis ventralis medialis of Friedlander, is somewhat larger, and forms a more or less triangular field, of which the fissura ventralis forms one side. The fibres of this field are all large and average 1.5 /x, many of them being over 2 fi. In the Imnbo-sacral enlargement the enormous increase in size of the ventral and lateral funiculi seems due to an accession of smaller fibres which are added to the inner zone, and this increase is more marked in the ventral than in the lateral funiculus.
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A commissura alha anterior of ol)liquely crossing fibres is present at all levels of the cord, connecting the two ventral funiculi. It is greatly increased in size between the 28th and oGth segments. A sagittal section through the commissure in this region does not show any segmental grouping of these fibres. In Weigert preparations strands of fibres can be traced through the commissure coming from the outer zone of tlie ventral funiculus and extending to the opposite ventral horn. We have here doubtless a motor tract from higher centers, the fibres of which decussate before ending about the cells of origin of the motor nerve roots. The large number of ventral horn-cells in the lumbosacral enlargement woidd thus partly explain the large size of the commissure which here prevails. No trace of commissural fibres dorsal to the grey commissure was found in any of our sections. A posterior white commissure is apparently lacking.
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resume'.
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In looking back at the more important characteristics presented by the spinal cord of the ostrich, a feature to be first referred to is that in its mass the cord forms by far the largest part of the central nervous system. In other words, then, we have here an animal the various parts of whose body receive their principal innervation from the spinal cord, and the influence of the brain on these parts is secondary and remote — an animal that works chiefly with its primary apparatus.
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This suggestion as to the important part played by the primary nervous complex is further confirmed by the fact that the grey substance and associating collaterals vary in amount at different levels accordinfj to the demands made by the parts supplied. Thus throughout the cervical cord where there is a small and uniform number of neck muscles to be supplied the primary apparatus presents a correspondingly small
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George L. Streeter 27
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and uniform size. It is increased in the region supplying the wing musculature. A relatively greater increase would he expected in flying hirds, the comparison of the ostrich with one of the large hirds of prey would he interesting. When we go farther caudalwards and come to the increase of the primary apparatus corresponding to tlie massive leg musculature we find a great tumor-like enlargement, or Locomotor Brain, which demonstrates, as perhaps nowhere else in the animal kingdom, the close interdependence hetween a section of the central nervous system and the area innervated.
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An interesting feature of the lum1)o-sacral enlargement is the manner in which the neuromeres are marked off on the ventral surface of the cord by the hill-like prominences, calling to mind the segmental appearance presented by the well-known Trigla cord.
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The marked development of the sinus rhoml)oideus offers favorable conditions for the study of this characteristic feature of the bird cord. We are enabled to contribute some facts as to the nature of the peculiar tissue with which this sinus is filled.
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In studying the finer structure of the cord, the grouping of the cells into defined columns could be followed, some of which extend throughout the length of the cord. Two particularly interesting groups were found, one limited to the thoracic region in the posterior grey commissure, the other a group of " giant " cells occurring in the lumbo-sacral and cervical enlargements. The segmental groups of cells or nuclei occurring on the periphery of the cord, which have recently been the subject of much attention, are found in the characteristic way, and moreover are here present as macroscopic structures.
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Our material was not such as to allow us to say anything of especial importance concerning the fibre tracts that would be new for the bird spinal cord. In this direction we can only look for advancement from experimental work such as was begun by Friedlander in this laboratory. Attention, however, is to be called to the short course taken by the fibres in the dorsal funiculi, and to the small proportion of these fibres that eventually reach the higher centers through this path directly.
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THE DEVELOPMENT OF THE HYBEIDS BETWEEN FUNDULUS HETEEOCLITUS AND MENIDIA NOTATA WITH ESPECIAL EEFEEENCE TO THE BEHAA^OE OF THE MATEENAL AND PATEENAL CHEOMATIN.
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BY
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WILLIAM J. MOENKHAUS, Ph. D. With 4 Plates.
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contents.
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page pace
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I. Introduction 29 4. General Review of Literature 41
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II. Material and Methods 30 5. Conjugation of Pronuclei and the
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III. Nomenclature 31 First Cleavage 43
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1\ . Fertilization 32 6. Second Cleavage 44
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V. Development 33 7. The Rotation of Nuclei 46
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1. Cleavage 33 8. Third Cleavage 47
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a. Form of Cleavage 33 9. Fourth Cleavage 48
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b. Rhythm of Cleavage 35 10. Later Cleavage *8
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2. Development of Dispermic Eggs. 36 11. Comparison With Other Forms. . 50
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3. Later Development 37 12. Maternal and Paternal Nucleoli. 52
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VI. The Individuality of the Maternal 13. The Persistence of the Individual
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and Paternal Chromosomes 39 Chromosome 53
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1. Introduction 39 Summary 54
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2. Material and. Methods 39 Papers Cited -.-.... 56
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3. Description of the Chromosomes. 40 Explanation of Plates 58-64
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• I. Inteoduction.
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During the summer of 1899, while endeavoring to find two species of fishes that I could readil}^ hybridize with the view of making certain variation studies on hybrids, I began what has since grown into a rather extensive series of experiments on the limits of crossing in fishes. Among many crosses effected, the one which proved of special interest both then and since, is that between Fnndulus heteroclitus and Menidia notata. The results of the other crosses I have reserved for another paper. In the following pages only the results obtained on the above-named hybrid are considered.
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In addition to their availability, the long period over which they spawn and the ease with which they can be hybridized, the reason for making a special study of the hybrids between Fundulus heteroclitus and Menidia notata, is the fact that the cliromosomes of the two species can be readily distinguished morphologically. This fact is a distinct advantage in following out the nuclear history with reference to the im Ameeican Journal op Anatomy. — Vol. III.
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30 The Chromatin in the Development of Hybrids
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portant question of the individuality of the maternal and paternal chromosomes during the development of the hybrids. Before taking up this question, a brief description of the impregnation, cleavage and later development of these crosses will be given.
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Among the many to whom I am under obligations for favors, I wish especially to mention Professor Charles B. Davenport, not only for much help during the progress of the w^ork, but also for first directing my attentions to the possibilities in this line of experimentation. I wish also to especially thank the United States Commissioner of Fish and Fisheries, Geo. M. Bowers, for continual privileges at their Woods Hole Marine Station.
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II. Materia^ and Methods.
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Fundulus heteroclitus and Menidia notata are among our most common coast fishes. Both species can be obtained in any desired number in the bays along our eastern coast. They spawn over a period of about six weeks, beginning the latter part of May. The spawning period must be about the same, since I have always been able to obtain ri]5e individuals of both species at the same time during the period above mentioned. The eggs of Fundulus heteroclitus are the larger, measuring 13-14 to the inch. I have taken as many as 599 eggs from a single large female, but the number obtained is usually considerably smaller than this. The eggs of Menidia notata measure on the average 26 to the inch. It is easy to get several hundred eggs from a single female. A rather large, well-filled female yielded, by actual count, 1413 eggs. The eggs of both species flow readily if properly handled. Those of Menidia often flow so easily as to make it difficult to handle a ripe individual without losing a portion of the spawn. Menidia notata has a much greater abundance of milt, so that it can easily be expressed as a thickish, perfectly white fluid. It is less easy to express the milt from Fundulus heteroclitus, so that I have usually found it preferable in my experiments to cut out the testes and tease them apart over the eggs. The two species belong to two distinct orders, Fundulus to the Haplomi and Menidia to the Acanthopteri.
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The eggs were, in all cases, fertilized in small watch-glasses. All the eggs desired for any given experiment were first expressed into this watch-glass. Sometimes the eggs of a number of females were placed together when a large lot was desired. The milt was then added and after ten or fifteen minutes the contents were emptied into a fingerbowl of fresh sea-water. By a series of washings the excess of milt and the defective eggs were removed. The water was renewed two or three
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William J. Moenkhaiis 31
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times a clay and the eggs were allowed to develop as far as they would. During the two-, four- and eight-cell stages the per cent of eggs impregnated and the character of the impregnation was determined. The normally impregnated eggs were isolated and their further development watched from time to time, and the desired stages preserved.
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The necessity of proper precautions is, of course, evident, to prevent contamination by the introduction of other sperm than that desired. These, in all the experiments, consisted in (1) carefully sterilizing all the vessels and instruments that were used in the experiments; (2) keeping the two sexes of the same species in separate aquaria; (3) carefulh^ washing the hands and the fish at the time of the experiment ; and (4) carrying a control lot of eggs, taken from the same lot used for the experiment, in a separate fingerbowl containing water from the same source as that used on the eggs that were hybridized. I may say here, that in none of the experiments did I find a single egg of the control lots that showed any signs of development; so that there is no doubt that in all of the experiments the possibility of an error from contamination has been eliminated.
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Not all the lots of eggs that may be obtained during the spawning season of a species will show the same per cent of impregnation, even with sperm taken from the same species. In order, therefore, to obtain a more reliable estimate of the percentage of hybrid eggs impregnated, it was essential in each experiment to fertilize a sufficient number of eggs from the same lot to serve as an index of the normal condition. Furthermore, it was most essential in determining whether the development of the hybrids was going on under favorable conditions, to carry along with them some normal eggs under the same conditions.
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The developing eggs were kept under close observation from the time of impregnation to their death. Extensive notes were taken on the living eggs and desired stages were preserved in a variety of killing fluids — Perenyi's, Picro-acetic, Zenker's; and for surface study Child's method, of first placing the eggs for about a minute in a corrosive-acetic solution and then, after rinsing in water, in 10 per cent formalin, was used. This latter method in both species of eggs leaves the egg membrane and yolk beautifully clear while it turns the protoplasmic portions white, thus making an ideal preparation for surface study.
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III. Nomenclature.
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For clearness and brevity's sake in the following discussion I have found it desirable to adopt certain expressions which should here be
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32 The Chromatin in the Development of Hybrids
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defined. An egg or embryo obtained by using Fundulus heteroclitus as the female, is designated as the Fundulus egg, embryo or hybrid, as the case may be. The reciprocal, with Menidia notata as the female, will then be a Menidia egg, embryo or hybrid. A normal egg, embryo or cross is one in which both parents belong to the same species, in distinction from a hybrid egg or embryo in which the two parents belong to different species.
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IV. Fertilization.
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1. Fundulus lieteroditus, female, and Menidia notata, male.
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The cross in which Fundulus heteroclitus was used as the female was made ten times. In three of the experiments the males had died before the milt was taken. In one of these the male had been dead for an hour but the milt was normally white and the results of the experiments could not be told from those in which the males were alive and vigorous.
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The per cent of eggs impregnated was not determined for all the ten experiments. In all the experiments, however, it was considerably above 50 per cent. Below are given the per cents based on actual count of four experiments. The per cents in these range from 70 to 93.
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 +
Experiment No. 24b 87 per cent.
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" 25b 80
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" " 29b 93 "
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"120 70
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 +
Of the eggs impregnated, approximately 50 per cent in each experiment were normally impregnated, the remainder were, with a very few exceptions, disperic.
 +
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2. Menidia notata, female, Fundulus liet^roclitus, male.
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 +
The cross in which Menidia notata M'as used as the female was made 8 times. Two of .these experiments were made at Cold Spring Harbor during the summer of 1898, and the remaining six at Woods Holl two years later. The results obtained at the two places were not the same. At the former place the per cent of eggs impregnated was very small, namely 14, while at the latter place the impregnation was nearly perfect, 96 per cent. The difference may be due to the fact that at Cold Spring the mother fish had to be transported for half a mile in a bucket, so that they were dead or almost so by the time that the eggs were procured. The experiments at Woods Hole were, on the other hand, carried on under the most favorable circumstances ; the mother fish being alive
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William J. Moenkhaus 33
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and vigorous at the time the eggs were taken. I think, therefore, that the eggs of Menidia notata can be almost perfectly impregnated by Fundulus heteroclitus.
 +
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The character of impregnation is in striking contrast to that of the reciprocal cross, in that practically all the eggs fertilized are normally impregnated. There was an occasional dispermic and some polyspermic eggs. This is true whether the per cent of eggs impregnated is small or large. None of these dispermic or polyspermic eggs were isolated to see how far they would develop, nor preserved for the study of their internal character. This difference in the character of impregnation in reciprocal crosses I have found nowhere so strongly marked in any of the many other crosses I made among fishes. The dispermic condition of 50 per cent of the eggs when Fundulus is used as the female, is regular and occurs in every experiment, so that this diffrence is a constant one.
 +
 +
At the time these experiments were made for the first time, I was not aware that any crosses between so distantly related species of fishes had been recorded. Subsequently I found that Appellof, 94, had made an equally remarkable cross between two European species : Labrus rupestris, female, and Gadus morhua, male. He says nothing about the percentage of eggs impregnated except that " ein Anzahl " were found in regular cleavage the following day, nor about the character of impregnation — whether any of the eggs were dispermic or polyspermic in addition to the normally impregnated ones. Pfiiiger's experiment, 82, in which he succeeded in impregnating the eggs of an anuran, Eana fusca, with the sperm of a urodele, Triton alpestris and Tiiriton tgeniatus is in some respects even more remarkable. However, he succeeded in obtaining only polyspermic impregnation. Morgan, 94, succeeded in impregnating the eggs of Asterias with Arbacia. Mathews, 02, repeated the experiments and concludes that Morgan's impregnations were probably a species of parthenogenesis consequent upon shaking the eggs and not a true impregnation by the sperm of a sea urchin. My experience with many othe? crosses between fishes as distantly related as Fundulus and Menidia incline me to the belief that the normal impregnation of these two classes of Echinoderms is perfectly possible. This remarkable experiment deserves to be repeated with all possible precautions.
 +
 +
V. Development. 1. Cleavage.
 +
 +
a. Form of Cleavage. — The cleavage of the eggs normally impregnated goes on in a perfectly normal manner. The eggs all pass regularly 3
 +
 +
 +
 +
34 The Chromatin in the Development of Hybrids
 +
 +
through the two, four, eight, sixteen, etc., cells which in no way differ from the corresponding stages of the normal eggs. The cleavage in the hybrid eggs might show differences (1) in the irregularities in the size of the cleavage cells or in the stages of different eggs in any given lot, and (3) in the rate of cleavage.
 +
 +
In a lot of Fundulus eggs, which have been taken from a single mother fish and normally impregnated, all the eggs, except in rare instances, will remain nearly perfectly abreast in their time of cleavage. I have observed, however, that in a composite lot taken from a number of females such perfect concert in the rate of cleavage may not obtain. If the eggs are impregnated by sperm from a strange species even so distantly related as Menidia this concert of cleavage is not affected so far as I am able to detect. The same can be said for the reciprocal cross. Three hybrid eggs came under my notice which should be mentioned in this connection. These had stopped in their development, the one at the two-cell stage and the other two in the four-cell stage. The blastomeres were in each case perfectly formed. The eggs were all found in the same lot. The three abnormal ones were isolated to watch their further fate. The eggs all died without dividing further. I have endeavored to determine whether there was a greater irregularity in the size of the blastomeres of the different cleavage stages, in the hybrids than in the normals. My observations go to show that this is not the case in the stages below the 32-cell stage. Beyond this I was unable to make any comparison on account of the complexity of the cell mass. In a lot of normal eggs of Fundulus there are always to be foimd a number of .eggs in which there is more or less variation in the size of sister blastomeres. One cell in the 2-cell stage, in an extreme case, may be several times smaller than its mate. From this condition to that of perfect equality in the size of the blastomeres there are all intergradations. This inequality may be begun in the second or third cleavage where, in addition, the cleavage planes may vary considerably in their direction, giving rise to irregularities in the arrangement of the blastomeres. Such irregularities everyone has noticed who has watched any considerable number of cleaving fish eggs. In the hybrid eggs I was unable to make out any difference in the extent to which such irregularities occurred. This was certainly contrary to my expectations, since I had considered it unlikely that two different chromatins of such diverse origin would work so perfectly together through all the complicated activities incident to cell division. That this may be, is evident not only from the above consideration, but also from a closer study of the internal phenomena described further on and from similar observa
 +
 +
 +
William J. Moenkhaus
 +
 +
 +
 +
35
 +
 +
 +
 +
tions on a great many other equally distant crosses which were made among fishes.
 +
 +
6. Rhythm of Cleavage. — In .general, as already pointed out by Born, 83, the hybrid egg develops slower than the normal. I have observed this repeatedly in fishes. In most of the crosses that have come under my observation, however, the difference in the rate is very slight and cannot in most cases be detected in the early cleavage stages. In the following table is given a comparison of a lot of hybrid eggs with a lot of normals. The eggs were taken from the same mother at the same time, fertilized at the same moment and kept under exactly similar conditions. The observations were made at the same time on both batches of eggs and the stage at which each was found was indicated as accurately as possible.
 +
 +
 +
 +
Time of
 +
 +
 +
Observation.
 +
 +
 +
Fund. X Fund.
 +
 +
 +
Fund. X Men.
 +
 +
 +
9.10 P.
 +
 +
 +
M.,
 +
 +
 +
June 26.1
 +
 +
 +
In 2 cells.
 +
 +
 +
In 2 cells.
 +
 +
 +
9.40
 +
 +
 +
 +
 +
 +
 +
Beginning 4 cells.
 +
 +
 +
Beginning 4 cells.
 +
 +
 +
10.00
 +
 +
 +
 +
 +
 +
 +
Completion 4 cells.
 +
 +
 +
Completion 4 cells.
 +
 +
 +
10.15
 +
 +
 +
 +
 +
 +
 +
Beginning 8 cells.
 +
 +
 +
Beginning 8 cells.
 +
 +
 +
10.20
 +
 +
 +
 +
 +
 +
 +
Well begun on 8 cells.
 +
 +
 +
Well begun on 8 cells.
 +
 +
 +
10.30
 +
 +
 +
 +
 +
 +
 +
In 8 cells.
 +
 +
 +
In 8 cells.
 +
 +
 +
11.00
 +
 +
 +
 +
 +
 +
 +
Beginning 16 cells.
 +
 +
 +
Beginning 16 cells.
 +
 +
 +
9.00 A.
 +
 +
 +
M.,
 +
 +
 +
" 27.
 +
 +
 +
Well along in segmentation.
 +
 +
 +
Well along in segmentation.
 +
 +
 +
9.00 P.
 +
 +
 +
M.,
 +
 +
 +
" "
 +
 +
 +
Well begun on gastrulation.
 +
 +
 +
First trace of gastnilation.
 +
 +
 +
9.00 A.
 +
 +
 +
M.,
 +
 +
 +
" 28.
 +
 +
 +
2/8 + over the yolk.
 +
 +
 +
V2 or less over the yolk.
 +
 +
 +
3.00 P.
 +
 +
 +
M.,
 +
 +
 +
" "
 +
 +
 +
Blastopore closed.
 +
 +
 +
2/3 over the yolk.
 +
 +
 +
5.30
 +
 +
 +
 +
 +
 +
 +
Blastopore closed, the embryo long and narrow.
 +
 +
 +
Blastoderm closing or nearly closed; embryo much shorter than normal.
 +
 +
 +
9.00 A.
 +
 +
 +
M.,
 +
 +
 +
" 29.
 +
 +
 +
Embryo with optic vesicle.
 +
 +
 +
Blastopore closed, embryos short, no optic vesicle; apparently dead.
 +
 +
 +
 +
1 Eggs fertilized at 7 P. M., June 26.
 +
 +
 +
 +
From the table it will appear that the retardation in the development does not appear until the close of cleavage. If the development of the hybrid is slower than the normal during the first four or five cleavages it is so slight that it cannot be detected. From this time until the time of gastrulation the hybrids fall considerably behind. From the time of gastrulation on they fall increasingly more behind the normals. It is probable that the slowing-up process does not take place at the same rate ■ but that it becomes increasingly rapid as development proceeds. In the reciprocal cross, if compared with the normal eggs of Menidia, the same conditions obtain. The normal Menidia eggs cleave a little
 +
 +
 +
 +
36 The Chromatin in the Development of Hybrids
 +
 +
more rapidl}'^ than those of Fundulus. This increased rate is also maintained for the Menidia hybrid eggs.
 +
 +
This law of the rate of cleavage in hybrids I have considered elsewhere but the following facts are of interest here. When the two species crossed have eggs that cleave at a different rate the cleavage is still that of the egg species. The eggs of Fundulus heteroclitus can very easily be impregnated by Tautoglahrus adsperus. The eggs of the former cleave ordinarily in about two hours after the addition of the sperm. Those of the latter, under similar conditions, cleave in about fifty minutes. In the hybrid, however, the rapid sperm is unable to alter the rate of the cleavage and vice versa. This law is further strikingly illustrated in the cross between Batrachus tau and Tautoglahrus. The eggs of the former species can be impregnated by the sperm of the latter. 'The cleavage furrows, however, do not appear until 8 hours after impregnation, approximately that of the egg species.
 +
 +
Stassano, 83, maintained that he was able to hasten or retard the cleavage of Echinoderm eggs by sperm of another species. Driesch, 98, however, by extended experiments in the same group of animals, has, shown just the reverse and it is probable that Stassano erred in his experiments.
 +
 +
2. Development of Dispermic Eggs. — The dispermic eggs fall at once into four cells. The cleavage takes place synchronously with the cleavage of the normal eggs, so that when the normal eggs are in the twocell stage, about an equal number of eggs will be found in the four-cell stage. This correspondence in the rhythm of cleavage is not strictly maintained after the first cleavage, in that the rate is slightly slower in the normals. The form of the cell cannot be distinguished from those in the four-cell stage of the normals. The four-cell stage, or the first cleavage is followed by the eight-cell stage, this by the sixteen-cell stage, etc., in a normal manner.
 +
 +
Such dispermic eggs continue their development to a late stage of cleavage, when they invariably die. I have isolated, in the aggregate, many hundred dispermic eggs and followed their development but have never seen an egg that showed any signs of forming the germ ring or the embryonic shield. They form a normal heap of cells and the blastoderm may even spread to a slight extent, but beyond this they do not go.
 +
 +
That such eggs which fall at once into four cells are dispermic, i. e., eggs whose nucleus conjugates with two male pronuclei, is clearly shown
 +
 +
 +
 +
William J. Moenkhaus 37
 +
 +
in sections of such eggs. Figure 1 (Plate I) shows the three pronviclei before their fusion. This egg would in all probability have fallen into four cells at once. In the metaphase there are two spindles placed at right angles to each other, an aster at each of their poles and at the point of intersection the chromosomes are being distributed. I am unable to say whether in such dispermic eggs more than two spermatozoa enter of which only two would then succeed in conjugating with the egg pronucleus.
 +
 +
Having such an easy way of producing dispermy I isolated large numbers of such eggs for further development to see whether I might be able to obtain any evidence on the question of the relation of double impregnation and double monsters or double embryos. Fol., 83, was the first to raise this question in connection with his studies on Echinoderm eggs. He obtained from a lot of polyspermic eggs a considerable number of double and multiple gastrulse. He maintained that the polyspermic condition was responsible for this result. In 1887 Oscar and Eichard Hertwig reared many thousand polyspermic Echinoderm eggs and obtained only about ten double gastrula^, a proportion entirely too small to lend any support to the hypothesis of Fol. Further observations of Oscar Hertwig, 92, on isolated polyspermic frog eggs and of Driesch, 93, on isolated dispermic Echinoderm eggs speak against this hypothesis. In the dispermic fish eggs I hoped that the double character might show itself, in the first place, in the double grouping of the cells in early cleavage and, in the second place, in the appearance of a double embryonic shield which could be taken as an indication of an attempt to produce two embryos. In regard to the first, it was found, as already intimated, that the early cleavage stages, so far as the form and grouping of the blasto meres are concerned, do not differ from the normal eggs. In regard to the second, none of the eggs went beyond the late cleavage stage. A careful search failed to reveal any sign of even a beginning of an embryonic shield. Inasmuch as the majority of the normally impregnated hybrid eggs develop far enough to form an embryonic shield and many of them considerably beyond, the fact that none of the dispermic eggs formed such shields must be taken as evidence against the theory of any relation between dispermy and double embryos.
 +
 +
3. Later Development. — When cleavage has well progressed in the Fimdulus hj'brid the blastoderm spreads and the germ ring with a faint indication of an embryonic shield, forms. From this stage on a variety
 +
 +
 +
 +
38 The Chromatin in the Development of Hybrids
 +
 +
of conditions become apparent. The blastoderm may continue to spread in an apparently normal fashion, encompassing the yolk, and the embryonic shield enlarges correspondingly. The " blastopore " closes and the rudiments, of the embryo are laid down. The three germ layers, the chorda and neural cord are differentiated (Fig. 2, Plate I). The eyes were seen in only two out of all the specimens that were obtained. Sections of one such embryo showed that the optic cup is forming and the lens, composed of a mass of cells, is constricted off from the ectoderm (Fig. 3). No definite arrangement of the cells in the lens can be raade out. In the retina the cells were arranged into more or less distinct transverse rows. In both structures the cell boundaries are seldom to be made out; the nuclei, on the contrary, are large, distinct and provided with one or more very large nucleoli.
 +
 +
The proportion of embryos that thus normally close the " blastopore " is small. During the growth from the germ-ring stage to the closure of the " blastopore " the failures in the developmental processes especially show themselves in the variety of abnormalities which occur. The embryo may stop at the early embryonic-shield stage and, after two or three days of apparent life, die. Others endeavor to lay down the embryo so that the normal processes may go on for a time. The blastoderm may more or less completely enclose the yolk with the result that the embryos are too short in varying degrees and the " blastopore " may remain as a long slit or an open cleft of varying form (Figs. 4, 5, 6, 7, Plate I). The number of embryos dying at these varjdng stages is not the same. Comparatively few die during the early embryonic-shield stage. The bulk of the embryos starting on gastrulation succeed in encompassing the yolk to two-thirds or more of the extent giving rise to the variety of " blastopore " formations above described.
 +
 +
In the reciprocal hybrid the development beyond the cleavage stage is markedly less successful. A large per cent of eggs will form the germ ring and the early stages of the embryonic shield, but of these only an occasional one closes the " blastopore " in an approximately normal manner. The earlier stages of gastrulation not uncommonly form normally. Beyond this the abnormalities occur. These are of the same general character as those described in the Fundulus hybrid. Figures 8 to 10 (Plate II) show some typical cases.
 +
 +
In both hybrids the developmental processes come to a standstill at various stages during gastrulation and doubtless also during cleavage stages. That the latter is true is evident from the fact that there are always a number of eggs in cleavage that never form any germ ring. From the table given on page 35, it appeared that the rate of development of the hybrid eggs became increasingly slower than the normals as de
 +
 +
 +
William J. Moenkhaus 39
 +
 +
velopment proceeded. This slowing-up process is to be interpreted as an increased weakening in the developmental energy. Either the miequal givins: out of, or the unequal draft upon, this energy in different portions of the developing eml^ryo, may result in the various abnormalities above described.
 +
 +
The bearing of these abnormalities upon the question of embryo formation is not to be discussed here. Other crosses have yielded material much more instructive and the subject will be taken up in connection with a description of those crosses.
 +
 +
VI. The Individuality of the Maternal and Paternal
 +
 +
Chromosomes.
 +
 +
1, Introduction. — As stated in the introduction of this work, one of the points of especial interest in the hybrids between Fundulus heteroclitus and Menidia notata is the fact that the chromosomes of the one may be distinguished, morphologically, from those of the other. I was introduced into the importance of this through the study of a section of a hybrid egg which was in the anaphase of the first cleavage. In this spindle two kinds of chromosomes appeared, easily distinguishable. Subsequent comparison with the chromosomes of the parent species showed that one of the kinds of chromosomes belonged to one and the other to the other parent, and that the introduction into a strange egg did not modify their characteristic form. With these conditions obtaining it has been possible for me to follow the history of the maternal and paternal chromosomes in these hybrids to a late stage of cleavage. The phase of this subject which has engaged me especially is that of the individuality of the two parental chromosomes during development.
 +
 +
2. Material and Methods. — Appropriate stages were preserved from the moment of impregnation to a late cleavage stage. Corresponding stages of both hybrids and of normal eggs were taken. The killing fluids used were Flemming's, Zenker's, Perenyi's, and picro-acetic. The last two have been of most service to me. The eggs were directly placed into the fluids without first removing the membranes. The most convenient method for manipulating the eggs in the paraffin and one which I adopted altogether is as follows: the membrane was removed and the yolk with the protoplasmic cap or embryo surmounting it was imbedded with the cap directly upward. By properly mounting the paraffin block I could lay the protoplasmic cap into horizontal sections until the yolk was reached. This very much simplified an, at best, very laborious task. The sections were in practically all cases made 7^2 or 10 micra thick. It
 +
 +
 +
 +
40 The Chromatin in the Development of Hybrids
 +
 +
was found that such sections might include the entire thickness of the spindles in the earlier stages of cleavage. This was a matter of considerable importance since it was desirable to get all of the chromosomes of a given spindle in the same section. All the staining was done with Haidenhain's hsematoxylin. This stain was found perfectly satisfactory, find because of its simplicity and the ease with which it can be controlled was used exclusively.
 +
 +
3. Description of the Chromosomes. — The chromosomes of Fundulus heteroclitus are long, slender and usually straight. They measure 3.18 micra in length. In a given anaphase all the chromosomes are of practically the same length. Just before breaking up into the chromosomal vesicles the constituent chromomeres can commonly be made out. These number four in nearly all cases that I have counted. In one instance one of the chromosomes had five. The number of chromosomes is 36.^ In their migrations to the poles they lie alongside of each other parallel, for the most part, with the spindle fibres so that at the anaphase their form can be easily made out (Fig. 11, Plate II).
 +
 +
The chromosomes of Menidia notata are short and usually more or less curved. They are sometimes straight and in some cases slightly sigmoid. They measure 1.00 micron in length. As in Fundulus, they have a uniform size in any given anaphase (Fig. 12, Plate II). I have tried to make out the component chromomeres, but without success. The number of chromosomes is about 36. I have not been able to count them definitely.
 +
 +
In Figure 13 (Plate II), are given the two kinds of chromosomes drawn to the same scale. Both the relative size and the difference in form are shown. The difference in the two chromosomes comes out very strikingly also when seen side by side in the same cell [Figs. 14, 15 (Plate II) and 29, 30 (Plate IV)]. The small chromosomes are here grouped together on one side of each half of the spindle and the long ones on the other side of the spindle.
 +
 +
There can be no doubt that these two diverse forms of chromosomes occurring in the hybrid eggs are the chromosomes of the two diverse parents which, notwithstanding their association with strange chromosomes in a strange cytoplasm, are evidently functioning in a perfectly normal manner. A description of their behavior from the time of conjugation to a late cleavage stage is the purpose of the present section. Before en 2 In only one instance was I able to count the number satisfactorily. Every chromosome was definitely distinguished and counted in this case. I had, however, concluded that 36 was the approximate number from numerous counts made on crosssections of anaphase spindles.
 +
 +
 +
 +
William J. Moenkhaus 41
 +
 +
tering upon this, however, for reasons to be stated afterwards, it will be important to give a brief general account of the character of the work thus far done on this subject.
 +
 +
4. General Review of Literature.— Since the first discoveries by van Beneden, 83, Boveri, 90, Guignard, 91, and others, of the numerical equality of the maternal and paternal chromosomes in fertilization, mucii interest has developed in the question whether these might not retain their individuality throughout all the cells of the developing embryo. Van Beneden, who worked with Ascaris, in which the pronuclei may not fuse before the formation of the first cleavage spindle, was able to follow the maternal and paternal chromosomes into the resting nucleus of the first two daughter cells. Here they were lost. Although unable to follow them beyond the first cleavage, he expresses his conviction that the two chromatin masses probably remain distinct throughout subsequent divisions. Boveri, 91, working with the same animal, made similar observations and was led to formulate his well-known hypothesis " that in all cells derived in the regular course of division from the fertilized egg, one-half of the chromosomes are of strictly paternal and the other half of maternal origin." He further endeavored to follow out the fate of the individual chromosome during the resting period of the nucleus. Boveri differed from van Beneden in this important respect, that he found that not only the maternal and paternal chromatin remained distinct but also that the individual chromosomes retained their individuality. With this interesting and important question thus clearly pointed out so long ago, one should consider it remarkable that so few researches have siru:e been directed toward its solution, were it not for the evident difficulties attending any effort to distinguish the exactly similar parental chromosomes beyond the first cleavage. Extension of our knowledge to a large number of forms showed that three conditions obtained in regard to the fusion of the pronuclei during fertilization : (1) Animals in which the two pronuclei are so completely fused as no longer to be distinguishable. (2) Animals in which the pronuclei do not fuse but remain more or less separated by a membrane. (3) Animals in which both conditions may occur.
 +
 +
In 1893 Hacker pointed out that in Cyclops tenuicornis also, the two pronuclei do not fuse in fertilization and, furthermore, that in the twocell stage the nuclei are composed of two closely united but distinct halves, one of which he identifies with the male, the other with the female pronucleus.
 +
 +
Eiickert, 95, extended these observations to Cyclops strenuous and pub
 +
 +
 +
42 The Chromatin in the Development of Hybrids
 +
 +
lished the first research directed specifically toward the solution of this question. Riickert found, in the first place, that the condition of doublenuclei could be followed considerably beyond the late cleavage stages and, in the second place, that the chromosomes might be arranged in two groups upon the spindle, more or less distinctly separated. From the bilobed nuclei of the one, two, four, etc. cells a double group of chromosomes might arise and these two groups could be followed each into one of the two halves of the subsequent resting nucleus. Such bilateral grouping of the chromatin in the spindle occurred only in the earlier cleavage but the double nuclei could be found, although in constantly decreasing number, in later stages. The strong probability that in the early stages the two halves of the double nuclei represent the double source of the chromatin, makes the assumption that in the later stages such double nuclei have a similar significance, justifiable. It is worth while to state in this connection Euckert's cautious conclusions in his own words, " Jedenfalls geht aus der vorstehenden Untersuchung hervor, dass in der ersten Entwickelungszeit mindestens bei einem Theile der Kerne eine Vermengung der vaterlichen und miitterlichen Halfte nicht statt hat, das ein soldier Vorgang fiir den normalen Verlauf der Entwickelung somit nicht erforderlich ist. Das Chromatin kann seine urspriingliche Vertheilung beibehalten trotz wiederholter mitotischer Theilungen und Aufiosungen in ein feinfadiges Geriist, und obwohl die iibrigen Lebensvorgange innerhalb seiner Substanz, die Assimilation . und das Wachsthum, gerade zu dieser Zeit der rasch aufeinanderfolgenden Theilungen lebhaftere sind als sonst."
 +
 +
V. Hacker, 95, 02, Avorking also on Cyclops, carries the observations of Euckert considerably further. In addition to the double nuclei and the bilateral distribution of the chromatin on the spindle, he observed a physiological difference in the maternal and paternal chromatin masses. This physiological difference showed itself in the different stages in which the two masses of chromatin may be within the same cell. It enabled him often to distinguish two groups when otherwise there was no spatial separation or no nuclear membrane to separate them. In Cyclops brevicornis, however, he could not recognize the double distribution of the chromatin beyond the eight-cell stage except in the primordial germ cells from the beginning of gastrulation.
 +
 +
Eecently, Conklin, 01, has shoMm that in Crepidula, even more clearly than in Cyclops, the double character of the nuclei during certain phases quite commonly obtains during earlier cleavage (first 5 or 6 generations), and gives this the same interpretation that others had given it.
 +
 +
Conklin called attention to the fact that in these double nuclei each
 +
 +
 +
 +
William J. Moenkhaus ^ 43
 +
 +
half -11811311}' contained one nucleolus, so that these might also be regarded as maternal and paternal. Hacker, 02, in a recent preliminary, summarizes the results of his comparative study of species of Cyclops. He endeavors to show that the maternal and paternal chromatin masses are each represented by a nucleolus in certain phases of the cell cycle. Taking these as an index he is able to establish the individuality of the parental chromatin masses throughout the cycle of an individual.
 +
 +
Among plants Miss Ferguson, 01, has shown that in Pinus strobus the male and female chromatin remains distinct during the first two cleavages, as far as she has followed them, and suspects from sections of later stages that this individuality may persist.
 +
 +
The two important papers by Herla, 93, and Zoja, 95, on Ascaris hybrids treat the subject from a standpoint so similar to that of my own work that it will be advantageous to consider them later.
 +
 +
The above brief survey of the work thus far done on this subject enables me to point out the following facts: (1) The evidence upon which the authors base their conclusions rests on the assumption that the two halves of the double nuclei occurring beyond the two-cell stage represent, the one, the mat-ernal, and the other, the paternal chromatin. (2) The chromatin of the two parents not only retains its individuality but also remains spatially separated or bilaterally distributed in the nucleus during the various phases of division. In the following detailed consideration of my own results I shall have occasion repeatedly to refer to these facts.
 +
 +
5. Conjugation of Pronuclei and the First Cleavage. — The time elapsing between the moment of entrance and the time of conjugation of the pronuclei is the same as that for the normal eggs. Thus, in the Fundulus cross, at a temperature at which the first cleavage furrow forms just two hours after impregnation, the male pronucleus has become apposed to the female pronucleus at 55 minutes after fertilization. At 65 minutes, they have usually become well fused. During its migration the sperm has become metamorphosed into a vesicle which cannot be told from the female pronucleus. I have taken much pains to find some distinguishing mark between the maternal and paternal chromatin at this stage, hoping such might serve in distinguishing them in subsequent resting stages. The size, form and arrangement of the chromatin granules in the two pronuclei, however, are, so far as I have been able to make out, altogether similar.
 +
 +
■ In a late metaphase of a Fundulus hybrid egg, 73 minutes after fertilization, two chromatin masses can readily be made out (Fig. 16, Plate II). The one is evidently made up of the long chromosomes which, in
 +
 +
 +
 +
44 The Chromatin in the Development of Hybrids
 +
 +
the process of splitting, already extend their free ends for some distance on each side of the equatorial plane. The other group is made up of the short chromosomes which, also in division, appear as large granules rather than elongated structures.
 +
 +
In the anaphase [Pigs. 14, 15 (Plate II), 29, 30 (Plate IV)], two , groups of chromosomes occupy each half of the spindle. Their form here comes most distinctly to view. Careful examination of each group shows that it comprises chromosomes of only one type, so that the chromatin material has not become mingled during the fusion of the pronuclei. The two groups do not occupy the same position with reference to the pole or the equatorial plane, i. e., they are not equidistant. In other words, the two kinds of chromosomes are not in the same stage of migration. This physiological difference is not so well marked in the Fundulus hybrid where the small ones become the stragglers [Figs. 14 (Plate TI) and 29 (Plate IV) ], but is very distinct in the Menidia hybrid [Figs. 15 (Plate II) and 30 (Plate IV)].
 +
 +
As the chromosomes become transformed into the resting nucleus each is converted into a vesicle in a manner essentially similar to that described for Crepidula by Conklin. In an early stage the two groups of vesicles can be distinguished by the difference in the size of the vesicles. These fuse at first into larger ones, giving rise to a lobed nucleus. At this stage it is no longer possible to tell the two kinds of vesicles apart. The fusion continues until a single well-rounded resting nucleus results, with all traces of its double character lost.
 +
 +
6. Second Cleavage. — Although all traces of the maternal and paternal chromatins are lost in the resting nucleus of the two-cell stage there is very little doubt that they have really remained spatially distinct. That this is so, is shown by the fact that when the chromatin forms into the chromosomes of the next cleavage the two kinds again appear, and in all the spindles examined they were again bilaterally distributed on the spindle.
 +
 +
The kinds of chromosomes can, naturally, be best distinguished during the anaphase, but even in the metaphase this may be done. In such a metaphase of a Menidia hybrid, for instance, Fig. 17, where the long chromosomes are the introduced ones, there may be seen on the one side of the spindle the long ones with the ends of a part of the chromosomes already extending toward the poles, while on the other side the short ones may be seen confined with characteristic strictness to the equatorial plane. Figures 18 (Plate II) and 31 (Plate IV) represent one of the groups of the migrating chromosomes of an anaphase of a second cleavage. Here the chromosomes come most distinctly to view. The
 +
 +
 +
 +
William J. Moenkhaus 45
 +
 +
long ones all grouped together on one side of the spindle (left in figure) and the short ones on the other side. Whether all of each kind that entered the resting nucleus have again appeared I cannot say, inasmuch as it has been impossible thus far to recover all the chromosomes of each parent. This is due to the complexity of the chromosome mass. The chromosomes are so small and numerous that their number cannot be determined even in the clearest preparation. In any given section some of the long chromosomes are usually cut, making the pieces indistinguishable from the short ones, so that it is practically impossible to follow out all of the chromosomes of each kind. That we have here to do again, however, with the maternal and paternal chromosomes, there cannot be the shadow of a doubt.
 +
 +
Riickert, 95, in his Fig. 6, gives the lateral view of the anaphase of the second cleavage spindles of Cyclops strenuous, both of which, but especially the spindle to the right in the figure, sliow two groups of chromosomes spatially separated in each half of the spindles. Kiickert takes these to be the maternal and paternal groups of chromosomes, and he gives, it would seem, very good reasons for thinking so. That the two chromatin masses of the first cleavage represent the two parental chromatins is beyond question. He is able to follow them from the time of the conjugation of the two pronuclei through the various phases of the division to the reconstruction of the resting nucleus. In this reconstruction the first appearance of the nucleus is that of a double grou}) of small vesicles. The vesicles of each group, it appears, fuse with each other, the halves remaining distinct at first by the presence of a more or less distinct wall and a corresponding constriction in the outer membrane but later only by the latter. The two halves of the resting nucleus, therefore, are to be identified as the maternal and paternal portions. The emergence of two chromatin masses from this double nucleus in the following division distributed on the spindle in the manner above described, would strongly favor the view that the two substances had not mingled during the resting stage. Conversely, the strong probability that the two chromatin masses remain distinct in the previous resting nucleus argues strongly in favor of Riickert's supposition that the two groups of chromosomes appearing in the subsequent division represent the maternal and paternal chromosomes. Conklin has given very strong evidence for the same thing in Crepidula. It is apparent to every one who has closely followed through the researches described above that the one thing to be desired is better evidence that the two groups of chromosomes emerging from the two bilobed resting nuclei of the first two blastomeres are derived from the two lobes of the nuclei
 +
 +
 +
 +
46 The Chromatin in the Development of Hybrids
 +
 +
and really represent the materna] and paternal chromatin, Conklin expresses the situation in the following words : " It still remains to show that these double nuclei really represent the egg and sperm nuclei which have not yet lost their individuality. This cannot be demonstrated in Crepidula, for the reason that this double character is not apparent at every stage in the nuclear cycle, but it is extremely probable, as the following observations will show : " The detailed reasons given need not be repeated here.
 +
 +
There are but two ways to demonstrate this with certainty, namely, either to follow the process in the living egg, or to be able to distinguish the two kinds of chromosomes, as I have been able to do in the hybrid under consideration.
 +
 +
Herla, 93, and Zoja, 95, made some observations which bear directly upon this point. In the study of an Ascaris containing eggs in various stages of early cleavage they found that the number of chromosomes in the cells was only three, one of which was slightly smaller and like the chromosomes of the variety univalens. The eggs, they conclude, were hybridized by the sperm of univalens. They were able to trace the independent maternal and paternal chromosomes to the 12-cell stage. With only three chromosomes it is not possible to determine with certainty very much about their distribution in the spindle. Zoja, in fact, says that the small chromosome may vary its position with reference to the other two, sometimes being between the two latter. These observations, therefore, can throw little light on the particular question of the distribution of the two chromatins in the nucleus.
 +
 +
In my own hybrids, however, where the number of chromosomes is great, any disturbance of their grouping can be readily made out. The conditions described for these hybrids, taken in connection with the observations of Herla and Zoja, demonstrate in the clearest manner that the two chromatins may remain distinct in these resting nuclei and that the chromosomes in the subsequent division may be and are grouped spatially. They, furthermore, lend the strongest support to the belief that in the other forms described (Cyclops, Crepidula, Pinus) the two groups of chromatin or chromosomes arising from the bilobed resting nuclei of the two first blastomeres may really represent the distinct parental chromosomes.
 +
 +
7. The Rotation of the Cleavage Nuclei During the First two Cleavages. — In the first cleavage spindle the chromosomes lie side by side in a horizontal plane. In this same plane they can be followed into the early resting stage of the two daughter nuclei. Evidently, inasmuch as the second cleavage plane forms at riglit angles to the first, the nucleus will
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 +
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William J. Moenkhaus 47
 +
 +
have to rotate through an arc of 90° in order that both kinds of chromosomes may again be halved. This rotation takes place between the vesicular stage of the nucleus and the metaphase of the following division. Just when during this period it takes place or mostly takes place I cannot say. At the metaphase the rotation is probably completed. When the rotation is completed both chromosome groups again occupy a horizontal plane.
 +
 +
In all but one of the cells of the Menidia hybrid examined the small chromosomes bore a definite relation to the cleavage plane, that of a position in the spindle away from the plane of division. In the single exception it was found that the small chromosomes were above the large ones and, hence, occupied a vertical plane.
 +
 +
The behavior of the chromosomes in the third and subsequent cleavages is different from that of the first two. It will, therefore, be advisable to describe these stages in detail before entering into a comparison with the conditions found in other forms.
 +
 +
8. Third Cleavage. — The two groups of chromosomes of the second cleavage spindles pass into a resting condition of the four-cell stage, in which it is again impossible to distinguish the two kinds of chromatin. There is no constriction or partition to divide the nucleus into two lobes or parts, as is common in Cyclops and Crepidula. Since, so far, the chromatin in these hybrids had behaved essentially like that in the other forms described by other authors, I expected that in the metaphase and anaphase of the third division the short and long chromosomes should again appear in two groups on the spindle. If the conditions here should run parallel with the conditions in Cyclops and Crepidula, this is what should be expected. But in this I was disappointed. Whereas in the second cleavage every cell which I examined shows the two kinds of chromosomes bilaterally distributed, the third cleavage spindles, for the most part, do not show such distribution. An occasional spindle occurs in which the grouping has not been completely destroyed. Figure 19 (Plate III), it will be seen, shows the short chromosomes to the rigtit and the long ones largely to the left. It is to be noted, however, that each kind is not restricted to its group but a few of each kind have become mingled with those of the other. The mingled condition is the prevailing type where it is impossible to make out any grouping.
 +
 +
The position of the parental chromosomes with reference to the cleavage plane which could be so readily followed out during the second cleavage is during the third cleavage, naturally, largely destroyed since the bilateral distribution of the chromatin has been destroyed. In three spindles in which the position could be made out with reasonable cer
 +
 +
 +
48 The Chromatin in the Development of Hybrids
 +
 +
tainty the small chromosomes were placed in a horizontal plane toward the side away from the last cleavage plane.
 +
 +
9. Fourth Cleavage. — When in the fourth cleavage the chromatin has. resolved itself into chromosomes the two kinds are again mingled. The mingling has evidently gone farther, because in very few of the cells can even a partial grouping be discovered. I have found only one cell in which the two kinds of chromosomes were bilaterally distributed upon the spindle. In the sectioning of this cell the knife cut in such a way as to pass between the two groups so that in the one section nearly all short ones were found, and in the other section nearly all long ones. In my study of these sections I had well in mind the possibility that the short ones might be the ends cut from the long ones of the other section. That this is not the case, however, is evident from the fact that the long chromosomes of the .one section have the characteristic length of the one species, and those of the other section that of the other species. There cannot be any doubt that we have to do here with two kinds of chromosomes, and that we can be perfectly certain these represent the distinct maternal and paternal groups. The usual condition is for the chromosomes to be well mingled on the spindle. In such an anaphase. Fig. 20 (Plate III), the two kinds of chromosomes can be clearly made out. In endeavoring to recover all of the chromosomes of each kind, I have found it convenient to draw each chromosome as I followed it, retaining its relation to some other one or more chromosomes but not its position in the spindle. Figure 21 (Plate III) represents such a drawing of an anaphase of the fourth cleavage, Although, as stated above, I have been unable to recover all the chromosomes, the drawing which I made as faithfully as I could with only the partial aid of a camera, serves well to show the presence of two kinds of chromosomes and their mingled condition.
 +
 +
10. Later Cleavage. — I have followed the behavior of the maternal and paternal chromosomes from the fourth cleavage through successive stages to late cleavage. Here, with often several hundred cells in any given section, in all stages of division and cut in many different planes, the conditions for such study are favorable. I have carefully examined many thousand cells in both hybrids with the view of finding one in which the two kinds of chromosomes had remained grouped but I have not been able to find a single undoubted instance. On the other hand, nuclei showing the two kinds of chromosomes mingled together upon the s])indle are everywhere to be found. The two kinds of chromosomes, naturally, cannot be distinguished in the metaphase, not even when the chromosomes have begun to split. In the stage represented in Figure 32 (Plate IV) (lower cell to left), some of the long chromosomes may be seen
 +
 +
 +
 +
William J. Moenkhaus 49
 +
 +
characteristically extending their ends toward the poles. The short chromosomes still confined to the equatorial plane cannot be identified as such. In nearly every anaphase, however, the short chromosomes can be clearly distinguished among the long ones [Figs. 23 (Plate III) and 32 (Plate IV)].
 +
 +
The two kinds of chromosomes can be distinguished not only by their size but also physiologically. In Figures 22 and 32, it can be seen that the short chromosomes, as a whole, are nearer to the pole than the long ones. This shows most clearly in the further half of the spindle where the short chromosomes remain more abreast and are, as a whole, nearer the pole, forming a band across the spindle. Here, as in the earlier stages of the Menidia hybrid, the long chromosomes are the stragglers, being more irregular than and, as a whole, behind the short ones in their migration to the poles.
 +
 +
While this difference in the rate of migration comes out most strikingly in the spindles where the chromosomes have not yet become mingled [Figs. 29, 30, 31 (Plate IV)], it is just as truly present in the later cleavage cells where this mingling has taken place. The small chromosomes, in the more extreme instances, may in the telophase become completely separated from the long ones, as shown in Fig. 23 (Plate III). This figure represents the early telophase of the third cleavage. The group of small vesicles nearer the pole are doubtless the small chromosomes already well along in their transformation while the larger group would then represent the larger chromosomes not yet so far along in their transformation but that some of the longer chromosomes can be identified. It will occur to every one that notwithstanding the fact that the chromosomes may be thoroughly mingled during the active phases of the cell cycle, the two kinds may in this way become separated in the resting nucleus. The reasons for believing that this does not usually occur will appear below in connection with another matter.
 +
 +
I have considered it important to carefully compare the conditions in the hybrids with corresponding stages in the normal eggs of the two parent species. Although practically all of the points above brought out Avould be sufficiently evident taken by themselves they become doubly so through such comparison. The question to arise is whether the differences in the size of the chromosomes might not also be found in the normals. This is clearly not the case. In any given cell during a phase when the chromosomes come distinctly to view all the chromosomes are of practically the same size. Any variations in their size cannot be confounded with the differences obtaining in the hybrid egg?: Furthermore, the chromosomes, apart from their size, show a certain individual4
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 +
 +
 +
50 The Chromatin in the Development of Hybrids
 +
 +
ity in their behavior during various phases of division. In the equatorial-plate stage the chromosomes of Menidia notata arrange themselves in an even band across the spindle. In a corresponding stage in Fundulus, the plate presents a more ragged appearance, the ends of some of the chromosomes extending out towards the pole. This difference is especially well marked just at the time of splitting. In Figures 24 (Plate III) and 33 (Plate IV) is shown a cell in later anaphase of Menidia notata taken from about the middle cleavage stage of the embryo. The chromosomes are in a compact group without any stragglers along the spindle as is so common in the hybrid cells. Figure 25 (Plate III) is taken from an early cleavage stage of Fundulus heteroclitus. All the chromosomes are of the characteristic long form. In the later anaphase, [Figure 26 (Plate III)], corresponding approximately to the stages above given for Menidia notata (Figs. 24, 33), these chromosomes retain their characteristic length and extend along the spindle for some distance. If with these conditions in the normals corresponding stages in the hybrid are compared (Figs. 22 and 32), it will be seen that in the latter the conditions characteristic of both species are present. Here, as already stated, the short chromosomes appear as a band nearer the pole, extending across the spindle, and the longer ones, belonging to Fimdulus, extend further along the spindle toward the equator. This somewhat tardy migration of the longer chromosomes may be caused by their not being in their native cytoplasm, for in the reciprocal cross where the conditions are reversed this difference in the rate of migration does not obtain.
 +
 +
11. Comparison with Other Forms. — "WHien I first discovered that Menidia and Fundulus possessed two kinds of chromosomes and that these could be distinguished in the first cleavage spindle, I went at once to later cleavage stages for the further study of their behavior. In such stages I could easily get great numbers of cells in all stages of division in a single section. Those who had worked upon other forms had found that even in late cleavage stages the double nuclei, representing the maternal and paternal chromosomes, were more or less abundantly present. In sections of such stages I found no difficulty in finding evidences of the kind that had been employed by others in their studies upon other forms, namely, the grouping of chromosomes into two groups, during various stages of the division of the cell, and bilobed and double resting nuclei and the rather constant presence of double nucleoli in each nucleus (Fig. 27, Plate III). Knowing that the cells contained chromosomes of such different form as I had seen in the first cleavage, I was much disappointed at my inability to identify the two kinds here. In those cells where the chromosomes were grouped I had every reason to expect the
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William J. Moenkhaiis 51
 +
 +
one group to show one kind, and the other group, the other, but just this I was unable to do satisfactorily. One of two things must be true, (1) either the distinction between the two kinds of chromosomes had disappeared or (2) they liad become mingled in the course of development. The fact that I could make out two kinds of chromosomes which, however, were mingled upon the spindle, spoke directly for the latter view, but I still hoped that a study of successive stages from the first cleavage on might enable me to find conditions here similar to that found in other forms. As already indicated, they found in Cyclops and Crepidula that the maternal and paternal chromatin remained not only distinct but also spatially separated up to varying late stages of development. Except during the first and second and, in part, the third cleavage, this condition does not obtain in the hybrids under consideration. The chromosomes become mingled. This mingling probably has begun to a slight extent in the second cleavage and is clearly well along in the third. By the time cleavage is well along all the somatic cells have them mingled.
 +
 +
The evidence that has been given to show that the two kinds of chromatin remain spatially distinct in the forms referred to above is very strong. I have shown beyond any doubt that this may be the case for a very brief period in development. It is possible that the length of this period may differ in different forms even to the extent that they remain thus distinct throughout the entire embryonic period. It is interesting in relation to this subject to compare the results of Riickert on Cyclops strenuous and that of Hacker on Cyclops brevicornis. The former found double nuclei, although in constantly decreasing number, up to a late stage of development. The latter no longer found these double nuclei in the 16- and 32-cell stage nor in the stage just preceding the migration of the sex cells to the interior. He was, however, able to distinguish the two masses by physiological differences in the sex cells. This shows what a difference may obtain in two species of the same genus.
 +
 +
In the Menidia and Fundulus hybrids the bilateral arrangement of the chromosomes is destroyed at about the same stage as in Cyclops brevicornis, namely, at the third and fourth cleavage. According to Zoja 95, the two kinds of chromosomes in the Ascaris hybrid may be mingled before the 12-cell stage. These observations suggest that possibly the reason Hacker could not find the double nuclei beyond the 16-cell stage lay in the fact that in Cyclops tenuicornis the chromosomes also became mingled early in cleavage. Eiickert did not find any distinct grouping of the chromosomes during the active phases of cell division beyond the four- and eight-cell stage. In the light of Hacker's observations on a
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52 The Chromatin in the Development of Hybrids
 +
 +
form so nearly related to Eiickert's and that of Zoja's and mine, it may well be questioned whether the double and bilobed nuclei of Riickert really are any indication of the distinctness of the maternal and paternal chromatin. These conditions should at least make us cautious against accepting too readily the conclusions based on the mere presence of double and bilobed nuclei, double nucleoli and the like without any further means of identifying them with the maternal and paternal chromatin. Further work on a large number of forms is desirable to see whether it is the rule for the parental chromosomes to become mingled in these early cleavage stages.
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12. Maternal and Paternal Nucleoli.- — The recent studies of Hacker, 02, on parental nucleoli in different Copepod crustacean forms, has already been mentioned. The rather constant presence of two nucleoli in the nucleus he takes as an index of the separateness of the maternal and paternal chromatin. According to this idea, one of the nucleoli would represent the chromatin of the one parent and the other that of the other. At the time that his preliminary paper appeared I had been working on the nucleoli in fish hybrids. Most of the nuclei in the resting stage, when not too young, show two nucleoli. In the reconstruction of the nucleus the smaller chromosomal vesicles at first fuse into larger ones. In this stage one can often see a number of nucleoli in each nucleus. This multinucleolate condition is followed by a binucleolate condition as the fusion of these larger vesicles is finally completed. Each vesicle seems to forai a nucleolus so that the number of nucleoli present in a nucleus is in a general way an index of the number of vesicles composing the nucleus. Observations of this kind, it seems to me, strengthen Hacker's position. Two nucleoli would indicate that the nucleus is essentially composed of two vesicles or units of some kind, although these could not be distinguished in any other way. I have endeavored to make out some constant difference in the size or structure of these nucleoli in the hybrids but without success. In cells of the same section all conditions obtain in the size, from a strongly unequal to perfectly equal nucleoli within the same nucleus. This interpretation of the nucleoli by Hacker has much in its favor. In such forms as he studied, in which he maintains that the two parental chromosomes remain bilaterally distributed, it is easier to conceive how the nucleoli might represent the two paternal chromatins. In the fish hybrids under consideration, however, where the binucleolate condition is probably just as constant for the cells as in Cyclops, but where I have shown that the two chromatins do not remain bilaterally distributed but both kinds are scattered through the nucleus, it is difficult to believe that the scattered chromosomes of a given parent are represented by a common nucleolus.
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William J. Moenkhaus 53
 +
 +
13. The Persistence of the Individual Chromosome. — The question whether the individual cliromosome persists through the resting stage so that upon the resolution of the reticulum into the chromosome the same component chromatin granules again go together to make the same chromosome from which they were derived is a question first raised by Eabl, 85, and later definitely stated by Boveri, 88. Since that time so much evidence has accumulated going indirectly to support this conclusion that it has come to be rather generally accepted. Even a general review of this evidence is unnecessary here. Such a review would show that the fact has never been definitively demonstrated. Some of th.e most direct evidences yet given are the observations of Herla, 93, and Zoja, 95, on the Ascaris hybrids in which it was shown that the small chromosome of the variety univalens which entered the resting nucleus with the larger ones of the variety bivalens again emerged in its characteristic form. Equally stroag evidence is now afforded by my own observations on hybrid fishes. Here, as in the Ascaris hybrids, two kinds of chromosomes enter tJie resting nucleus from which each kind again emerges. As long as the two kinds remain grouped, as during the first two divisions, this fact has little added significance, since within each group it would be perfectly possible for the component chromosomes to exchange chromatin granules during the resting p'^riod. If, however, as occurs in later cleavage, the two kinds of chromosomes become mingled the chromatin granules of both kinds must lie mingled together within the resting nucleus. If from such a nucleus the two kinds of chromosomes again emerge it amounts almost to a demonstration that the chromatin substance of a given chromosome forms a unit and that this unit persists.
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 +
It should be mentioned here that the hypothesis of Boveri of the independence of the parental chromosomes has not received universal support. Prominent among those who have held in varying form the opposite view, namely, that the two parental chromatins become fused and mixed either at the time of fertilization or during development, is Hertwig. Hertwig, 87, maintained that fertilization demanded the thorough mixing of the sperm chromatin with the egg chromatin. Later, 90, he revised his view in that he no longer considered it essential that this fusion takes place at the time of fertilization but that it nevertheless took place later, during the earlier stages of development. Wilson and Mathews, 95, from their studies on the fertilization of Echinoderm species, concluded that because the fusion of the two pronuclei is here so thorough it would be impossible to maintain that the two chromatin masses remained distinct.
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54 The Chromatin in the Development of Hybrids
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These objections have largely been disposed of by the researches of Ruckert, Hacker, Herla, Conklin and others. These leave little doubt that the maternal and paternal chromosomes may remain distinct to a late stage in development, and I have shown that however thoroughly the chromosomes may lose their identity to our view during the resting period of the cell they nevertheless retain their individuality.
 +
 +
In view of its possible bearing on the theories of heredity just now becoming prominent through the recent rediscovery of the Mendelian laws of inheritance, it is highly desirable that this question of the individuality of the parental chromosomes be most thoroughly investigated. Further observations along this line on other hybrid fishes I have well under way, which I hope to be able to present in the near future.
 +
 +
SUMMARY.
 +
 +
The eggs of Fundulus heteroclitus can be readily impregnated with the sperm of Menidia notata. From 70 to 93 per cent of the eggs are fertilized. Of this number about 50 per cent are dispemiic, the remainder, normal.
 +
 +
The eggs of Menidia notata can be even more completely impregnated by the sperm of Fundulus heteroclitus. Under favorable circumstances 96 per cent of the eggs are fertilized. Of these only a few are dispermic or polyspermic.
 +
 +
The normally impregnated eggs of both crosses develop normally to varying stages of embryo formation. They never go beyond the closure of the " blastopore."
 +
 +
The embryos differentiate the three germ layers, the chorda and neural cord. In rare instances the eyes may begin to develop — the optic cup and the lens being formed.
 +
 +
The per cent of eggs that develop to the closure of the " blastopore " is comparatively small. The per cent is much greater in the Fundulus hybrids than in the reciprocals.
 +
 +
The more usual thing is for the embryos to show abnormalities. These appear during the process of gastrulation and are probably all the expression of a weakening of the developmental energy.
 +
 +
The abnormalities take the form of variously shortened embryos with the " blastopore " completely closed or imperfectly so, in which case the latter may take the form of a long slit or of a cleft of varying irregularity in shape.
 +
 +
The early cleavage stages are passed through in a perfectly normal manner. The blastomeres show no greater variation in form from the typical than do normal eggs.
 +
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William J. Moenkhaus 55
 +
 +
The rhythm of cleavage is that of the egg species. A spermatozoan from a species that normally has a different rate of cleavage cannot modify the rate of the hybrid egg.
 +
 +
Hybrid eggs may develop more slowly than normal eggs. This usually does not appear imtil later stages. As development proceeds the difference in rate grows increasingly great.
 +
 +
Dispermic eggs fall at once into four cells of the normal size and arrangement. This is followed by a normal 8, 16, 32, etc. cell stage.
 +
 +
The dispermic eggs of the Fundulus hybrid may develop to a late cleavage stage but never form a germ ring or embryonic shield.
 +
 +
The chromosomes of the two parent species, Fundulus heteroclitus and Menidia notata, are morphologically distinguishable, the rods of the former being long and straight in form, those of the latter, shorter, and commonly slightly curved. These retain their characteristic form when introduced into a strange egg through hj^bridization.
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 +
During the development of the hybrids they retain their individuality. During the first two cleavages each kind remains grouped and bilaterally distributed on the spindle. During the resting stage of the four-cell stage the chromatin becomes more or less mingled, so that when the third cleavage spindles are formed the grouping and the bilateral distribution of the chromatin has largely disappeared. During the following resting period the mingling has gone further, so that a complete grouping of the two parental chromosomes occurs very rarely in the following division. During the subsequent cleavages to a late cleavage only the mingled condition was observed.
 +
 +
This mingling of the chromosomes does not destroy their individuality for in stages of division favorable to bringing out the form of the chromosomes both kinds can be readily seen.
 +
 +
In these hybrids any nuclear conditions which would indicate that the chromatin is bilaterally arranged does not indicate any bilateral distribution of the two paternal chromatins in those nuclei.
 +
 +
The mingled condition of the maternal and paternal chromosomes in all but the very early stages of cleavages in these hybrids makes the bilateral distribution in the other forms described — Ascaris, Cyclops, Crepidula and Pinus — an open question.
 +
 +
The conditions obtaining in these hybrids are considered among the strongest evidences in support of Boveri's hypothesis that the individual chromosomes persist and do not mix in the resting stages of the nuclei
 +
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56 The Chromatin in the Development of Hybrids
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PAPERS CITED.
 +
 +
Appellof, a., 94.^ — Ueber einige Eesultate der Kreuzungsbefruchtung bei
 +
 +
Knochenfischen. Bergens Museuin Aarbog, No. 1, pp. 1-19, Taf. I. Beneden, E. van, 83. — Sur la maturation de I'oeuf et la fecundacion. Arch.
 +
 +
d. Biol., Tom. IV, pp. 265-640, PI. X-XIX. Beneden, E. van, and Neyt, A., 87. — Nouvelles recherclies sur la fecundation et la division mitosique ches I'Ascaride megalocepliale. Bull.
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 +
Acad. Roy. Belgique, 3me. Ser. Tom. XTV, pp. 215-295, PI. I-VI. Born, G., 83. — Beitrage zur Bastardirung zwischen den einheimischen Aiiur enarten. Arch. f. d. ges. Physiol., Bd. XXXII, pp. 453-518. BovE-Ri, Th., 87.— Zellen-Studien, Hft. I., Jen. Zeit., Bd. XXI, pp. 423-515,
 +
 +
Taf. XXV-XXVIII. BovEHi, Th., 88.— Zellen-Studien, Hft. II, Jen. Zeit., Bd. XXII, pp. 685-882,
 +
 +
Taf. XIX-XXIII. BovERi, Th., 90.— Zellen-Studien, Hft. Ill, Jen. Zeit., Bd. XXIV, pp. 314-401,
 +
 +
Taf. XI-XIII. BovERi, Th., 91.— Befruchtung. Ergeb. Merk. u. Bon., Bd. I, pp. 386-485. CONKLIN, E. G., 01. — The Individuality of the Germ Nuclei during the Cleavage of the Egg of Crepidula. Biol. Bull., Vol. II, No. VI, pp. 257-265. Driesch, H., 93.— Entwicklungsmechanische Studien V. Zeit. f. wiss. Zool.,
 +
 +
Bd. LV, pp. 29-34, Taf. III. Driesch, H., 98.— Ueber rein-mutterliche Charactere an Bastardlarven von
 +
 +
Echiniden. Roux' Arch., Vol. VII, pp. 6d-102. Ferguson, M. C, 01. — The Development of the Egg and Fertilization in
 +
 +
Pinus Strobus. Ann. Bot., Vol. 15, pp. 435-479, PL XXIII-XXV, GuiGNARD, L., 91. — Nouvelles etudes sur la fecondation. Ann. des Sci. Nat.
 +
 +
Bot., Tom. XIV, pp. 163-296, PL IX-XVIII. Hacker, V., 92. — Die Eibildung bei Cyclops und Canthocampus. Zool.
 +
 +
Jahrb., Abth. f. Anat., Bd. V, pp. 211-248, Taf. XIX. Hackek, v., 95. — Ueber die Selbstandigkeit der viiterlichen und miitter lichen Kernbestandtheile wahrend der Embryonalentwicklung von
 +
 +
Cyclops. Arch. f. Mikr. Anat., Bd. XLVI, pp. 579-618, Taf. XXVIII XXX. Hacker, V., 01. — Ueber die Autonomic der viiterlichen und miitterlichen
 +
 +
Kernsubstance vom Ei bis zu den Fortpflanzungszellen. Anat. Anz.
 +
 +
Bd. XX, pp. 440-452. Herla, v., 93. — Etude des variations de la mitose chez I'Ascaride megalo cephale. Arch, d, Biol., Tom. XIII, pp. 423-520, PL XV-XiX. Hebtwig, O. and R., 87. — Ueber den Befruchtungs- und Theilungsvorgang
 +
 +
des thierischen Eies unter den Einfluss ausserer Agentien. Jen.
 +
 +
Zeit., Bd. XX, pp. 120-241, Taf. III-IX. Hertwig, O., 90. — Vergleich der Ei- und Samenbildung bei Nematode a.
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Arch. f. Mikr. Anat., Bd. XXXVI, pp. 1-138, Taf. I-IV. Hertwig, O., 92. — Urmund und Spina Bifida. Arch. f. Mikr. Anat., Bd.
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XXXIX, pp. 353-502, Taf. XVI-XX. Mathews, A. P., 02. — The so-called Cross Fertilization of Asterias and
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Arbacia. Am. Journ. Physiol., Vol. VI, No. IV, pp. 216-218,
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William J. Moenkhaus _ 57
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Morgan, T. H., 93. — Experimental Studies on Echinoderm Eggs. Anat.
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Anz., Bd. IX, pp. 141-152. Pfluger, E., E!2. — Die Bastardzeugung bei den Batrachiern. Arch. f. gos.
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Physiol., Bd. XXIX, pp. 48-75. Rabl, C, 85. — Ueber Zelltheilung. Morph. Jahrb., Bd. X, pp. 214-330. RticKERT, J., 95. — Ueber das Selbststandigbleiben der vaterlichen und
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miitterliehen Kernsnbstance wahrend der ersten Entwickliing des
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befruchteten Cyelops-Eies. Arch. f. Mikr. Anat., Bd. XLV, pp. 339 367, Taf. XXI, XXII. Stassano, E., 83. — Contribuzione alia fiziologia degli spermatozoidi. Zool.
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Anz., Bd. VI, pp. 393-395. WiLSOX, E. B., and Mathews, A. P., 95. — Maturation, Fertilization and
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Polarity in the Echinoderm egg. Journ. Morph., Vol. X, pp. 319-342. ZojA, E., 95. — Sulla independenza della cromatina paterna e materna nel
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nucleo delle cellule embryonale. Anat. Anz., Bd. XI, pp. 289-293.
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58
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The Chromatin in the Development of Hybrids
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EXPLANATION OF PLATES. PLATES I TO IV.
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ABBREVIATIONS.
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bVpo., Blastopore. cd., Chorda. ec'drm., Ectoderm. emb., Embryo. en'drm., Endoderm. In^., Lens. nis^drm., Mesoderm. pi'bl.. Periblast. ylTc., Yolk.
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PLATE I.
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Fig. 1. Conjugation of pronuclei in a dispermic Fundulus hybrid egg.
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Fig. 2. Cross section of a Fnndulus hybrid embryo that was nearing' the closure of the " blastopore." It shows the ectodemi, endoderm, mesoderm, neural cord, chorda and periblast.
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Fig. 3. Horizontal section through the eye of a Fundulus hybrid embryo that had come to the close of its development. Very few of the cell boundries can be made out. Cells of the cup are arranged in more or less distinct rows. Nucleoli numerous and large. One cell in the lens in the process of division.
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Figs. 4, 5, 6 and 7. Caudal end of four Fundulus hybrid embryos which had come to the close of their development. Shows four types of abnormal " blastopore " closure.
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DEVELOPMENT OF HYBRIDS -WM. J. MOENKHAUS
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PLATE
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vj cJk/ . "Vvva' oLaayv
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{jC'cLayiA/.
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AMERICAN JOURNAL OF ANATOMY— VOL. II
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GO The Chromatin in the Development of Hybrids
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PLATE II. •
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Figs. 8, 9 and 10. Types of abnormal " blastopore " closure in the Menidia hybrids.
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Fig. 11. Late anaphase of the first cleavage of a normal Fundulus heteroclitus egg. All of the chromosomes are of the long- type.
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Fig. 12. Anaphase of the first cleavage of a normal Menidia notata egg. All of the chromosomes are of the short type.
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Fig. 13. Chromosomes of Fundulus heteroclitus and Menidia notata drawn io the same scale. Both are taken from the spindle shown in Figure 14.
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Fig. 14. Anaphase of the first cleavage of a Fundulus hybrid egg. To the right in each half of the spindle occur only the short chromosomes; to the left, only the long ones, c? Menidia. 5 Fundulus.
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Fig. 15. Anaphase of the first cleavage of a Menidia hybrid. To the right are all long chromosomes; to the left, all short ones. J' Fundulus. 5 Menidia.
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Fig. 16. Metaphase of first cleavage of Fundulus hybrid. The larg'e chromosomes to the right. J' Menidia. 5 Fundulus.
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Fig. 17. Metaphase of the second cleavage of a Menidia hybrid. The long chromosomes to the right. ^ Fundulus. 5 Menidia.
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Fig. 18. Half of an anaphase spindle of the second cleavage of a Menidia hybrid. All of the long chromosomes are to the left, c? Fundulus. 2 Menidia.
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DEVELOPMENT OF HYBRIDS
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WM. J. MOENKHAUS
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' CAvJlr
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12
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10
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■'■■'%
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15
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16
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YV«lj
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18
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2
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AMERICAN JOURNAL OF ANATOMY— VOL. Ill
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63 The Chromatin in the Development of Hybrids
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PLATE III.
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Fig. 19. Anaphase of the third cleavage of a Menidia hybrid. In the upper half of the spindle the two kinds are clearly bilaterally distributed. J' Fundulus. $ Menidia.
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Fig. 20. Early anaphase of the fourth cleavage of a Menidia hybrid. The two kinds of chromosomes are evidently mingled. (^ Fundulus. 5 Menidia.
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Fig. 21. Chromosomes from a fourth cleavage spindle of a Fundulus hybrid. The cell w^as in anaphase. The chromosomes of the drawing are such of one end of a spindle in a single section as could be distinctly made out. They are drawn by only the partial aid of the camera. Each chromosome is faithfully reproduced so far as its form and size are concerned but its position with reference to its neighbor is not in every instance retained in the drawing, c? Menidia. $ Fundulus.
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Fig. 22. Anaphase of cell from middle cleavage of a Menidia hybrid. The two kinds of chromosomes are mingled. The short ones, as a whole, are nearer the i^ole than the long ones, so that they form a band extending across the spindle. J' Fundulus. 5 Menidia.
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Fig. 23. Telophase of the third cleavage of a Menidia hybrid. Only one-half of the spindle is shown. Of the two groups of vesicles the smaller ones, nearer the poles, are probably from the short chromosomes. The group of larger vesicles is composed for the most part of the long chromosomes not yet so far along in their metamorphosis. ^ Fundulus. 5 Menidia.
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Fig. 24. Late anaphase of a cell from middle cleavage of a normal Menidia notata. Only the small type of chromosomes are present. Compare with corresponding stages in Figures 22, 26 and 32.
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Fig. 25. Early anaphase of cell from early cleavage of a normal Fundulus heteroclitus egg. Only the long chromosomes are present.
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Fig. 26. Late anaphase of a cell from later cleavage of a normal Fundulus heteroclitus egg. Only the long- type of chromosomes are present. Compare with corresponding stages shown in Figures 22, 24 and 33.
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Fig. 27. Cells from middle cleavage of a Menidia hybrid. They show the double nuclei in these cells.
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Fig. 28 is omitted on purpose.
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DEVELOPMENT OF HYBRIDS WM. J. MOENKHAUS
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PLATE III
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v»|
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X
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V^^***
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I
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21
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"A
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24
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x,irf
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19
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5<
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I
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cf'
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25
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25
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■■>c(
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<?<
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22
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26
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27
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AMERICAN JOURNAL OF ANATOMY— VOL. Ill
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64 The Chromatin in the Development of Hybrids
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PLATE IV.
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Fig. 29. Anaphase of the first cleavage of a Fundulus hybrid egg. The small Menidla chromosomes introduced by the sperm are grouped to the right.
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Fig. 30. Anaphase of the first cleavage of a Menidia hybrid egg. Here the long Fimdulus chromosomes have been introduced by the sperm and are grouped on the right in the spindle.
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Fig. 31. Half of an anaphase spindle of the second cleavage of a Menidia hybrid. The short and the long chromosomes are grouped to the right and left respectively. (^ Fundulus. 5 Menidia.
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Fig. 32. Late metaphase and late anaphase of cells from middle cleavage of a Menidia hybrid egg. In the latter the long chromosomes extend for a considerable distance along the spindle fibres while the short ones are nearer the poles and form a band across the spindle, c? Fundulus. 5 Menidia.
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Fig. 33. Late anaphase of a cell from middle cleavage of a Menidia notata egg. All the chromosomes are short. There are no long straggling chromosomes as in the hybrid cells (Fig. 32.) See also Figures 22 and 2G.
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DEVELOPMENT tDF HYBRIDS WM. J. MOENKHAUS
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PLATE IV
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AMERICAN JOURNAL OF ANATOMY— VOL. Ill 5
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ON THE LUNG OF THE OPOSSUM.
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BY
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JOHN LEWIS BREMER, M. D.
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From the Emhryological Laboratory of Harvard Medical School.
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With 11 Text Figukes.
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The lung of the new-born opossum in the pouch shows peculiarities, already partly described by Selenka, which make it appear tliat respira
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FiG. 1. Opossum, 10.5 mm. trans. Series 614, No. 305. Ao, aorta; oc.s, oesophagus; «M, auricle, ren, ventricle of heart; pr., new bronchial bud.
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tion is carried on in specially modified bronchi and bronchioles before the infundibular portion of the lungs is developed. Selenka's descrip A.MERicAN .Journal of Anatomy. — Vol. III.
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68
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On the Lung of the Opossum
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tion, found on p. 159 of his '^ Entwickel ungsgeschichte der Tiere," is as follows :
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" The lungs of opossum have to develop into functioning breathing organs within the last three days of uterine life. There is neither the available material nor the necessary time to make a very great number of alveoli and prepare them for breathing (as is completely done in
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Fig. 2. Opossum, 13.5 mm. frontal. Series 618, No. 838. Pul. art, pulmonary artery ; pul. ve, pulmonary vein ; oes, oesophagus ; pr, new bronchial bud ; card, part of cardiac lobe.
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foetal life in placentalia) . Only a few dozen large air-chambers, as a provisional breathing apparatus, can be made, which later, during the life iji the pouch, develop by the growth of partitions into a richly branched bronchial tree. The lung may be said to be of rapid growth inasmuch as the alveoli are ready for breathing in a remarkably short time; but its growth is slow if we consider the increase in the number
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John Lewis Bremer
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69'
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of alveoli as a gauge. Probably in this wonderful development of the (ipossum lung, the forces at work in evolution are reproduced, for the lung of a new-born opossum has exactly the form of a reptilian lung."
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In the main this description is correct, but it seems to me imperfect in some respects. The opossums examined by me were : first, six newborn, taken from the same pouch, ranging in size from 10.5 to 13.5 mm. , second, two of about 14 cm. ; third, two young adults and one old adult.
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The lungs of the smallest opossums correspond to the description of Selenka, as they are composed of a few large air-chambers, opening almost directly into the main bronchus, which is itself an elongated chamber. The appearance in section is shown in Figs. 1 and 2, a trans
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'card.
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Fig. 3. Fig. 4.
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Fig. 3. Cast of lung of 12.5 mm. opossum, seen from behind and from the left. pr, new bronchial buds.
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Fig. 4. Cast of lung of 12.5 mm. opossum, seen from the front and a little above. pr, new bronchial buds; card, cardiac lobe; It. ep, rt. ep, left and right eparterial bronchi; x, x, groove for pulmonary artery.
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verse and a frontal section of a 10.5 mm. and a 12.5 him. opossum respectively; and the general form is shown in Figs. 3 and 4, drawings of a cast of the lung of the 12.5 mm. opossum obtained by Born's waxplate method. Selenka is wrong, however, in speaking of alveoli ; the large chambers correspond to bronchi and bronchioles; infundibula and alveoli are lacking.
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In the lungs of placentalia growth in the embryo is accomplished by the branching of the small tubes of cuboidal cells and with narrow lumen, which represent the bronchi ; each new limb in turn sends out
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70
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On the Lung of the Opossum
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new buds, all of which are like the parent stem, pushing into the surrounding mesenehyma. Just before birth a new kind of bud, with a different system of division, is developed from the end of the last set of branches, and these form the infundibula and alveoli, the true breathing portion of the lung. In the young opossum, which is transferred to the pouch when only about 10 mm. long, breathing must be carried on at the same time as the growth and branching of the bronchial tree; so instead of the usual short buds of cuboidal epithelium, as found in placentalia, in the young opossum large chambers are found, representing the narrow tubes, but lined with peculiar epithelium so that
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they may serve as respiratory organs. These chambers are not, however, to be considered alveoli, but bronclii and bronchioles : and they retain their power of sending off new buds or branches, which may be seen in the model and in the sections as hornlike processes, hollow and slender at first (soon widening into large chambers), pushing into the surrounding mesenehyma, and giving evidence that Selenka's idea of division into a bronchial tree by means of newly forming partition walls is wrong. The lung of the newborn opossum is composed of a simple system of branching bronchi and bronchioles, dilated
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Fig. 5. Opossum, 13.5 mm. Series 618, No. 303. i linpd with modified enithe Ep, epithelium Uning air-chamber; mits, muscle; anci imeu W lUl luuuimu tpitiic
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'"'^^T'^^i^^'r^%:'i^iS^^f limn to allow for l)reathing,
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P^Km^. Lacerta. Series 604, No. 325. but retaining their pOWCr of
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Fig. 8. Opossum, 14 cm. Trans.section of lung. x'.„4-i,„t, ornwth Pr, new bronchial branches ; i»/, infundibula. luriner growui.
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On examining the epithelium lining these air-chambers, we find what seems to me a transitional stage between cuboidal and "breathing epithelium" (see Figs. 5 and 6). Directly over the capillaries, three of which are cut across in Fig. 6, the cells have become squamous, with a thin plate and the nucleus lying between the blood vessels : but the plates are not to be compared in thinness with those of the human lung, for instance (in one cell in Fig. 6 the nucleus lies in the plate) ; and the meshes of the capillaries are so
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Jolm Lewis Bremer
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71
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wide that many cells remain cuboidal, having no capillary over which to spread a plate. This peculiar epithelium extends not only over the inner surface of all the air-chambers, but also over the main bronchi as far up as the beginning of the rings of cartilage.
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If we compare these lungs with those of some reptiles we find that they are similar both in tlie arrangement of air-chambers opening into a dilated main bronchus, and in the character of the epithelium, as is shown in Fig. 7, drawn from the lung of Lacerta. Also in both the opossum lungs and reptilian lungs there are bands of muscle fibres running circularly around the central air-chamber or bronchus, whose probable function is to contract the lung and force the air out during expiration. Still further, in the reptilian lung the arrangement of the bronchial branches is symmetrical, both ' right and left bronchus being provided with one branch anterior to, and another posterior to the pulmonary artery; and if we examine the drawing (Fig 4) and the diagram (Fig. 10) made from the same opossum, we find that in the new-born opossum also there is one bronchial branch in front of and another behind the artery in both right and left lung. This was found in five out of the six new-bom opossums; one was rendered useless for serial work. In other words, the new-born opossum has an eparterial bronchus on both right and left sides; that on the left is always the smaller and slightly lower placed, and the air-chambers supplied by it do not form ttie apex of the lung; still in spite of its small size and relatively low position, it is distinctly above the first ventral bronchus and behind the artery and so corresponds to the eparterial bronchus of the right lung, and may be considered as making the two lungs symmetrical and reptilian in type, as no placental mammalian lungs are. This symmetry is marred by the presence of a large cardiac lobe on the right side, of which I can find no trace on the left. Still, as regards general appearance, character of the lining epithelium, and symmetry of bronchial branches (with this one exception),, these lungs are, as Selenka says, reptilian.
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Let us now trace the growth of this lung further. On looking at Fig. 8, a section of the lung of a 14 cm. opossum, we find the primary bronchi and their early branches now provided with a thick coat, partly due to the multiplication of the circular muscle fibres already mentioned, while
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Fig. 9. Photograph of cut surface of lung of young adult opossum.
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72
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On the Lung of the Opossnm
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pul.
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-'-'-'art.
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the lining epithelium has reverted to the cuboidal type or even become' cylindrical. We have found now the reason for the peculiar epithelium seen when these passages were breathing spaces; it was a compromise allowing enough oxygenation of the blood for an animal whose existence is passed in the mother's pouch, and yet not far enough removed from the cuboidal type to make it hard to revert to it.
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The bronchial tree has become quite complicated and at the surface of the lung may be seen in cross-section new hornlike processes (pr.) representing newly formed branches. But with these are terminal pieces of much larger size, often with triple branching, seen chiefly on the surfaces of the lung where growth has nearly ceased. They represent a new element in the opossum lung, but one found in placental lungs just before birth, namely the infundibular portion of the lung (inf.). They may be seen forming a cortex in Fig. 9, a photograph of the cut surface of the lung of a young adult opossum; but with age they become inconspicuous because more evenly distributed. They mark the end of the stage of rapid growth, for from these infundibula no
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branches, unless we count
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the alveoli, are given off;
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and so they are absent from
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all activity grov/ing portions
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(such as the borders in Fig.
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8), their places being taken ■^ep ^
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by the hornlike processes,
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which are capable of further growth. The lung has
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changed from a reptilian „ ,„ „ . to a mammalian type part FiG. 10. Diagram. ^ ^
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lung of opossum of ia.5 w j^y the multiplication of
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mm. seen from in front. J J ^^
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Pul. art, pulmonary ^he bronchial branches, but
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artery; card, cardiac ^ '
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i^n'cf^i'htTpI^t^^eV^fil Chiefly by the addition of a ^,,.1, ?ia.ram: Lung^of
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bronchi. VI, m-st ven- ^ew class of air chambers, op^fJi^^ °* ^* ^°^- ^^'^^ ^'^^^
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tral bronchial branch. ^ inrioui.
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growing from the ends of the bronchioles, but differing from them m that the spaces are dilated instead of horn-shaped or tapering, and are lined with true "breathing epithelium," with narrow-meshed blood vessels and very thin plates.
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The opossum lung changes from reptilian to mammalian also in the loss of the left eparterial bronchus. How this comes about I am unable to state for the lack of the necessary stages, for already in the opossum of 14 cm. the change is complete, as can be seen in Fig. 11, a diagram of the bronchial branches and the arteries of an opossum of that size, where no trace of a left eparterial bronchus remains.
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John Lewis Bremer ' 73
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Following Selenka's suggestion, then, it seems to me that we find in the lung of the opossum an epitome of the evolution from reptilian to mammalian lung, and that the chief points are the loss of the left eparterial bronchus in mammals, and the addition to the reptilian lung, which consists only of bronchi and bronchioles, of a new apparatus, with a different and more complicated system of branching, and with walls better adapted for breathing — the infundibular portion of the lung.
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ENAMEL IN THE TEETH OF AN EMBRYO EDENTATE (DASYPUS NOVEMCINCTUS LINN)/
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BY
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A. M. SPURGIN, M. D. With 2 Plates.
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Tomes was the first to work on the embryology of the teeth of the nine-banded armadillo {Dasypus novemcinctus L.). In 1874, he examined two embryos, one early and one relatively late. The exact length of these embryos I have been unable to ascertain, but in the early one a layer of dentine had been deposited. He said that the stellate reticulum or enamel pulp was absent, and that he failed to find any enamel or anything like it upon the teeth. He regarded the enamel organ as rudimentary, stating that an enamel organ was present in all tooth-germs, and that it was entirely independent of any subsequent development of enamel.
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In 1884, Pouchet and Chabry examined embryos of Oryderopus capensis, and Bradypus tridactylus. In an embryo of the former, of 32 cm., they found a typical rudimentary incisor with an enamel organ and dental papilla in which a layer of dentine had been deposited. An embryo of 12 cm. of Bradypus tridactylus showed an enamel organ covering the dental papilla in which a layer of dentine had appeared. In an embryo of the same animal of 23 cm. in which the teeth had erupted, they described the dentine, vasodentine, and outer coat of cement of the typical adult tooth. They found no enamel, and state that the stellate reticulum was absent in the enamel organ of the sloths.
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As early as 1828, A. Brants found a rudimentary incisor in the lower jaw of Bradypus tridactylus. P. Gervais in 1873 confirmed this discovery. Burmeister made a similar discovery in the fossil Scelidotherium leptocephaluni. Flower, in 1869, described a rudimentary incisor in the lower jaw of Tatusia Peha (Dasypus novemcinctus) , and in 1877 Eeinhardt observed as many as four in the lower jaw of the same animal.
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Hensel has shown that the armadillos, Dasypus novemcinctus and D. hybridus Desm. are diphyodont. In an examination of thirty five skulls
 +
 +
I Contributions from the Zoological Laboratory of the University of Texas. No. 51. American Journal of Anatomy. — Vol. III.
 +
 +
 +
 +
7G Enamel in tlie Teeth of an Embryo Edentate
 +
 +
of the former, he found some with milk teeth and some showing the change to the premanent set. 'J'he last tooth had no predecessor, and the teeth were not changed until the animal had nearly reached the adult stage. On examination of two skulls of Dasypus hyhridus a similar condition was found. A rudimentary incisor was also found in this animal. The milk teeth have been described as two-rooted, but Tomes holds that this appearance is due to the absorption set up by the pressure of the succeeding permanent teeth.
 +
 +
In 1889, 0. Thomas found in two young specimens of Orycteropus of fourteen and eighteen inches respectively, a complete though rudimentary set of milk teeth in each jaw, none of which were in the premaxillge. They were all minute, and this fact led him to think it very doubtful that they would ever have cut the gum. Unfortunately his material was limited, and he made no histological investigation, so we know nothing of tlie structure of the enamel organ at this stage. Thomas has also examined specimens of Bradypus, Choloepus, and Dasypus, apparently of a suitable age, and could find no trace of a milk dentition; he says, however, that the possibility still remains that in younger stages uncalcified tooth-buds of such teeth may be present. Tomes and Flower have also examined fu'tal Choloepus and Bradypus and have found no trace of any milk dentition.
 +
 +
In 1892, Rose (92% p. 507) observed in an embryo of Myrmecophaga (Udactyla 20 cm. long, at the point of the jaw, where in other cases the tooth-buds are connected with the mouth epithelium, a row of exceptionally high papilla?. He thinks that very probal^ly in younger, stages the tooth-buds were formed at this place but were not further developed. In the same year, in an embryo of 7.G cm. of Manis tricuspis and one of ]\f. javanica of 9 cm., Eose ° found well defined dental folds in both upper and lower jaws, and in the lower jaws rudimentary club-shaped toothl)uds. He has shown by examination of older specimens that they subsequently disappear.
 +
 +
The only work that has been done on the teeth of the armadillo since that of Tomes has been done by Eose and by Ballowitz who worked at the same time but independently of each other.
 +
 +
In 1892, Eose examined two embryos, one of Dasypus novemcinctus of 7 cm. and one of D. hyhridus of 6 cm. In the first-named embryo he found an enamel organ composed of an inner and an outer epithelial layer and a well developed stellate reticulum. He did not describe any
 +
 +
2 The mounted slides of these embryos had been furnished by Max Weber {'91) who had not found any indicatiou of the dental folds.
 +
 +
 +
 +
A. M. Spurgin 77
 +
 +
stratum intermedium. lie described the l)uds for the permanent teeth as arising from the outer layer of the enamel organ, and not, as Tomes has shown it in one of his figures, as coming from the mucous membrane of the mouth cavity. The bud for the eighth tooth, which has no predecessor, he described and illustrated as arising directly from the mucous membrane of the mouth cavity. With the exception of the first, the teeth are bicuspid. The tooth-buds of two rudimentary incisors were described but no dentine had been deposited. Eose found the same general condition in the embryo of Dasijpiis liyhridus. Besides two rudimentary incisors, there were seven back teeth, the first two having single cusps. On the whole the development wa« further advanced than in the other embryo, dentine having been deposited in the rudimentary incisors as well as in some of the back teeth. Rose found in connection with the enamel organ of both of these rudimentary incisors, secondary buds coming from the outer epithelial layer, the one from the second incisor being best developed. He remarks that this does not cut off the possibility that tliis tooth may also have a successor in the later change of teeth. He states that while the embryos examined by him bad no enamel, they did have, as a secretion product of the enamel cells. a thin structureless membrane lying directly against the dentine and exactly corresponding to the formation which in other animals we call Nasymth's membrane.
 +
 +
Ballowitz examined two embryos of Dasypns novemcinctiis of 6 and 8 cm. respectively. He found a typical enamel organ, with inner and outer epithelial layers, stratum intermedium, and well developed stellate reticulum. He describes the processes of the inner columnar epithelial cells, generally known as Tomes' processes, but says he has not been able to explain them. He states that very soon after the first layers of dentine have been deposited, the outer layer of cells disappears and the stellate reticulum is replaced by connective tissue. He says that while it is true that the inner epithelial layer and stratum intermedium remain over the calcified dentine in an unbroken layer, they have undergone a considerable change ; the inner layer loses its columnar shape and becomes flattened, the stratum intermedium is reduced, so that only two or three layers of flat cells can be found on the cusps. Whether these cells have anything to do with the development of Xasymth's membrane, or Avhether in these teeth such a membrane was present at all, Ballowitz says, it was impossible to decide. In the tooth-buds of the larger embryo, which were separated only by connective tissue, he found secondary buds coming off from the lingual side of the outer epithelial layer of the enamel organ. Xo rudimentary incisors were described, and I presume none
 +
 +
 +
 +
78 Enamel in the Teeth of an Embryo Edentate
 +
 +
were observed. The point Ballowitz lays most stress upon is the finding of an epithelial ring at the base of the dental papilla which is a portion of the enamel organ constricted off from the lower edge of that organ. He has shown this epithelial ring to persist in the adult, and he regards it as essential to the development of the dentine in these continuously growing teeth. He quotes from A. von Brunn's work on the enamel organ in support of this theory, but I have been unable to see this article. Ballowitz denies that the presence of the stellate reticulum and stratum intermedium have any close connection with the deposition of enamel, stating positively that at no time can enamel be deposited in the Dasypus novemcinctus, and that the only functions of the enamel organ are : to give form to the developing tooth, to stimulate the odontoblasts to deposit dentine, and to give off the epithelial ring which is necessary to the continued development of the dentine.
 +
 +
A year ago. Dr. W. M. Wheeler, of the School of Zoology, had the good fortune to secure four embryos of the Dasypns novemcinctus from an adult female which had been kept in the laboratory for several weeks. The embryos were removed immediately after the animal had been chloroformed, and were hardened for six weeks in Miillcr's fluid, primarily for studying the placentation. He found four placentae inclosed in one amnion (Plate I), but has not since had the time to study the subject further. Dr. Wheeler very kindly furnished me the material for working on the embryology of the teeth, but owing to the pressure of other work, nothing was done until this year.
 +
 +
The largest embryo of 9 cm.' and one measuring 8.5 cm. were selected. From the larger embryo, longitudinal sections of the lower jaw were made, and by making a sagittal section of the upper jaw, both longitudinal and transverse sections were obtained. They were imbedded in celloidin, cut 25 micra thick, and stained in hasmatoxylin and eosin, but were not kept in series. From an embryo of 8.5 cm. both longitudinal and transverse sections of the lower jaw and longitudinal sections of the upper jaw were made. They were imbedded in paraffin, cut 10 micra thick, mounted in series, and stained with iron hsematoxylin.
 +
 +
In the longitudinal sections of the lower jaw of the 8.5 cm. embryo, I found five rudimentary incisors and eight back teeth. The jaw measured 11 mm. from the tip to the posterior edge of the last toothbud. The first incisor was found 1.8 mm. from the tip, the width of
 +
 +
" In all cases the measurements jjiven are from the crown of the head to the base of the tail.
 +
 +
 +
 +
A. M. Spurgin 79
 +
 +
the jaw at this point being 1.5 mm. The incisors were separated from each other by about .5 mm. These rudimentary incisors diminished in size and degree of development from behind forward as shown in Plate II, Fig. 1. They were separated by connective tissue with the exception of the last two, which were separated by a rather large piece of cartilage. A similar piece of cartilage behind the last tooth and a somewhat smaller piece growing up between the third and fourth teeth (Plate II, Pig. I), would seem to indicate that sockets were to be formed for at least the last two. The shape of the first three teeth is that of a true incisor with a single cutting edge, while the shape of the last two is nearly that of a typical cuspid with a single somewhat prominent cusp.
 +
 +
On each of the rudimentary incisors a layer of enamel has been deposited. The relative thickness, which diminishes from the back tooth forward, is represented in Plate II, Fig. 1, by the black line. Under the low power, the enamel appears as a dark band which, in many sections, has been pulled away from the dentine and fractured in the direction of the enamel rods. This was due to the sectioning, since the tissue had not been completely decalcified by the Miiller's fiuid. Plate II, Fig, 2, shows this condition in a high power drawing of one of the incisors. With they'g-inch oil immersion lens the direction and structure of the enamel rods could be made out. In the fourth and fifth incisors the inner layer of the enamel organ had lost its columnar character; the stellate reticulum had disappeared, and only a few layers of flattened epithelial cells remained over the enamel layer. In the first three teeth, in which the enamel was not so thick, more of the enamel organ remained, and at places away from the central area of the cusp, the columnar cells of the inner layer could be seen. The cells in the immediate area of the cusps were flattened as in the case of the last two teeth. This clearly indicates that the enamel in the last two teeth has been completely laid down, while more may yet be deposited from the columnar cells in the three anterior teeth. No Nasmyth's membrane could be found, and no secondary buds were observed in any of the rudimentary incisors. These buds were not to be expected, since the development had advanced considerably further than in the 6 cm. embryo of Dasypus hyhridus, in which Eose demonstrated their occurrence. Although I carefully examined the sections from the upper jaws of both embryos, I failed to find any trace of Imds for rudimentary incisors.
 +
 +
From a study of the longitudinal and transverse sections from the lower jaw of the 8.5 cm. embryo, it could be seen that the tooth-buds of the eight back teeth were almost completely surrounded by cartilage. The two plates of cartilage forming the groove in which the teeth were
 +
 +
 +
 +
80 Enamel in the Teeth of an Embryo Edentate
 +
 +
developing sent prolongations between them which roughly followed the contour of the teeth. The tooth-buds, however, were close together and complete septa had not as yet been formed between them. In all the back teeth except the eighth, a thin layer of dentine had been deposited and in a few of them it was calcified. On the whole the development of the teeth in the lower jaw was in advance of that of the upper. In the embryo of 9 cm. the development was still further advanced, and calcified dentine was found in most of the teeth. Plate II, Fig. 3, shows the first back tooth with a well developed layer of enamel appearing under the low powder as a much darker band than the dentine, and broken at frequent intervals in the direction of the enamel rods in the process of sectioning. As in the first three rudimentary teeth, the columnar cells of the inner layer of the enamel organ have become flattened over the thicker portion of the enamel layer, while they still retain their shape over the thinner portions (Plate II, Pigs. 2 and 3, ce). The stellate reticulum and outer layer of the enamel organ were still present over the sides of the dental papilla as shown in Fig. 3. The portion marked eo, which has been torn in sectioning from the body of the enamel organ, shows the epithelial ring (er) in process of being constricted off. This has been described in full by Ballowitz and has also been observed by Pouchet and Chabry in the embryo of the sloths. I found this portion of the enamel organ in many sections of both rudimentary and back teeth. In some sections it has been separated from the enamel organ. Plate II, Fig. 4, shows a high power drawing of the enamel and dentine from the same section as Plate II, Fig. 3. The uncalcified dentine is easily distinguished from the darker calcified dentine, being cut off from the latter by a sharp line of demarcation, a condition which was not found in any of the rudimentary teeth. This may indicate that in these teeth the dentine has been completely deposited. If such proves to be the case in later embryos, Ballowitz's theory concerning the epithelial ring would have no weight.
 +
 +
In the fifth and sixth tooth-buds, in the longitudinal sections of the smaller embryo, the buds for the permanent teeth could be seen coming off from the outer epithelial layer of the enamel organ. Plate II, Fig. 5, which shows this, shows also a portion of the enamel organ with inner and outer epithelium, stratum intermedium, stellate reticulum, and Tomes' processes. A thin layer of uncalcified dentine has been deposited. I do not find that the outer layer of the enamel organ is broken through until after the enamel has begun to be laid down. Eose and Ballowitz both describe the breaking up of this layer as taking place shortly after
 +
 +
 +
 +
A. M. Spurgin 81
 +
 +
a thin layer of dentine lias been deposited and much earlier than is the case with most animals. Kose (93, p. 448) describes the same condition in the teeth of reptiles. The condition I find is exactly what we sliould expect. As is well known, the dentine is deposited first and the outer layer and stellate reticulum of the enamel organ do not disappear until after the first layers of enamel have been deposited (Sudduth, 86, p. 640). Eose and Ballowitz, however, found no enamel and Ballowitz describes the early degeneration of the entire enamel organ.
 +
 +
The bud for the last tooth, which has no predecessor in the milkdentition, was considerably smaller than the other seven. A well-rounded dental papilla was present, and the enamel organ was connected with the enamel organ of tlie seventh tooth by an epithelial band consisting of several layers of cells (Plate II, Fig. 6). I could trace this band distinctly through ten or twelve sections from the longitudinal series of both upper and lower jaws of the 8.5 cm. emljryo. I was also able to follow it in the transverse serial sections of the lower jaw of the same embryo.
 +
 +
As will be seen, the results of my work on the tooth embryology of the armadillo diifer in several important points from those of Eose and of Ballowitz. Eose described and figured the bud for the last tooth as coming from the mouth cavity direct, but it had not as yet expanded into the enamel organ and no dental papilla was present. What Eose had was probably a tubule of one of the glands which appear in the region behind the last tooth-bud.
 +
 +
As has been mentioned, Eose describes as the secretion product of the enamel cells, a thin structureless membrane which lies directly against the dentine and corresponds to the Xasmyth's membrane of other animals. It is very evident that such a membrane does not exist between the dentine and the enamel which I have shown to be deposited later. Eose may have seen a very thin layer of enamel.
 +
 +
I shall not enter into a discussion of the epithelial ring upon which Ballowitz lays so much stress, since I did not have access to the literature upon the subject, but he is certainly wrong in asserting that the only function of the enamel organ is to give the form to the developing tooth, and to give off the epithelial ring. In regard to the presence of the stellate reticulum, Eose offers no explanation. Ballowitz. while recognizing that the stellate reticulum is found only in tooth -buds in which a layer of enamel is afterwards deposited, denies that it has any connection with the deposition of this substance. It is indeed difficult to see how Ballowitz could have failed to see any significance in Tomes' processes, which he described in connection with the enamel cells. He 5a
 +
 +
 +
 +
82 Enamel in the Teeth of an Embryo Edentate
 +
 +
says that we could adopt Waldeyer's mechanical theory of the enamel pulp as merely serving to make room for the developing tooth, were it not for the fact that the entire enamel organ disappears so early. But I have shown that this is not the case ; the breaking up of the outer epithelial layer and disappearance of the stellate reticulum does not take place any earlier in the armadillo than in other animals. Wliile j-ecognizing the importance of the enamel organ in all animals as directing the growth of the dentine and giving the form to the tooth, I do not believe that the stellate reticulum merely subserves a mechanical functioii; but I regard the finding of enamel in the armadillo as strengthening the view that the stellate reticulum holds pabulum for the first layers of enamel.
 +
 +
I was unable to see Eeinhardt's article, but find through Eose's discussion of it that he describes the rudimentary teeth of Dasypus novemcinctus as having closed roots and states that they never cut the gum but are later absorbed. He says, however, that the last tooth is sometimes retaino^ in half-grown animals. I did not find the teeth showing any signs of absorption and, as can be seen from Plate II, Fig. 1, they have open roots which are typical of the persistently growing adult teeth. I believe that the teeth will be erupted and thus lost. I am led to this view by the fact that there are indications of the formation of sockets for the last two teeth, and that the teeth are all fairly well developed. Supporting this view, we know that in the Priodontes the teeth in the anterior portion of the Jaw are soon lost and that all traces of the sockets disappear. We also know that in Dasypus seiosus, and the fossil Chlamydotherium, incisors still function.
 +
 +
While Rose and Ballowitz very correctly state that the discovery of a well developed enamel organ in the armadillos tends to show that they are descended from animals whose teeth are more highly organized, I have shown that enamel is still present on the teeth of the milk dentition, and that the gradual reduction of the enamel, as well as that of the incisor teeth, is still taking place. I believe that older stages of the Dasypus liyhridus, in which, according to Rose, the enamel organ is equally well developed, will show enamel. The question as to whether or not any enamel is present in the tooth-buds of the premanent teeth, and the question as to how long the enamel remains on the milk teeth are matters for further study. The fact that the enamel organ is well developed in the eighth tooth (Plate II, Fig. 6), which has no predecessor in the milk dentition, would seem to indicate that this permanent tooth would have enamel. I attempted to demonstrate this by making dry sections of back teeth taken from several adult armadillos; but as
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 +
 +
A. M. Spurgin 83
 +
 +
I was unable to obtain a young animal, all the teeth at my disposal were more or less worn, and if there had been enamel on the teeth at eruption it had been worn oft'.
 +
 +
Although no enamel is present on the adult teeth of any of the living Edentates, the fossil forms Progmegatherium and Promylodon, from the infra-Pampean beds of Argentina, have been distinguished by Ameghino from the Megatherium and Mylodon as possessing bands of enamel. Burmeister, 91, however, who has also worked on the fossils of this region, disputes Ameghino's statement. Flower (91, p. 204), states that some Glyptodonts occurring in South American beds of an earlier age than the Pleistocene have enamel bands on the teeth. I consider this fact of great weight in showing a possible connection between the Glyptodonts and the living armadillos through the fossil Chlamydotherium, whose teeth resemble those of the Glyptodonts, but have no enamel.
 +
 +
University of Texas, Austin, Texas, May 15, 1903.
 +
 +
BIBLIOGRAPHY.
 +
 +
Ameghino, F., 92. — Repliques aux Critiques du Dr. Burmeister sur quelques Geners de Mammiferes Fossiles de la Republique Argentine. Bol. Ac. Arg., XII, pp. 437-469.
 +
 +
Ballowitz, E., 92. — Das Sclimelzorgan der Edentaten, seine Ausbildung im Embryo und die Persistenz seines Keimranaes bei dem erwachsenen Thier. Hert^vig's Archiv, Bd. XL, p. ]33.
 +
 +
Brandts, 28. — Dissert, inaug. de Tartigradis, Lugduni Batav.
 +
 +
Brunn, von A. — Ueber die Ausdehnung des Schmelzorgans und seine Bedeutung fiir die Zalmbildung. Arcli. fiir mikrosk. Anatomie, Bd. XXIX.
 +
 +
Burmeister, H., 81. — Atlas de la description physique de la republique Argentine. Mammiferes, p. 101.
 +
 +
Burmeister, H., 91. — Continnacion de las adiciones al Examen de los Mammiferos Fosiles Terciarios. An. Mus. B. Aires, III, pp. 401-461, pis. VII-X.
 +
 +
Flower, 68, eg.—Proceed Zool. Soc. 1868, p. 378; 1869, p. 265.
 +
 +
Flower and Lydekker, 91. — An introduction to the Study of Mammals, Living and Extinct, pp. 173-211. London, A. & C. Black.
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Gervais, p., 55. — Histoire naturelle des mammiferes, p. 254.
 +
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Gervais, p., 73. — Journal de Zoologie, 1873, p. 435.
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Hejstsel, 72. — Beitrage zur Kenntniss der Saugetiere Siid-Brasiliens. AbhandL d. Kgl. Akad. d. Wissensch. in Berlin, 1872, pp. 103-107.
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 +
PoucHET, C. et Chabry, L., 84. — Contribution a I'odontologie des mammiferes. Journ. de I'anatomie de la physiol., 1884, T. XX, pp. 173-179.
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Reinhardt, 77. — Vidensk. Meddel. Naturhist. Foren. Kjoebnhavn, 1877.
 +
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Rose, C, 92.^. — Beitrage zur Zahnentwickelung der Edentaten. Anatom. Anzeiger, Bd. VII, p. 495.
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Rose, C, 92^. — Ueber rudimentare Zahnanlagen der Gattung Manis. Anatom. Anzeiger, Bd. VII, 618.
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84 Enamel in the Teeth of an Embryo Edentate
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Rose, C, 93. — Ueber die Zahnentwickelnng der Kreuzotter (Vipera berus
 +
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L.). Anat. Anzeiger, Bd. IX, p. 448. SuDDUTH, W. X., 86. — American System of Dentistry, j)p. 640-645. Lea Tiros.,
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Phila. Tomes, C. S., 74, — On the Existence of an Enamel Organ in the Armadillo.
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Quarterly Journ. of Microsc. So. 1874, p. 48. Thomas, O., 89. — A Milk Dentition in Orycteropus. Proceed. Eoy. Soc,
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Vol. XL VII, 1889-90, p. 246. Weber, Max., gi. — Beitrage zur Anatomie und Entwickelung des Genus
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Manis. Zoologische Ergebnisse einer Reise in Niederlandisch Ostindien, Bd. II, 1891.
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EXPLANATION OF PLATES I AND II. EEFEEENCE LETTERS. c, cartilage. iep., inner laj^er of enamel organ.
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ct., connective tissue. mm., mucous membrane.
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ce., columnar cells over thinner oep., outer laj^er of enamel organ.
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portion of enamel layer. 0., odontoblast cells.
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d., dentine. sr., stellate reticulum.
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dp., dental papilla. si., stratum intermedium.
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e., enamel. sb., secondary bud.
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CO., enamel organ. stb., portion of second tooth-bud.
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er., epithelial ring. nd., uncalcified dentine.
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eb., epithelial band. *., space left by shrinkage
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fep., flattened cells of enamel of specimens.
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organ. All figures (except Plate I) were made with the aid of the Camera Lucida and in the process of reproduction were reduced about one-third.
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PLATE I.
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Photograph of embryos Dasypus novemoincttis L., showing placentation. Reduced about one-third.
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PLATE II.
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Fig. 1. Longitudinal section, lower jaw 8.5 cm. embryo, showing rudimentary incisors. Leitz obj. 3, Oc. 1.
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Fig. 2. Rudimentary incisor of Fig. 1 enlarged, showing enamel separated from the dentine and fractured in the direction of the enamel rods. Leitz obj. 7, Oc. 1.
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Fig. 3. Longitudinal section, lower jaw 9 cm. embryo, showing enamel in the first back tooth. Leitz obj. 3, Oc. 1.
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Fig. 4. Enamel and dentine of Fig. 3 enlarged, showing uncalcified dentine and odontoblast cells. Leitz obj. 7, Oc. 1.
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Fig. 5. Longitudinal section, lower jaw 8.5 cm. embryo. A portion of the tooth-bud of the fifth back tooth, showing the secondary bud conaing from the outer epithelial layer of the enamel organ. Note Tomes' processes of the inner epithelial layer directed toward the dentine. Leitz obj. 3, Oc. 4, tube drawn out to 20 cm.
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Fig. 6. Longitudinal section, lower jaw 8.5 cm. embryo, showing the enamel organ of the eighth tooth-bud still connected by an epithelial band to the enamel organ of the seventh tooth-bud. Leitz obj. 3, Oc. 1.
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ENAMEL IN THE TEETH OF AN EDENTATE (ArmadiUo).
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A. M. SPURGIN
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PLATE
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AMERICAN JOURNAL OF ANATOMY—VOL. Ill
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ENAMEL IN THE TEETH OF AN EDENTATE (Da>^ypus noremcinctiis L.).
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A. M. SPURGIN U_
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AMERICAN JOURNAL OF ANATOMY—VOL. Ill
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PLATE II
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^f *.
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9
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ii
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i*?
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■■!»
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THE EMBEYONIC DEVELOPMENT OF THE OVAEY AND TESTIS OF THE MAMMALS.
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BY
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BENNET MILLS ALLEN.
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From the Hull Zoological Laboratory, University of Chicago. With 7 Plates and 5 Text Figures.
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This work was carried on with the aim of solving the following problems: (1) The origin and development of the seminiferous tubules and their homologues in the ovary; (2) the origin, development and homologies of the rete tubules together with their relations to the Malpighian corpuscles of the mesonephros on the one hand, and to the seminiferous tubules of the testis on the other; (3) the origin, development and homologies of the connective tissue elements and interstitial cells of the ovary and testis.
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Incidental to the solution of these problems, the work has involved to a greater, or less extent a consideration of the following allied problems: (1) The development of sex cells; (2) the morphological phases of sex differentiation; (3) cell degeneration in the sex gland and rete; (4) the degeneration of the mesonephros and the development of the Wolffian and Miillerian ducts.
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This work, covering as it does a very broad field, naturally touches upon many points that have already been treated by previous workers. Although much has been written upon this subject there is a singular lack of unanimity in the results attained. This is largely due to the fact that only in a very few cases has the process of development of the sex gland been followed in an extensive series of stages. Su6h work has naturally resulted in giving rise to many false and contradictory views upon these subjects.
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The difficulties in the investigation of these problems are further enhanced by the fact that the sex glands are composed entirely of mesodermal tissue, in which a large part of the cells are without definite cell boundaries.
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2. Material and Technique.
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The material employed includes numerous stages in the development of the ovary and testis of the rabbit, from the 13-day embryo to and including adult stages. The pig material includes only embryonic stages, but is more complete for the period covered than is the rabbit material.
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American Journal of Anatomy. — Vol. III. 8
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90
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Embryonic Development of Ovary and Testis of Mammals
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The two forms studied — rabbit and pig — are complementary in their points of special suitability for this work. Although there are minor differences in the development of the sex glands in these two forms, yet the general process is essentially the same. In general the pig is the more instructive form for a study of the early stages of embryonic development, while the rabbit furnishes material better suited for the study of the post-embryonic stages.
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The following tables indicate the stages studied, the sex of the specimen and the number of series cut in each case. The stage of development is indicated in the rabbit by the number of days and in the pig by the length of the embryo. Rabbit.
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f 13 D.
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14KD.
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16 D.
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17 D. ! 18 D.
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19 D. 21 D. 23 D. 25 D. {2& D.
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At Birth.
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Indifferent. 1
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Female. Male.
 +
 +
3 2 2 2
 +
 +
4 2
 +
 +
 +
 +
f 3D.
 +
 +
8 D.
 +
 +
10 D.
 +
 +
13 D. .. 1
 +
 +
17 D. .. 1
 +
 +
24 D.
 +
 +
25 D. .. 1 31 D. .. 1
 +
 +
m -{ 37 D. .. 1
 +
 +
45 D . . . 1
 +
 +
50 D . . . 1
 +
 +
78 D. .. 1
 +
 +
85 D. .. 2
 +
 +
93 D. .. 1
 +
 +
100 D. .. 1
 +
 +
130 D. .. 1 1
 +
 +
[ 140 D . . . . . 1
 +
 +
One each of the following stages of adult rabbit ovaries ; 6-months-old virgin. Old individual, 3 months since last
 +
 +
pregnancy. 3K da.ys pregnant.
 +
 +
s^2
 +
 +
 +
 +
7 13
 +
 +
14K
 +
 +
16
 +
 +
17
 +
 +
 +
 +
During lactation.
 +
 +
 +
 +
1st pregnancy.
 +
 +
 +
 +
Stag-es. 0.6 ■ cm. 0.7 cm. 0.8 cm. 0.9 cm. 1 cm. 1. 1 cm. 1.25 cm. 1.33 cm.
 +
 +
1.4 cm.
 +
 +
1.5 cm.
 +
 +
 +
 +
Pig Embryos
 +
 +
Indifferent.
 +
 +
1
 +
 +
1
 +
 +
1
 +
 +
1
 +
 +
1
 +
 +
1
 +
 +
1
 +
 +
1
 +
 +
4
 +
 +
 +
 +
Female. Male.
 +
 +
 +
 +
1.6
 +
 +
1.7
 +
 +
1.8
 +
 +
2.5
 +
 +
3
 +
 +
3.5
 +
 +
4
 +
 +
5
 +
 +
5.7
 +
 +
7.5
 +
 +
8
 +
 +
8.5
 +
 +
10
 +
 +
13
 +
 +
13.5
 +
 +
15
 +
 +
18
 +
 +
20
 +
 +
 +
 +
cm. cm. cm. cm. cm. cm. cm. cm. cm. cm. cm. cm. cm. cm. cm. cm. cm. cm . cm.
 +
 +
 +
 +
Note The pig embryos were measured from the cervical to the tail bend in all stages up to 5 cm. length, when the measurement was taken from the base of the tail to the top of the head. This change was made for practical reasons of precision. The 5 cm. stage would be about equivalent to the 4 cm. stage.
 +
 +
 +
 +
Bennet Mills Allen
 +
 +
 +
 +
91
 +
 +
 +
 +
The material Avas, in practically all cases, fixed in Flemming's fluid and stained with Heidenhain's iron hematoxylin with a counter stain of Siiurefuchsin, Orange G. or Bordeaux red. Sections were cut to a thickness of 6f /j or in a few cases 10 /u and were mounted in series. It was, of course, necessary in all cases to section the anterior part of the mesonephros together with the sex gland itself.
 +
 +
An account of the earlier literature upon this subject would be useless repetition, since we have such valuable and extensive reviews as those given by Waldeyer, 70 and 02, Born, 94, Coert, 98, Winiwarter, 00, Bouin, 00, Mihalkovics, 85. In the general summary reference will be made to some of the more recent and important works bearing upon these problems; but no attempt will be made to attain completeness in a consideration of the earlier literature, a very large part of which is of merely historical value.
 +
 +
 +
 +
II. GENERAL TOPOGRAPHY.
 +
 +
The orientation of the various organs to be considered may be well understood from, a study of the pig embryo of 2.5 cm. length (Text Fig. 1 and Plate I, Fig. 1). The mesonephra are a pair of elongated laterally compressed bodies attached to the dorsal body wall on each side of the mesenter}'. Their long axes diverge anteriorly and converge posteriorly. They are prominent structures, extending three-fourths of the length of the abdominal cavity, being closely united to the dorso-median part of its wall by their short, broad mesenteries. Each mesonephros is flattened on its median face while the lateral face is convex. A sharp ridge extending the entire length of the medio-ventral face marks the course of the Wolffian and Miillerian ducts, the latter being ventral to the former. The genital ridge is situated on the median surface of the mesonephros and extends its entire length immediately ventral to the mesentery. It is covered by a thickened layer of epithelium continuous with the general peritoneal lining of the abdominal cavity, yet differing from it in that its component cells are columnar instead of fiattened, and are closely crowded together,
 +
 +
 +
 +
 +
Fig. ]. Mesonephros and associated structures. Pig embryo of 3.5 cm. length. a. r., adrenal body ; e. p., epithelial plate ; m. r., mesenteric ridge ; r. c, rete ridge ; t., testis ; W. M., WoliBan and Mullerlan ducts.
 +
 +
 +
 +
92 Embryonic Development of Ovary and Testis of Mammals
 +
 +
Three distinct regions may be distinguished in this genital ridge, each of which occupies, roughly speaking, one-third of its length. Named in their order, they are: (1) the rete; (3) the sex gland; (;^) the mesenteric ridge.
 +
 +
The anterior end of the rete is a low plate of thickened epithelium in which lies the opening of the Miillerian duct. Posterior to this plate, the rete assumes the form of a slender, low ridge thot terminates at the anterior end of the sex gland.
 +
 +
In both male and female of this stage, the sex gland is cyliAdrical, and rounded at both ends. It projects well into the body cavity, being united to the mesonephros along its entire length by a relatively narrow mesentery.
 +
 +
For the posterior third of the genital ridge I suggest the term mesenteric ridge. This diminishes in height from its anterior to its posterior end, which grades off into the general peritoneal covering of the mesonephros.
 +
 +
A transverse section of the epithelial plate in which the ]\riillerian duct takes its origin, shows it to be similar to that investing the remainder of the genital ridge. At the dorsal edge of the plate are seen more or less solid invaginations, the rete cords, while the opening of the Miillerian duct is situated in the ventral part. It appears as a hollow invagination clothed with cells much like those of the epithelial plate, from which they are undoubtedly derived, as can be easily seen from a study of earlier stages.
 +
 +
The rete consists of a series of cords embedded in a loose stroma. Their proximal ends are directly continuous with the peritoneum while their distal extremities lie deep in the stroma, in some cases reaching to the Malpighian corpuscles with which they are frequently in direct contact.
 +
 +
The rete cords penetrate into the sex gland a short distance behind its anterior end. This point, termed the hilum, is morphologically the anterior end of the sex gland, although it appears to be situated more posteriorly in the testis, owing to a secondary flexui'e of that organ. The ovary, on the other hand, retains the primitive condition in this regard.
 +
 +
At this stage the ovary and testis can be readily distinguished, although they contain essentially the same structures, viz: Sex cords, albuginea and germinal epithelium. (1) The sex cords of the testis develop into the seminiferous tubules which, at this stage, appear as long contorted anastomosing and branching cords of cells. Their homologues in the ovarv are termed the medullarv cords. These have all
 +
 +
 +
 +
Bennet Mills Allen 93
 +
 +
the essential characters of the seminiferous tubules save for the fact that they are by no means so well-developed nor so extensive as those structures. (2) A zone of connective tissue separates the sex cords from the peripheral peritoneal investment of the sex glands. It is compact in the testis, while in the ovary it is loose, broad and irregular in outline, forming only an incomplete barrier between the peritoneum and medullary cords. In both ovary and testis this peripheral connective tissue zone is continuous with masses of loose connective tissue (stroma) packed in between the sex cords. I shall refer to it as the albuginea in both testis and ovary, although that term is usually applied to it in the testis alone. (3) The peritoneal layer is thin in the testis and its component cells are flattened. Quite a different condition prevails in the ovary where it is decidedly thickened and is seen to be giving off cords of cells from its inner edge. These are the so-called egg-tubes of Pfliiger. They are in some instances continuous with the medullary cords, although such cases are rather rare, the two sets of structures being usually distinctly separated by the albuginea.
 +
 +
The posterior third of the genital ridge (mesenteric ridge) need not be considered further save to note that it hecomes more elevated in later stages and takes on a more decided mesenteric character.
 +
 +
In the mesonephros there soon appear processes of degeneration that "bring about decided changes. Even in the embryo 3.5 cm. in length there is seen a commencement of degeneration in certain tubules in its anterior portion. This process continues during succeeding stages, chiefly affecting the Malpighian corpuscles, but sparing from 10 to 12 of the tubules destined to form the rete efferentia of the testis, but which later degenerate in the female. To such an extent has this degeneration process been carried on in the 10 cm. embryo that the portion of the meso^iephros lying anterior to the hiluni is shrunken and the investing peritoneum thrown into wrinkles. Degeneration of the portion posterior to the hilum has just begun at this stage. In the female, the shrinkage of the anterior part of the mesonephros has caused the anterior ends of the Miillerian and Wolfiian ducts to be bent over the ovary in a dorsal direction to such a degree that sections through this region show these ducts to be cut through twice (Fig. 3). After this, the degeneration process rapidly reduces the mesonephros, until, in the 20 cm. embryo, it consists of little more than a mere mass of connective tissue containmg a few scattered glomeruli and uriniferous tubules, the vasa efferentia in the testis alone being spared.
 +
 +
 +
 +
94 Embryonic Development of Ovary and Testis of Mammalt
 +
 +
 +
 +
III. OBSERVATIONS UPON SUCCESSIVE STAGES OF DEVELOPlSrENT
 +
 +
IN THE PIG.
 +
 +
0.7 cm. Embryo. — The mesonepliros is covered by a more or less distinct peritoneal layer, which is not clearly differentiated from the stroma, except in the dorsal and lateral portions, but becomes increasingly distinct on the medio-ventral surface, where the genital ridge later takes its origin. The transition is, however, a very gradual one and the differences slight. There is a rather loose vascular mesenchyme tissue that fills in the space between the peritoneum on the one hand and the Malpighian corpuscles and mesonephric tubules on the other.
 +
 +
The cells of both the peritoneum and underlying mesenchyme do not have definite boundaries, appearing in this, and in later stages as well, to form a continuous protoplasmic network, to which the nuclei give character by their more or less definite arrangement. A region of the peritoneum extending from the base of the mesentery one-third the distance to the Wolffian duct is of particular importance, since it is the rudiment from which the genital ridge takes its origin (Text Eig. 2 and Plate I, Eig. 3). A point about opposite the twentieth glomerulus marks the boundary between the future sex gland and the rete. In the region of the genital ridge (Plate I, Eig. 3), as defined above, the greater part of the nuclei are of various shapes and sizes and stain rather deeply with ha3matoxylin. The nuclei of the peritoneum are closely packed together and are usually elongated by mutual pressure. They rest upon a loose felt-like basement membrane, which is formed by the interlacing of numerous slender branching protoplasmic fibrils given off by both the peritoneal and stroma cells. The peritoneal origin of the stroma is clearly indicated at many points where mutual pressure of the peritoneal cells is crowding them through the basement membrane, which has, in fact, disappeared at such spots as a result of this process. The positions and angles of inclination of the columnar nuclei give satisfactory evidence on this point. The presence of numerous mitotic figures in the peritoneum indicates a
 +
 +
 +
 +
 +
Fig. 2. Transverse section of mesonephros and associated structures. Pig- embryo 1.4 cm. Jengtti. d. a., dorsal aorta: </., glomerulus; %. m., mesentery of the intestine ; Ji., liver; m. /., mesentei-ic fundament ; 8. {/., sex gland; u. t., uriniferous tubule; W. a.. Wolffian duct, x 26.
 +
 +
 +
 +
Bennet Mills Allen 95
 +
 +
rapid multiplication of its cells. On the other hand the stroma cells divide with far less frequency.
 +
 +
As might be expected from the above, the stroma cells are practically identical with the peritoneal cells from which they are originating. In general their nuclei tend to assume a more rounded shape.
 +
 +
Here and there in both peritoneum and stroma one finds cells quite different from those described above. These have clearly marked boundaries, lightly staining cytoplasm, a centrosphere and a centrosome. The large, round nucleus contains prominent nucleoli, usually two in number, and also a chromatin network of slender strands quite different in appearance from the rather granular irregular chromatin masses of the peritoneal and stroma nuclei. These primitive ova are so rare that one must hunt through as many as seven or eight sections in order to find one. They divide by mitosis, as a result of which division they are found to occur in small groups.
 +
 +
The inner boundary of the mesenchymal portion of the sex gland rudiment is formed by the capsules of the Malpighian corpuscles. The component cells of the capsules resemble those of the stroma in their lack of definite boundaries and in the character of their nuclei, being distinguished from the latter chiefly by the darker color of their cytoplasm.
 +
 +
The rudiment of the rete (Plate I, Fig. 2) is essentially like the sex gland rudiment save for the fact that the basement membrane of the peritoneum is somewhat less distinct in the former than in the latter and the primitive ova are not quite so numerous; these differences are probably due to the fact that the tissue of the sex gland rudiment is more dense than that of the rete rudiment.
 +
 +
0.8 cm. Embryo. — In this stage, the mesonephros is found to have almost doubled in size; for this reason there has been little thickening of the rete and sex gland rudiments. The number of primitive ova has greatly increased and many clear cases of mitosis are found among them. The basement membrane of the peritoneal layer has become roore clearly defined in both rete and sex gland rudiments, yet it is still broken in spots where cells are being proliferated into the underlying stroma, sometimes forming chains of two, three or four cells.
 +
 +
1.0 cm. Embryo. — The mesonephros has become half again as broad in this stage as in the preceding one, and has also increased in the dorso-ventral dimension.
 +
 +
The rete rudiment has not grown in thickness, yet the peritoneal cells are seen to be rapidly dividing by mitosis. This results in a
 +
 +
 +
 +
96 Embryouic Development of Ovary and Testis of Mammals
 +
 +
crowding which in some places is so great as to bring about the formation of actual peritoneal invaginations which extend into the stroma and frequently come in contact with the attenuated capsules of Bowman.
 +
 +
Peritoneal invaginations arising in the sex gland rudiment (compare Plate II, Figs. 5 and 6) by the same process of crowding are more diffuse, and more numerous than in the rete. Another difference between these two regions of the genital ridge is found in the fact that the sex gland invaginations do not in any case reach as de^p as the capsules of Bowman, the stroma being thicker in this region than in the rete rudiment.
 +
 +
In many cases the stroma cells are assuming the character of connective tissue. Primitive sex cells are present in the peritoneum and in the peritoneal invaginations and stroma of both rete and sex gland rudiments. Their number has increased in the sex gland rudiment, while they have shown little or no numerical increase in the rete region.
 +
 +
1.25 cm. Embryo. — Although the rete rudiment has increased but little in thickness, the peritoneal invaginations of the rete region, which may now be termed rete tubules, are much further developed than in the preceding stage. The sex gland rudiment, on the other hand, has increased greatly in thickness. Its peritoneal invaginations (sex cords) have also increased in length and in number. Their nature can be best understood by referring to Plate II, Fig. 5. In this and in later stages the nuclei are still attached to the basement membrane which is in fact formed, as we have seen, from protoplasmic processes connected with them. So closely are the sex cords placed that there are very few stroma cells between them. No clear cases of such are seen in this figure. Such, however, are present and form in part the rudiment of the intertubular stroma so prominent in later stages. There is no doubt tbat this stroma from time to time receives additions from cells which pass through the investing membrana propria of the sex cords.
 +
 +
The sex cords are tubular invaginations of the peritoneum and their membrana propria are accompanying infoldings of the basement membrane as seen in earlier stages (Plate I, Fig. 4 and Plate II, Fig. 5). The sex cord nuclei are connected with the membrana propria by fine fibrils which apparently hold them in position.
 +
 +
In its earliest stage, this is a true process of invagination, but in the later stages it is only apparent because of the fact that the sex cords grow at their points of attachment to the peritoneum (centrifu
 +
 +
 +
Bennet Mills Allen 97
 +
 +
gal growth). Without doubt the sex cords are homodynamous with • the rete tubules.
 +
 +
Llf. cm. Embryo. — The nuclei of the peritoneum covering the rete are more numerous than in the preceding stage, being even more closely crowded. This has resulted in a further increase in the number of rete cords. Primitive ova may or may not occur in any given rete cord (Plate II, Fig. 6), there being apparently no regularity in this matter.
 +
 +
Immediately ventral to the peritonemn of both rete and sex gland there is a thickened area of stroma to which addition is constantly being made by proliferation from the peritoneum. This thickening is of no especial importance in the rete, but in the sex gland it, together with a similar but less important area dorsal to the sex gland, furnishes the connective tissue that goes to form the mesentery. The peritoneal nuclei of these mesenteric rudiments (Plate II, Fig. 7) are cylindrical, with their long axes perpendicular to the long axis of the sex gland.
 +
 +
The rudiment of the sex gland shows very little advance over the preceding stage in point of structure. There has been a continued growth of the sex cords, "resulting in such a thickening of the sex gland rudiment as to cause it to appear hemispherical in cross-section (Text Fig. 2). As in the previous stage, the peripheral layer of cells is not marked off from the underlying sex cords attached to it because of the fact that it is still adding to the latter by rapid proliferation.
 +
 +
The capillary blood-vessels and stroma cells already noted are quite evident in the interspaces between the sex cords. Here and there the walls of these capillaries show spindle-shaped nuclei which are far more attenuated than are the stroma cells. The latter are most nmnerous at the distal (inner) ends of the sex cords where they form a loose layer separated from the capsules of Bowman by a layer of attenuated, deeply-staining connective tissue cells which have their origin in the mesenteric fundaments already described. Posteriorly the sex gland gradually shades off into the mesenteric ridge, the sex cords becoming fainter and fainter and the primitive sex cells decreasing in number. The last named are found to occur ahnost at the posterior extremity of the mesonephros, where the genital ridge exists only as a strip of tissue along which the peritoneum and underlying stroma are thicker and denser than ordinary. There is an equally gradual transition- from the sex gland to the rete. In following the sex gland into the rete region, the first sign of transition from the
 +
 +
 +
 +
98 Embryonic Development of Ovary and Testis of Mammals
 +
 +
former to the latter is noted in the nearer approach of the peritoneal invaginations to the capsules of Bowman. They become less crowded and the genital ridge decreases in height.
 +
 +
1.5 and 1.6 cm. Embryos. — The sex gland increases in volume to such an extent that in the 1.6 cm. stage it appears circular in cross-section. It is attached to the mesonephros by its mesentery which is now much narrower than the sex gland. The latter appears to have been constricted off from the surface of the mesonephros by lateral furrows. This, however, is not the case, because measurements show the mesentery to be as broad as the base of the sex gland of the 1.4 cm. stage. The constriction is apparent, not real. In reality the centrifugal growth of the sex cords has caused the sex gland to expand on all sides until it is now cylindrical in shape, instead of appearing as a slight elevation above the surface of the mesonephros as in preceding stages.
 +
 +
The sex cords are becoming longer and more contorted. Together with the growth in extent they become more clearly defined. The increase in the surface of the peritoneum caused by the expansion of the organ has not been accompanied by the formation of new cords, hence space is left in which many sex cords become arranged parallel to and immediately beneath it.
 +
 +
There appears the beginning of a most important process by which the sex cords become separated from the peritoneum through the development of the albuginea (Plate III, Pig. 8). The nuclei of the cords at their points of attachment to the peripheral cell layer (peritoneum) begin to elongate and in many cases to assume the appearance of connective tissue nuclei, while their cytoplasm is drawn out into slender strands that stretch across the necks of the sex cords. At the same time, a basement membrane is forming beneath the peritoneum dividing it from the elongated cells just described.
 +
 +
The mesentery is composed of cells derived from the dorsal and ventral mesentery fundaments, their rather irregular arrangement being disturbed by the ingrowth of a number of blood-vessels.
 +
 +
At this stage appears the peritoneal invagination that forms the Miillerian duct. It arises in the ventral part of a plate of thickened epithelium which forms the anterior end of the genital ridge. Exactly similar invaginations forming the most anterior rete tubules arise in the same epithelial plate immediately dorsal to this rudiment of the Miillerian duct. Prom the foregoing it seems fair to assume that the Miillerian duct is homodynamous with the rete tubules and sex cords.
 +
 +
1.7 cm. Emhryo. — In this stage the sex cords become completely
 +
 +
 +
 +
Beniiet Mills Allen 99
 +
 +
separated from the peritoneum (Plate III, Fig. 9). As we saw above, this was foreshadowed in the 1.6 cm. stage l)y the transformation of the basal nuclei of the sex cords into elongated connective tissue elements. They remain attached to the membrana propria, which in almost all cases becomes ruptured and allows them to lie free between the peritoneum and the intact inner portions of the sex cords. They now form a connective tissue layer (albuginea) separating the sex cords from the peritoneum. Here and there one can see a sex cord that still remains attached to the peritoneum, and it is not at all difficult to find portions of the membrana propria to which a number of the connective tissue cells are still attached. The l^asement membrane shown in the preceding stage to be forming at the places where these sex cords are breaking away, has become completely formed except at a few points where the sex cords are still attached. The connective tissue nuclei formed in the manner above described are very similar to the mesenteric nuclei. This fact has led many to claim that the albuginea is composed of nuclei that immigrate from the mesenteric fundaments. We cannot hold this view in the face of the facts above noted. Furtlier substantiation of the view of development in sihi is furnished by the fact that nuclei exactly like those forming the albuginea are found in the peritoneum, being no doubt formed by the same process that produced the albuginea tissue.
 +
 +
The two sexes cannot be clearly distinguished from one another at this stage, the process above outlined taking place in both ovary and testis. The separation of medullary cords is, however, not quite so complete in the ovary as in the testis, yet this can hardly serve to sharply distinguish the sexes at the stage now under consideration.
 +
 +
The rete strands are quite well developed at this period, being long and somewhat contorted (see Plate lY, Fig. 11), They usually take a course more or less nearly parallel to the peritoneum to which they still remain attached at their points of origin. In general, they grow posteriorly, those found at the posterior end of the rete regions extending into the anterior part of the sex gland. Along their course they frequently touch the capsules of Bo^vman, some of them growing straight inward from the peritoneum in such a manner that their tips come directly in contact with the IMalpighian corpuscles, thus appearing to form, in some cases, a part of their epithelial walls. This closeness of union is frequently sufficient to deceive one into considering them to take their origin from the capsules of Bowman.
 +
 +
At the boundary between sex gland and rete there is a transition area in which the peritoneum becomes considerablv thickened. Pos
 +
 +
 +
100 Emb];youic DeveloiDineut of Ovary and Testis of Mammals
 +
 +
terior to this it is found to send numerous sliort projections into the underlying stroma, and further back, these assume the character of closely-crowded sex cords. At this place the rete tubules are few in number and arise exclusively along a line very close to the mesentery. They can be distinguished from^sex cords only by their isolation and by their greater length.
 +
 +
In general the rete tubules are made up largely of cells without definite boundaries and in all other regards like those of the peritoneum from which they originate. Only occasionally does one find a primitive sex cell.
 +
 +
1.8 cm. Embryo. — Sexual dift'erentiation is not yet clearly established, although, in a vague way, the general distinctions mentioned in connection with the 1.7 cm. stage are to be taken as criteria.
 +
 +
The sex cords stand out in greater contrast to the stroma owing to the fact that the cytoplasm of the component cells is much denser than in the preceding stages (Plate IV, Fig. 13). In some places these cords show a central lumen. This is not due to any regiilar process of lumen formation, but has significance only in showing that there is in each sex cord a line of weakness or rudimentary lumen which owes its existence to the fact that these cords originated by a process of invagination of the peritoneum.
 +
 +
Exclusive of the primitive sex cells, there are two extreme types of nuclei common to the sex cords, intercordal stroma and albuginea. Those of the one kind are small and elongated, taking a deep diffuse stain (Plate IV, Pig. 13). The nuclei of the other type are larger, clearer and more rounded. There are all intermediate forms between these two extremes. The larger nuclei predominate in the peritoneum, the smaller variety characterize the albuginea, while the two kinds appear in about equal number in the sex cords and in the intercordal stroma. The marked similarity between the nuclei of the sex cords and stroma is not surprising when one considers the fact of their common origin. In the sex cords we shall term these the gerrainative cells in contradistinction to the primitive sex cells. Transition forms are found to unite the two distinct types of cells, thus showing that certain of the germinative cells are being transformed into primitive sex cells. The medium-sized germinative cells are probably the most primitive; these form the sex cells on the one hand and on the other the connective tissue cells. It is interesting to note that the nuclei of many genninative cells are dividing by amitosis.
 +
 +
The germinal epithelium of the sex gland of the 1.8 cm. embryo does not contain any primitive sex cells clearly differentiated as such.
 +
 +
 +
 +
Bennet Mills Allen 101
 +
 +
It is significant to note that one finds here certain transition forms which link the usual type of peritoneal cells with the primitive sex cells found in the sex cords. In some cases these transition nuclei show much the same characters as regards chromatin and nucleolus as do the nuclei of the primitive sex cells, yet they differ in shape and size. It should be noted, in this connection, that the peritoneal layer is almost completely separated from the sex cords by the albuginea.
 +
 +
There are numbers of spherules of fat in the peritoneum covering the sex gland. These evidently indicate a process of fatty degeneration that seems to attack the cytoplasm of the cells and later to destroy a few of the nuclei, resulting in giving to the peritoneum a ragged appearance, there being large gaps where the cells have been destroyed.
 +
 +
2.5 cm. Embryo. — Sex differentiation is very strikingly shown in this most important stage. In the testis the albuginea has become thicker and denser than in the preceding stage. At the same time, the peritoneum has become flattened and is definitely separated from the albuginea by a distinct basal membrane. It contains no primitive sex cells. The peritoneal covering (germinal epithelium) of the ovary has become thickened and has even begun to send a few slender cords of cells into the loose underlying albuginea. These are the cords of Pflliger. They are, in many cases, loosely connected with the ovarian sex cords which we shall hereafter designate as medullary cords.
 +
 +
The process of fatty degeneration noted in the peritoneum of the preceding stage is still taking place in both ovary and testis, and has even extended to the sex cords in which large numbers of fat spherules appear. These occur almost exclusively in the syncytial cytoplasm of the germinative cells. They are most numerous in the portion of the sex cords furthest from the mesentery. Their fatty nature seems pretty evident from the fact that they are stained black by osmic acid and also from the spherical form that they assume.
 +
 +
The medullary cords are quite shrunken, being in most part clumped together in an irregular mass lying near to the mesentery. Cell degeneration occurring in them is not balanced by sufficiently rapid cell division. The primitive sex cells are surrounded by the undifferentiated cells that we have been terming germinative cells. This term, however, should henceforth be applied strictly to these cells in the seminiferous tubules alone. All resemblance to a tubular condition is lost, the medullary cords appearing in the form of masses of cells with no evidence of a very regular arrangement.
 +
 +
 +
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102 Embryonic Developmeut of Ovary and Testis of Mammals
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Numerous stroma cells are found to have become highly modified. The cytoplasmic portions increase in amount, becoming clearly marked off from surrounding cells, a centrosphere and centrosome appear, and the nucleus becomes rounded, while its chromatin network stains deeply. In general they assume a certain resemblance to primitive sex cells, yet the nucleus shows marked differences in its smaller size and more deeply-staining chromatin network. They differ also in the fact that their cytoplasm becomes granular and in later stages contains droplets of fat. These modified stroma elements are the interstitial cells. They are very numerous in the testis and very rare in the ovary. In both sex glands they divide by mitosis. A large portion of the stroma nuclei do not undergo this transformation into interstitial cells, but become elongated and take on the character of connective tissue. In all iDrobability these are the cells whose nuclei were smallest in the preceding stage where a difference in size and appearance of the stroma nuclei was noted. Particular stress should be laid upon the fact that the interstitial cells appear contemporaneyiusly with the process of fatty degeneration in the sex cords and that they show points of resemblance to the primitive sex cells. The latter point is particularly significant in view of the fact that in the 1.8 cm. embryo the nuclei of the stroma were to all appearances similar to those of the germinative cells of the sex cords which were in some cases developing into primitive sex cells. This would lead to some very attractive hypotheses; but one should be cautious about drawing hasty conclusions from such points of mere resemblance.
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3 cm. Embryo. — There are no essential differences between the rete of ovary and testis. The rete cords are still being formed, their points of connection with the peritoneum persisting along the entire length of the rete rudiment in both sexes. Another point common to both sexes is the degeneration of certain Malpighian corpuscles of the anterior part of the mesonephros. Those lying nearest to the rete cords are especially affected, suffering a decrease in size and a consolidation of the capillaries contained in them.
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Primitive sex cells are being formed in the rete cords from the syncytial cells that have retained the primitive character exhibited by the peritoneal cells, from which these cords arise. This development of undifferentiated peritoneal derivatives to form primitive sex cells is probably homologous with the process by which the germinative cells of the seminiferous tubules of the testis and the cells of the cords of Pflliger of the ovary are being transformed into primitive sex cells. The sudden impulse to renewed activity in the
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Bennet Mills Allen 103
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formation of these cells apparently affects both rete and sex gland at the same time. It is barely possible that the presence of fatty spherules so evident in the 2.5 cm. stage may be in some manner correlated with the active formation of primitive sex cells in the seminiferous tubules, cords of Pfliiger and rete cords, all of which structures have been shown to be homodynamous. Such a hypothesis would, however, require more evidence for its proof than we have yet found.
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The fat globules so numerous in the sex gland of the 2.5 cm. embryo have almost entirely disappeared from all save the interstitial cells of the testis, in the fat globules of these there has been an increase which may or may not be correlated with their disappearance in the seminiferous tubules. Whatever loss of cells may have taken place in the serniniferous tubules at the preceding stage has been compensated for by a process of rapid cell division. This, together with the transformation of germinative cells, has resulted in a decided increase in the number of primitive sex cells.
 +
 +
The peritoneum of the testis has become still further flattened, and its fatty spherules have almost wholly disappeared.
 +
 +
The albuginea nuclei have become more attenuated than in previous stages, yet they do not differ essentially from certain other connective tissue elements of the stroma, many of the more attenuated of which are seen to become applied to the membrana propria of the seminiferous tubules in such a manner as to form thin connective tissue sheaths.
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The interstitial cells are an extremely important constituent of the testis, occupying the interspaces between the seminiferous tubules (see Plate IV, Fig. 13). In the ovary, on the other hand, they are very sparse. In the place of them one finds great masses of loose connective tissue, filling the interspaces between the other ovarian tissues.
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 +
3.5 cm. Embryo. — This stage will be noted chiefiy to record the reduction in the cytoplasm of the interstitial cells of the ovar}^ jSTot only has the cytoplasm of these sparse cells become shrunken, but the centrosome and centrosphere have almost disappeared. Both primitive sex cells and follicle cells of the medullary cords are suffering extensive degeneration. This continued process of degeneration is even more marked in the cortex and cords of Pfliiger, which are now just beginning to assume importance.
 +
 +
4- cm. Embryo. — There is an interesting process of karyolytic degeneration that appears in the rete cords of this stage. The chromatin of the nuclei so affected gathers together in a rounded solid
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104 Eml)rvonic Development of Ovary and Testis of Mammals
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mass which is finally set free in the cytoplasm by the rupture of the nuclear wall. It now breaks up into irregular fragments which finally become more or less rounded and eventually disappear. Here and there one finds nuclei of the seminiferous tubules and cords of Pfliiger which degenerate in the same manner. This process takes place very extensively in later embryonic stages.
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In both sexes the rete cords along at least three-fourths of the length of the rete region have become separated from the peritoneum by a layer of stroma. It was impossible to determine whether this process is analogous to the separation of the sex cords from the peritoneum covering the sex gland.
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Attention has already been called to the degeneration of certain Malpighian corpuscles of the anterior end of the mesonephros in the 3 cm. stage. In the particular specimen' now under consideration (4 cm. embiyo) this process has continued, affecting eight of the most anterior corpuscles. The remaining ten or twelve corpuscles between the degenerate ones and the hilum of the testis are, as a whole, quite normal. Certain of the more peripheral of these intact Malpighian corpuscles send out short evaginations that come in contact with corresponding processes from the mass of rete cords and fuse with them (Plate V, Fig. 15). In this manner preparation is made for the establishment of a subsequent connection between the mesonephric tubules and the rete cords. Connection is also no doubt established without the aid of these evaginations in cases where the rete tubules press tightly against the capsules of Bowman. The number of the above described evaginations arising from each Malpighian corpuscle varies decidedly. In many cases there are none at all; in others there are as many as three.
 +
 +
Smaller evaginations from the capsules of Bowman were found in a 3 cm. embryo.
 +
 +
5.7 cm. Embryo. Ovary. — One is struck by the very close resemblance between the medullary cords and the cords of Pfliiger. They are practically identical, position alone serving to distinguish them. The medullary cords (Plate III, Fig, 10) lie in the central axis of the sex gland, separated by a zone of connective tissue from the cord^ of Pfliiger which project inwards from the peritoneum. Both elements are in large part composed of primitive sex cells — in fact there are but few small, deeply-staining nuclei which may be identified as those of rudimentary granulosa cells. The latter have no well-defined limits, being in every regard similar to the cells of the peritoneal layer from which the cords of Pfliiger arise.
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Bennet Mills Allen 105
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Fatty degeneration has almost ceased in the medullary cords and cords of Pfliiger.
 +
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7.5 cm. Embryo. Ovary. — A few of the ^:x cells ct the cords of Pfliiger have undoubtedly developed into the condition of oocytes because of the fact that their chromatin threads have taken on the synapsis form described by Winiwarter, oo, in the rabbit. Corr'3Sponding synaptic stages are also found in the medullary cords, thus bringing out the close homology of the two structures.
 +
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The cords of Pfliiger have become elongated and have at the same time branched and anastomosed to form a network in a manner quite like that of the seminiferous tubules in the testis. The resemblance is still further heightened by the fact that the cords of Pfliiger are invested with a connective tissue layer formed by attenuated connective tissue cells of the stroma. The same is true of the medullarscords.
 +
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We might homologize these three structures by considering the seminiferous tubules and medullary cords as exactly homologous structures, while the cords of Pfliiger constitute a second series of invaginations in all respects homologous with the medullary cords save as regards the time of origin.
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Testis. — The structure of the testis is essentially the same as in earlier stages. There has been a progressive increase in the extent of the system of seminiferous tubules, which has been brought about by the continued growth and branching of those already laid down previous to their separation from the peritoneum (1.7 cm. embryo). The nuclei of the germinative cells are attached to the basement membrane by strands denser than the surrounding cytoplasm. This relation to tlie basement membrane is exactly similar to that of the peritoneal cells in the earliest stages (0.7 cm. embryo). The primitive sex cells are increasing in number by two processes, namely: (1) division by mitosis of those already present in earlier stages; (2) transformation of germinative cells into sex cells. All stages in this transformation process can be noted, any transverse section of the testis at this stage (Plate IV, Fig. 14) showing a complete series of transition forms. The same may be seen in embiyos earlier and later than this, namel}', from 3 cm. to 13 cm. in length. Primitive sex cells occur in the rete cords of both male and female, those of the male being apparently in the same stage of development as are those of the seminiferous tubules. This is not true in the female at this stage, owing to the fact that the primitive sex cells of the cords of Pfliiger and medullary cords have developed precociously, outstripping those of the rete ovarii. 9
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106 Embryonic Development of Ovary and Testis of Mammals
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8.5 to 10 cm. Emhrijo. — Up to the stage when the embryo is 8.5 cm. in length, the rete cords extend but a short distance straight in from the hiliim in the case of both ovary and testis, and are similar in both sexes, the primitive sex cells of the rete ovarii becoming larger than those of the rete testis.
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The embryo of 10 cm. length shows the rete testis to have rapidly developed an axial core of loose connective tissue that fills in the space central to the free tips of the radially directed seminiferous tubules. In the female, on the other hand, the rete ovarii extends no further into the ovary than in the preceding stages, remaining in contact with the anterior end of the irregular mass of medullary cords. The rete ovarii and rete testis now follow different courses of development.
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13 cm. Embryo. Male. — The cords of the rete testis have in most cases undergone a process of lumen formation. This is brought about by the drawing apart of the cells from the axis of the cords. As already shown, these rete cells are attached to the ensheathirrg membrana propria, hence the lumen is formed by a very simple process by which they are made to separate along the line of greatest weakness (axis of cord). These rete tubules branch and anastomose quite like the seminiferous tubules. Their homology to the latter is still more clearly shown by the great similarity in the component cells of the two structures.
 +
 +
The rete tubules contain a few primitive sex cells (Plate VI, Fig. 21) exactly like those found in the seminiferous tubules. These are nothing new, as we have seen them to occur in the rete tubules of the very earliest stages. Another point of similarity is found in the character of the epithelial cells of the rete tubules which are in every regard similar to the germinative cells of the seminiferous tubules. Transition forms between epithelial cells and primitive sex cells do not exist in the former, while they are quite plentiful in the latter.
 +
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The rete tiibules send out side branches (tubuli recti) that fuse with the inner ends of the seminiferous tubules. In this manner one rete tubule may come into direct connection with a large number of seminiferous tubules. In one section, a rete tubule was seen to send out four tubuli recti connecting with as many seminiferous tubules. The point of Junction of tubulus rectus and seminiferous tubule (Plate V, Fig. 18) is easily recognized by the difference in diameter of the two elements, by the difference in arrangement of their component cells and by the presence of a lumen in the rete tubules as contrasted with the absence of such in the seminiferous tubules. In cases where connection has not been completely established between these two struc
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Bennet Mills Alien
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107
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tures, the point of junction is marked by the persistence of the hasement membrane of tubulus rectus and seminiferous tubule. These membranes are soon absorbed and the two structures are in direct continuity with one another.
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Female. — The primitive sex cells are found in all parts of the rete ovarii, yet their distribution is in no sense uniform. The intraovarian portion contains great numbers of primitive sex cells, which show a close resemblance to those found in certain regions of the cords of Pfliiger. Associated with these sex cells of the rete are other and smaller cells which are practically identical with certain cells of the cords of Pfliiger destined to form the granulosa of the Graafian follicles.
 +
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As stated above, the primitive sex cells are not by any means confined to the intra-ovarian portion of the rete tissue, yet their number in the portion of the rete lying within the mesonephros is found to become less and less as the distance from the ovary increases. The same principle holds true in the male.
 +
 +
The medullary cords are greatly reduced (Text Fig. 3), consisting of clumps of cells containing sex cells in various stages of development, the most advanced being large oocytes with a well-formed layer of granulosa
 +
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cells. Such young follicles are rare j,,^, 3. Transverse section of ovary nnrl icnlafcirl and niesonei>hrJc structures of pigr em duu ifeUidLeu. ^j.yQ Length 13 cm. c, cortex ;/(. p.,
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The irinprmo^t pnrl^ of tbp porrls; of hollow cord of Ptiflger: ni., medulla; m. Xiie mueimosi enu^ 01 ine COras 01 ^^ medullary cord; M. d.. Mullerian
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Pfluger are being broken up to form 'ir.^'i', woith^n'duct.^T2^!'"^*^ follicles. These follicles are young,
 +
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each consisting of a large oocyte and a single layer of granulosa cells. The oocytes almost invariably contain numbers of fat globules situated in their cytoplasm and especially numerous about the centrosphere, where they appear to congregate, eventually combining to form a single large mass. This appears to be without doubt a process of degeneration, leaving clumps of granulosa cells which persist for some time after the oocytes have disappeared. Not only are these oldest sex cells being destroyed by fatty degeneration, but there is an independent process of karyolysis which destroys great numbers of younger sex cells. In addi
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108 Embryonic Development of Ovary and Testis of Mammals
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tion to these two processes is that by which the fine chromatin threads in the nuclei of the oocytes at the synapsis stage of their development frequently break down into a powder-like mass of very fine granules. This is no doubt another process of degeneration.
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On the side of the ovary facing the mesonephros, there are a number of invaginations of the peritoneum (Text Fig. 3). These often appear as hollow tubules that extend for some distance into the ovary. At points along their extent they are found to be solid, their cells being similar to those of the cords of Pfliiger. In fact they are to be interpreted as such. Transition regions are found in which the peritoneal lining of these hollow tubules is found to contain a greater and greater percentage of primitive sex cells (Plate VII, Fig. 25) up to the condition of the solid portions of the tubules where the lumen is entirely obliterated by the enlargement of the peritonealcells to form primitive sex cells.
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15 cm. Embryo. Female. — In this ^stage the above described hollow egg-tubes of Pfliiger, while still most common around the hilum, are also found in the region of the cortex furthest from the mesentery. They penetrate more deeply into the tissue of the ovary than in the preceding stage, some of them extending into its very center, where they could be readily mistaken for" rete tubules by persons who might have studied these structures in ether forms, such as the cat. There is no mistaking their identity, however, because they bear no resemblance to the true rete tubules and because they were readily followed through the series to their point of union with the peritoneum. These invaginations may arise either from deep grooves or from the smoother surfaee of the peritoneum. At this stage the medullary cords are still further reduced, no young follicles being found among them.
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The mass of rete tissue is now found to be constricted at its point of entrance into the ovary. Further development of the sex cells and of the intra-ovarian rete has caused the boundaries of the rete cords to become obscured.
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Male. — In the male, the glomeruli connected with the rete tissue have degenerated to such an extent that the rete tubules are now found to be in almost direct contact with the mesonephric tubules. A minute description of the seminiferous tubules of this stage will serve to unify the points touched upon in the preceding pages (see Plate 'Y , Fig. 18). They are still solid, yet their tubular nature is shown by the arrangement of the dense peripheral layer of nuclei belonarinff to the fferminative cells. Each nucleus is attached to the
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Beniiet Mills Allen 109
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nieinbrana propria by a cylindrical condensation of cytoplasm, frequently so short that the nucleus appears to rest directly upon the membrana propria. The axial portion of the tubule is occupied by a loose network of protoplasm. At no time do these germinative cells have definite boundaries.
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There are at this stage no transition forms between the germinative cells and primitive sex cells.
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IS cm. Embryo. Female. — This stage shoAvs some interesting points in the development of the intra-ovarian portion of the rete tissue. Lying in the mesentery at the hilum, it extends but a short distance into the ovary, not reaching the inner ends of the adjoining cords of Pfliiger. In this intra-ovarian portion of the rete, the oogonia have in some cases developed so far as to be surrounded by well-defmed follicles (Plate VI, Fig. 20). These young follicles have but a single layer of granulosa cells and are exactly like the follicles formed in the inner portions of the cortex, at this stage. With these rete follicles are found sex cells in all stages of development, likewise resembling corresponding sex cells in the cords of Pfliiger. The resemblance is made more complete by the fact that the sex cells of the rete are undergoing the same process of degeneration as are corresponding sex cells in the cords of Pfliiger.
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The portion of the rete lying in the mesonephros has become distinctly separated from the intra-ovarian portion just described. Most noteworthy, however, is the fact that the few primitive sex cells found in it have not developed beyond the original condition which they exhibited in the early stages of development.
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Few of the open tubes of Pfliiger exist as such at this stage, most of them having become transformed into solid cords of cells such as characterize the cortex as a whole.
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20 cm. Emhryo. Female. — A careful study of the inner ends of the cords of Pfliiger shows that many of the oocytes of the young primitive follicle! have disappeared as a result of the process of degeneration already described. The granulosa cells are apparently not affected, but persist in solid elongated clumps, similar to the remains of the medullary cords found scattered through the axial portion of the ovary.
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All the primitive sex cells and oocytes of the rete tissue have disappeared, leaving only the granulosa cells and their homologues.
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25 cm. Emhryo. Female. — At this stage the surface of the ovary is found to have become wrinkled and irregular. The cords of Pfliiger form a thick, dense, cortical layer, within which is the medullary por
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110 Embryonic Development of Ovary and Testis of Mammals
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tion of the ovary, made up of loose connective tissue (stroma) -which extends between the cords of Pflliger in the form of strands and plates having a texture denser than that of the central mass. These strands are continuous with a sub-peritoneal layer of connective tissue that separates the cords of Pfliiger from the peritoneum, thus putting an end to their further growth at the expense of the latter. Here and there a slender ingi'owth from the peritoneum is still found to pierce the connective tissue layer, yet these are of slight importance. I am not prepared to say whether they assume greater importance in later stages.
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The cortex contains sex cells in all stages of development, from the very young oogonia of the peripheral region to the small follicles in its innermost edge. The remains of the medullary cords and of the intra-ovarian portion of the rete are still present in the medullary region and are fomid to be in practically the same condition as in the 20 cm. embryo. Not a sign of sex cells is to be seen in either the intra- or extra-ovarian portions of the rete.
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The rete tissue is much more extensive in this stage than in the 20 cm. stage. There it was again more extensive than in the preceding (18 cm.) stage). Although these observations would seem to point to its growth after the degeneration of the sex cells, one should not lay too much stress upon this point. These seemingly conclusive facts may be conditioned l^y the great variability universally seen to exist in vestigial structures. A study of the rete cells failed to reveal extensive nuclear division in the above stages.
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IV. DETAILS OF DEVELOPMENT IN THE EABBIT.
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13-Day Embryo. — There is at this stage no obser^^able difference between the structure of the sex gland and rete rudiments. This stage corresponds with the 0.8 cm. stage of the pig. One is struck with the vagueness of the basement membrane of the peritoneum both in this and in succeeding stages of the rabbit, yet it is as tridy present as in the pig embryos where it appears with remarkable distinctness. Many of the cells of both stroma and peritoneum are found to be quite -irregular in shape, in many cases even amoeboid. Frequently they appear to be dividing by amitosis. It is very difficult to decide whether this be merely apparent or real. This point deserves special study, as it is of prime importance. A few figures of mitosis appear here and there. Primitive sex cells are present, though rare, occurring either in the peritoneal layer or beneath it. They are to be distinguished from the surrounding peritoneal and stroma cells l)y the same
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Bennet Mills Allen 111
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criteria noted in the pig embryo. Both rete and sex regions are found to contain them.
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lJ^y2-Day Embryo. — At this stage the sex gland rudiment is easily distinguishable from the rete portion of the genital ridge. It is hemispherical in transverse section, having attained a marked increase in height over the preceding stage by multiplication of the cells of the peritoneum and of the stroma cells which are manifestly derived from it. Sex cords are well formed, as in the 1.4 cm. stage of the pig, being likewise continuous with the peritoneum from which they were formed by a process of invagination. Although the cords appear with a fair degree of clearness, the rabbit is by no means so favorable a subject for the determination of the manner of their formation, as is the pig.
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The rete portion of the genital ridge is quite low in comparison with the rudiment of the sex gland. Here one finds certain scattered diffuse cords projecting from the peritoneum into the. underlying stroma, each invested by a membrana propria continuous with the basement membrane of the peritoneum. There are a few primitive sex cells in these rete tubules, but the predominating type of cells comprises those with small, oval, deeply-staining nuclei without cell boundaries, such as compose the peritoneum. These cells are attached to the membrana propria or basement membrane, as the case may be, by slender strands of cytoplasm, showing the same relation in this regard as do the corresponding cells in the pig embryo.
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The nuclei of the stroma in both rete and sex gland rudiments are found to be irregular in shape, giving the appearance of undergoing division by amitosis.
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16-Day Embryo. — The rete tubules of the region anterior to the sex gland can be readily detected. They lie in a mass triangular in transverse section. This is limited by the mesentery of the mesonephros, the capsules of Bowman and the peritoneum. In places the rete tubules can be seen growing in from the peritoneum and branching in the stroma. Each has a rudiment of a lumen which opens into the body cavity on the one hand, and on the other extends for a short distance into the interior of the tubule. These rete tubules can be found along the entire length of the rete rudiment and back beneath the sex cords of the rudimentary sex gland. In this region — the anterior end of the sex gland — it is difficult to distinguish the rete tissues from the underlying layer of connective tissue cells that separates them from the Malpighian corpuscles. The rete nuclei differ from those of overlying sex cords in that they are slightly smaller, more irregular and more deeply-stained than the latter. These rete nuclei are not to be dis
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112 Embryonic Development of Ovary and Testis of Mammals
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tinguished from the stroma nuclei nor from those of the peritoneum, all of the above named being amoeboid in shape, and giving the appearance of dividing by amitosis.
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A few large, well-marked, primitive sex cells are found in the rete tubules, beneath the sex gland and in those lying well within the anterior portion of the mesonephros in front of the sex gland.
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Testis. — In the anterior end of the testis the sex cords are still attached to the peritoneum. They reach a considerable length, and are often seen to branch once or twice in their course. More posteriorly one finds cords in process of separation from the peritoneum. As in the 1.7 cm. pig embryo, the nuclei of the proximal ends of these separating sex cords are becoming elongated and are assuming the character of connective tissue elements. Finally they break away from the basement membrane to form the albuginea dividing the peritoneum from the sex cords. More posteriorly still, this process is found to have been completed.
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Ovary. — In the ovary, the sex cords have not begun to separate from the peritoneum, although the introductory stages of such a process are seen. The sex cords are not so definite in outline as are 'those of the testis, owing to the fact that the stroma tissue separating them is not so dense as in that organ. The cells of the ovary are in all regards quite like the corresponding ones in the testis, the same small, amoeboid nuclei being found in the sex cords, rete tubules, stroma and albuginea, in addition to the primitive sex cells of rete tubules and sex cords.
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The peritoneum of the ovary is much thicker than that of the testis, being three cells thick in many places. Primitive sex cells are found occasionally in the innermost portions, together with intermediate forms connecting them with the ordinary peritoneal cells. The inner edge of the peritoneum is more or less irregular in outline, showing a number of short, rounded protuberances — the rudiments of the cords of Pfliiger.
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17-Day Embryo. Testis. — The seminiferous tubules of the testis contain primitive sex cells and germinative cells, with many transitional forms between the two. Both kinds divide by mitosis. The stroma nuclei are now more regular in shape, and no longer give the appearance of dividing by amitosis. As in the pig embryo, an investing membrane of connective tissue is found around the seminiferous tubules. This tissue has undergone a decided 'increase.
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Cases of karyolytic degeneration are seen here and there among the cells of the seminiferous tubules and rete tubules, although it is by no means common.
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Bennet Mills Allen 113
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The rete tubules are separated from the peritoneum save only at the anterior end of the mesouephros. They lie in a direction parallel to the long axis of the sex gland, being in some places closely applied to the capsules of Bowman. Many Malpighian corpuscles have given ' out evaginations that have fused with the rete tubules in the manner described in the pig embryo. The latter are distinctly separated from one another by their clear-cut membrana propria. No primitive sex cells are, at this stage, found in the rete tubules anterior to the sex gland, but they occur here and there in those underlying the anterior part of the sex gland. ,
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Ovary. — The description of the rete testis applies to the rete ovarii, there being no essential differences between the two.
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All except a very few of the medullary cords have broken away from the peritoneum. These resemble the seminiferous tubules to a certain extent, yet they have a tendency to form spherical clumps of cells which remind one of follicles.
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21-Day Embryo. Testis. — The rete tissue extends beneath the testis for over half its length. It occupies the space between the somewhat excavated inner face of that organ and the mesentery. A transverse section of the testis would show the mass of seminiferous tubules to appear as a crescent between the horns of which lie the rete tubules. These anastomose, forming a mass of tissue in which the boundaries of the component cords are largely obscured. From this unified mass slender branches (tubuli recti) pass to the seminiferous tubules, with which they unite. The nuclei of the rete cells are strikingly like those of the germinative cells, this resemblance being heightened by the fact that neither kind possess cell boundaries. Here and there in the seminiferous tubules, nuclei are found to degenerate by karyolysis. Clear transition forms are found to connect the primitive sex cells with the germinative cells (Fig. 23). This stage shows the interstitial cells to be well developed. They are characterized by having well-defined limits, granular cytoplasm, centrosphere and centrosome, and a spherical nucleus somewhat smaller than that of the primitive sex cells.
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OvAEY. — The chief advance over the preceding stage is found in the extension of the cords of Pfliiger into the loose connective tissue of the albuginea. It will be remembered that these cords were mere rudiments in the 17-day stage ; now they are quite well-developed.
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The medullary cords are but indistinctly separated from one another by rather sparse stroma cells which resemble the follicular cells of the medullary cords.
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114 Embryonic Development of Ovary and Testis of Mammals
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23-Day Embryo. Testis. — In the testis of this stage there is no important advance over the preceding stage.
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Ovary. — The rete tubules have assumed the appearance of the medullary cords save for the fact that they contain no primitive sex cells — a fact which might have been noted in the 21-day embryo. There has been little essential change in their general character. The nuclei of the rete tubules and their homologues in the medullary cords are still very irregular in form, giving the appearance of being in process of division by amitosis. Undoubted amitosis occurs among similar nuclei in the cords of Pfliiger (Plate VI, Fig. 22). These are destined to become the follicular (granulosa) cells of the Graafian follicles. It is possible that some of them may develop into oogonia — this point should be studied further. The cords of Pfiliger are connected with the peritoneum by slender necks.
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26-Day Emhryo. Testis. — The rete tissue has extended the entire length of the testis. Scattered primitive sex cells are found here and there in the part lying within the testis, but are not present in the mesonephric portion.
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The interstitial cells are found to occasionally divide by mitosis.
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There is an extensive karyolitic degeneration of sex cells of the seminiferous tubules. Not only is the nucleus affected in the manner already described, but the cytoplasm undergoes modification a,s well, in that it assumes the property of staining more deeply in these degenerating cells than it does in the normal ones.
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Ovary. — The medullary cords are now clearly separated from one another by rather wide intervals filled with stroma tissue. The cords of Pfiiiger have increased in extent and have, to a large extent, fused with one another until their original limits are marked only by dense plates and strands of connective tissue. As in the pig, we shall hereafter refer to this zone of densely-packed cords of Pfliiger as the cortex, in contradistinction to the inner core of looser tissue made up of stroma and medullary cords.
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Rabbit at Birth. Testis. — The rete tubules become more distinctly limited from one another and have begun the process of lumen formation simultaneously in all parts of the rete tissue. In this process the cells pull apart from the central axis of the cord which is a line of weakness due to the manner in which these cords are formed. The lumen of any given rete tubule is not continuous at first, being formed disconnectedly along the course of the cord. Each tubule is provided with a connective tissue sheath. Tlie typical epithelial cells of these tubules form a syncytium in which the deeply-staining, columnar
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Bennet Mills Allen
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115
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nuclei are arranged side by side, usually in a single layer, although one, at times, finds a superposed layer. A few primitive sex cells occur in the portion of the rete tissue lying within the testis. A complete series of transitional forms are found to connect the primitive sex cells with the germinative cells in this as in the 17, 21, 23 and 26day stages.
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In both male and female the mesonephros proper has almost wholly disappeared, leaving a connective tissue network in whose meshes lie masses of fat. The caput epididymis (Text Fig. 4), made up of contorted tubules (rete efferentia) lined with epithelial cells, still remains. These are the persistent uriniferous tubules of the anterior part of the mesonephos, the great bulk of the tubules posterior to these having degenerated, together with a few of those which were formed in the most anterior end of the mesonephros. A few shrunken glomeruli still persist in connection with the rete efferentia.
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Rahhit 3 and 8 Days after Birth. Testis. — These stages are interesting chiefly because they show a marked diminution in the number of interstitial cells which, however, are still found to be in process of division by mitosis.
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Rahhit 10 Days after Birth. Ovary. — The rete tubules are in all cases devoid of a lumen. They are frequently joined to hollow tubules of larger diameter which extend into the general rete mass. These are the rete efferentia. Their identity is shown by the large size of the lumen, and by the fact that the nuclei are larger than those of the rete tubules. These rete efferentia have been brought to lie within the hilum by the extension of the ovary to partially enclose the shrunken mesonephros. They probably grow into the ovary of their own account as well. The differences between medullary cords and rete tubules disappeared as far back as the 23-day stage of embryonic life.
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The cortical layer is distinctly bounded from the medullary substance by a dense layer of connective tissue which follows a zig-zag
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■"Fir.. 4. .Sagittal section of the testis of the rabbit, 3 days after birth, al., albuginea ; m., mesonephric remains; ?•., rete ; r. e., rete efferentia ; s. tu., seminiferous tubule ; W. d.. Wolffian duct, X28.
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116 Embryonic Development of Ovary and Testis of Mammals
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course to accommodate itself to the large rounded projections comprising the cords of Pfliiger. These cords are still attached to the peritoneum by their narrow basal portions.
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The medullary and rete tubules are usually two cells wide and without any trace of a lumen. Their nuclei are oval and ra'ther uniform in size. Here and there one finds primitive sex cells singly or in groups of two to four. They are frequently in the same stage of synapsis as are the more advanced nuclei of the cortex.
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In the 26-day female embryo the Wolffian duct has almost disappeared by degeneration, together with the great bulk of the urinifer'ous tubules. In the female, 10 days after birth, the mesonephric structures are found to have completely degenerated save for a few vestiges of uriniferous tubules lying within the rete tissue and the mesentery, posterior to the hilum. Aside from these vestiges, the mesonephros consists of loose connective tissue enclosing great masses of fat — the remains of former Malpighian corpuscles and mesonephric tubules.
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13 Days after Birth. Ovary. — Follicles are forming in the cortex at this stage. They are, of course, very s^ple, each consisting of a large oocyte surrounded by a single layer of granulosa cells. Exactly similar follicles are also found in the medullary cords.
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Very many nuclei in all parts of the cortex and medullai-y cords are suffering karyolytic degeneration.
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17 Days after Birth. Ovary. — The process of follicle formaition has continued, resulting in the breaking up of the inner ends of the cords of Plliiger. No sex cells are found in the medullary cords from this stage on. The ova of certain of the innermost follicles have disappeared, leaving clumps of follicle cells such as are found in corresponding stages in the pig. Frequently one finds two or even more oocytes in the same follicle.
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24- Days after Birth. Testis. — This stage shows the testis to have pretty largely assumed the characters prominent in adult life. The caput epididymis is made up of the much-contorted tubules of the rete efferentia, the latter being traceable down to their points of connection with the rete testis. This, as already stated in the" description of previous stages, is made up of a mass of anastomosing rete tubules from which proceed the tubuli recti that connect Avith the seminiferous tubules. In the posterior two-thirds of the testis, the rete is wholly surrounded by the seminiferous tubules that have closed in around it. Here and there primitive sex cells (spermatogonia) can still be found in the rete testis.
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Bennet Mills Allen 117
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Up to the stage of 8 clays after birth, there were found numerous transition forms connecting the germinative cells with the primitive sex cells. This is by no meins true of the 2-i-day stage (Plate YI, Fig. 24), where there is a very sharp distinction between the two types of cells which are singularly uniform among themselves. This is true not only in the characters already enumerated, but also in the staining reaction as Avell. The germinative nuclei take the iron hematoxylin stain with avidity while the primitive sex cells (spermatogonia) are not affected b}^ it at all.
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25 Days after Birth. Ovary. — The cords of Pfiilger are now almost entirely broken up to form large numbers of small follicles surrounded by connective tissue that has permeated the entire mass of each cord of Pfiilger.
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31 Days after Birth. Ovary. — It was noted in an earlier stage (17day ovary) that certain ova of the innermost follicles degenerated leaving clumps of follicular cells. These clumps are quite evident in the 31-day stage, lying along the border between the medulla and cortex.
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Jf5 Days after Birth. Ovary. — Many of the follicles have increased in size until they have acquired as many as three layers of granulosa cells, among which appears an incipient follicular cavity. Already the stroma forms a capsular investment (theca) about each follicle, and this investment has begun to show a ditferentiation into a theca interna and a theca externa. The nuclei of the cells of the theca interna are roimded and the cell body has become fuller in contrast to the attenuated fibrous character of the cells of the theca externa, whose nuclei have remained elongated and in every regard like those of the general stroma tissue of which they are an integral part. Transition forms l^etween both varieties of theca cells can be readily found. It might be Avell to call attention to the fact that there is a thin layer of attenuated stroma cells between the theca interna and the membrana propria of the granulosa layer. It will be termed the follicular capsule.
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At this period many cells of the theca interna have developed into interstitial cells similar to those described in the testis of the pig embryo. Each has a rounded nucleus, clear cell outlines, centrosphere. centrosome and numerous fatty granules deposited in its cytoplasm. The formation of these interstitial cells is genetically connected with a process of follicle degeneration which continues from the time of the earliest formation of follicles on through adult life.
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60 Days after Birth. Ovary. — The medulla becomes still more reduced in this sta2:e bv the invasion and growth of follicles in its bor
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118 Embryonic Development of Ovary and Testis of Mammals
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der. The ground substance is a rather compact connective tissue in which are imbedded the slender transversely-placed medullary and rete tubules, which are quite inconspicuous and devoid of a lumen. Large open lymph spaces are formed in the stroma. Typical interstitial cells are not at all uncommon.
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75 Days after Birth. Ovary. — Certain of the innermost follicles have increased greatly in size, having in some cases almost reached maturity. They encroach upon the limits of the medullary region to such an extent as to make the latter band-like in cross-section. In Plate VII, Fig. 26, is represented a portion of the ovary showing the more important tissues composing it. The granulosa cells have welldefined boundaries, being polygonal in shape as the result of mutual pressure. The nuclei are rounded and are found to divide by mitosis. The follicle is bounded externally by a clearly-defined membrana propria, external to which one finds a very thin layer of attenuated connective tissue cells. This investment (follicular capsule) is similar to that which surrounds the seminiferous tubules.
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Outside of this occurs the theca interna, which is from one to four cells thick, the component cells being elongated in a direction parallel to the surface of the follicle. They are rich in cytoplasm. The large, rounded or oblong nuclei stain more lightly than do the nuclei of the granulosa ceils.
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The slender branching and anastomosing medullary and rete cords are distinguishable from the surrounding stroma by the clear cytoplasm and small oblong, deeply-staining nuclei of their cells which have apparently remained unchanged in character from the earliest stages onward.
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Certain young follicles of a stage just before the formation of the follicular cavity have begun to degenerate, the process first afllecting the oocyte which in some cases has disappeared wholly or in part. The mass of follicular cells becomes irregular in outline, but shows no signs of degeneration. It is quite likely that certain cords of cells with nuclei larger than the true nuclei of the medullary cords in the midst of which they lie, have originated from these degenerating follicles, the resemblance between their nuclei and those of the normal follicles being very striking.
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85 Days after Birth. Ovary. — The ovary as a whole has changed but little in form and size, this stage being most remarkable on account of the great increase in the number of interstitial cells. This increase is due to a very extensive process of follicle degeneration which seems to be at its height, affecting follicles in all stages of de
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Bennet Mills Allen 119
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velopnient. The granulosa cells are the first to show signs of degeneration, the nucleus drawing up into a small globular homogeneous mass in the center of the cell. The cytoplasm changes in such a manner as to become more deeply stained than formerly and the whole cell becomes rounded. This is no doubt the process of chromatolysis described by Flemming, 85. It certainly results in the eventual liquefaction of the cells affected.
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Large numbers of connective tissue elements from the capsule and theca interna penetrate into the follicular cavity. As the granulosa cells degenerate further, the follicular cavity becomes smaller and the capsule, theca interna and theca externa contract, thus encroaching upon the follicular cavity until the latter has become greatly reduced. The above mentioned elements that have migrated into the follicular cavity from the capsule and theca interna persist after the granulosa cells have all disappeared. The cells from the theca interna later undergo fatty degeneration and finally disappear, leaving the slender connective tissue cells that had migrated from the capsule; these persist and probably remain to form part of the general stroma tissue, lying between the columns of interstitial cells, whose method of formation will be described later.
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A series of fine connective tissue fibres join the follicular capsule with the theca externa passing rather obliquely between the cells of the theca interna. When the capsule closes in upon the follicular cavity these threads are drawn taut and arrange the cells of the theca interna in radial rows. The whole mass may become laterally compressed by the growth of neighboring follicles. In very advanced stages of atresia, when the follicular capsule has become reduced to a crumpled remnant lying in the midst of the cells of the theca interna, the latter lose their cell walls and become irregular in shape. In this condition they undergo a process of rapid amitotic division (Plate VII, Fig. 27), the resulting nuclei being much smaller than before this process took place. These will develop into the prominent interstitial cells as seen in later stages.
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6 months' Virgin. — In the ovary of this animal (Text Fig. 5) it is possible to trace out the further development of the interstitial cells. They cease to divide and undergo a process of growth in size both of the nucleus and of the cell body. At this stage^ — just after division has ceased — there occurs a deposition of a substance occurring in the form of small spherules, which stain deeply with haematoxylin. They were found only in this one specimen and are in no wise to be confounded with the very numerous fat granules which now begin to fill the cytoplasm of these interstitial cells.
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120 Embryonic Development of Ovary and Testis of Mammals
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These fat grannies are very characteristic of the interstitial cells. In this stage certain of these cells which have become isolated from the general mass are found to have enlarged greatly and to have become stuffed full of large fat spherules which are very readily dissolved by xylol. Each of these cells contains a large excentric nucleus with
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a centrosphere and centrosome (Plate VII, Fig. 28). Most of the interstitial cells are crowded together by mutual pressure and are hence prevented from attaining the full size of the one figured, which lay free in the connective tissue.
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8 Months Old; 1st Pregnancy; 1^ Days Pregnant. — The corpora lutea appear to be in the height of their development. The lutein cells composing them are rounded and suffer very little mutual pressure, being separated by fair intervals in many cases. Between them is a loose mass of fibrous connective tissue. The interstitial cells on the other hand, lie in dense masses between the corpora lutea. They are arranged in parallel strands in the manner already noted. Certain interstitial cells that become separated from the general mass are found to be rounded and of almost the same size as the lutein cells of the corpora lutea.
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One is struck by the great resemblance of the interstitial and lutein cells (Plate VII, Figs. 29 and 30), a resemblance that practically amounts to identity aside from the matter of size, which difference can, in large part, be attributed to the factor of external pressure. The description of the interstitial cells of the 6-months' virgin practically applies to the interstitial cells of this pregnant rabbit and to the lutein cells of the corpora lutea as well.
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Ovaries of Older Pregnant Rahhits. — The ovaries of a number of animals in various stages of pregnancy were examined. In the older of these animals the lutein and interstitial cells are apparently indistinguishable in the deeper-lying regions where the cells are the oldest. In the most central zones they are found to be undergoing a process of hyaline degeneration, the cell limits becoming indistinct, the cytoplasm ragged, and the nucleus very faint. Finally the innermost regions are found to contain the shrivelled remains of these cells.
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Fig. 5. Transverse section of ovary of a six-months old virgin rabbit. /'., follicle ; d. /., degenerate follicle ; y. c, germiriative epithelium; i. »!., masses of interstitial cells ; I. s., Ijmpli spaces ; m., mesonephros remains. X 24.
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Bennet Mills Allen .121
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Corpora lutea and masses of interstitial cells are successively forming at the periphery and disintegrating in the interior of the ovary.
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Atretic follicles were found in adult pregnant females of various ages.
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V. DISCUSSION OF RESULTS.
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1. IxDiFFEEENT Stage. — TliB genital ridge first appears as an area of thickened peritoneum and underlying mesenchyme (stroma), extending the entire length of the mesonephros, and situated on the ventromedian face of that organ. The tissues composing it are in no wise different from those forming the remainder of the investment of the mesonephros. In section, the peritoneum is found to be separated from the stroma by a more or less distinct basement membrane formed by the interlacing of protoplasmic fibrils proceeding from the nuclei of peritoneal and stroma cells (Plate I, Figs. 2 and 3).
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The cells composing the peritoneum and stroma tissues are almost wholly without evident boundaries. Only here and there does one find scattered cells with distinct cell boundaries, centrosphere, centrosome, large nucleus, and clear c^-toplasm — the so-called primitive sex cells. They occur in all parts of the genital ridge Init are most numerous in that region in which the sex gland will form.
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The distinction between peritoneum and stroma is not based upon any essential difference in the character of their component cells at this early period, but is based upon their arrangement, the nuclei of the peritoneum being arranged with their long axes parallel to one another and perpendicular to the basement membrane, while those of the stroma tissue lie with their long axes usually parallel to the basement membrane of the peritoneum which is very faint at certain points where active division of the peritoneal cells is taking place. This is due to the fact that peritoneal nuclei are being crowded through the membrane by mutual pressure, caused by their rapid multiplication.
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In the 10 cm. stage, a regional differentiation begins to appear in the genital ridge. This is marked by the formation of numerous crowded peritoneal invaginations (Plate I, Fig. 4) in the middle third; less numerous and deeper invaginations in the anterior third; and the almost total lack of them in the posterior third. The regions from front to rear, as thus marked off, are the rudiments of the rete, sex gland and mesenteric ridge, respectively.
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These invaginations are caused by a progressive multiplication of the peritoneal nuclei. Although the first formation of these cords is a true process of invagination, further gi'owth is centrifugal, the peritoneum 10
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123 Embryonic Development of Ovary and Testis of Mammals
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moving outward, and at the same time adding to the cords already laid down, by a continuation of the invagination process.
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These cords are truly of a tubular nature, a lumen, though not present, being conditioned by the arrangement of the cells. Transverse sections of these incipient tubules show a bounding meml)rana propria which is continuous with the basement membrane of the peritoneum. Inside of this is a single layer of peritoneal cells with their bases attached to the membrana propria, while their apices meet at a common central point — the rudiment of the future lumen.
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The invaginations are much fewer in the rete region than in the sex gland rudiment, and also differ from those of the latter region in the fact that they penetrate through the stroma to the walls of the Malpighian corpuscles (Plate II, Fig. 6), from which the rudimentary sex cords are separated by a layer of stroma. The limit between the rete and sex gland rudiments may be roughly placed at a point opposite the 12th glomerulus, in the rabbit, and opposite the 20th, in the pig ; however, the sex gland rudiment slightly overlaps the rete region; hence the impossibility of drawing a sharp limit between the two.
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There has been a great diversity of opinion in regard to the origin of the sex cords and rete tubules. Practically all writers except Egli, Janosik, Coert and von Moller have derived the rete tubules from the Malpighian corpuscles. The above named, considered them as products of the peritoneum covering the mesonephros. There has been greater unanimity in regard to the derivation of the sex cords. We shall not enter into detail upon this subject, but shall simply point out the fact that Waldeyer, 70 and 02, Kolliker, 98, Balfour, 78, Rouget, 79, and others hold that the sex cords arise from the Malpighian corpuscles, and that they receive primitive sex cells which migrate to them from the peritoneum.
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According to Mihalkovics, 85, the cells of the sex cords arise from the germinal epithelium, not through direct invagination but in an indirect manner, through infiltration of the stroma by peritoneal cells which later become segregated to form strands.
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Schulin, 81, and Coert, 98, hold a somewhat different view, namely that the entire sex gland is formed from a homogeneous mass of cells blastema) derived from the peritoneum. This view differs from that of Mihalkovics, 85, in that the latter does not consider the stroma to be derived from the peritoneum, while Coert considers such a peritoneal origin of the stroma to be very probable, and extends this idea to explain the formation of the rete tubules and the sex cords as well.
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The continued growth of the sex cords at their bases and the accom
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Bennet Mills Allen 123
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panying outward movement of the peritoneum results in a thickening of the genital ridge in the sex gland region. This becomes more and more pronounced until the sex gland appears hemispherical in transverse section, later appearing as a disc attached to the mesonephros by a relatively slender bridge — the mesentery. It gives the impression of having become constricted from the surface of the mesonephros. This appearance is, however, delusive, as the mesentery is in reality slightly broader than was the base of the rudimentary sex gland.
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The cells composing the indifferent sex gland (Plates III and IV, Figs. 9 and 11) may be classed under three heads: (1) Primitive sex cells; (2) syncytial cells with small nuclei; (3) syncytial cells with nuclei of various sizes.
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The primitive sex cells, already described, are found chiefly in the sex cords, although they occur sparingly in the connective tissue of the mesentery. They divide infrequently by mitosis throughout these early stages.
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The nuclei of cells of class (2) stain deeply and are often attenuated. They form the albuginea and occur in the stroma, sex cords and, to a limited extent, in the peritoneum.
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Cells of the third class occur together with those of the second class, which they resemble in that they are without definite boundaries. Their nuclei are larger and usually stain less deeply, showing all gradations between those of the primitive sex cells and the small, deeply-staining syncytial cells of class ( 2 ) . It is almost certain that both forms originate from these cells of intermediate character of which the peritoneum is almost exclusively formed.
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In the basal portions of the sex cords at the time when the latter are being separated from the peritoneum, there is a direct transition of the nuclei of class (3) into connective tissue nuclei of class (2), which forms the mesentery and the albuginea (Plate III, Fig. 9). Certain small nuclei of the sex cords resemble tliese connective tissue nuclei in a most striking manner and probably arise by a similar process of differentiation. These last named belong to the germinative cells of the seminiferous tubules and to the follicular cells of the medullary cords.
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The albuginea and mesentery of the sex glands are derived from the peritoneum of regions immediately dorsal and ventral to the sex gland (Plate II, Fig. 7). The cells of these mesentery rudiments proliferate rapidly throughout the early stages, causing a rapid growth of the mesentery. Here and there primitive sex cells are formed in these regions and are carried down into the mesentery with the connective tissue in the midst of which they lie.
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124 Embr3^onic Development of Ovary and Testis of Mammals
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As stated above^ the albuginea is formed by the transformation of the cells occupying the basal parts of the sex cords, into connective tissue elements, which are liberated l^y the rupture of the membrana propria encasing them (Plate III, Fig. 9). In this manner the sex cords become separated from the peritoneum and undergo further growth and differentiation independent of that layer.
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The formation of the mesentery and albuginea and the separation of the sex cords from the peritoneum are far more clearly shown in the pig than in the rabbit, yet I have been able to verify these processes throughout in the latter animal. Coert, 98, has come to essentially the same conclusions, but is cautious in expressing himself in regard to these points, as he well may be, because of the difficulty of following these processes in the rabbit, upon which form he worked.
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The separation of the sex cords from the peritoneum takes place at a slightly earlier period in the female than in the male. Coert, 98, has laid considerable stress upon this fact in the case of the rabbit. However it is not of primary importance in the pig. In the latter animal, it takes place in embryos of 1.6 to 1.7 cm. in length.
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As previously stated, the rete tubules are serially homologous with the sex cords, differing from them at this stage chiefly in the fact that they are less numerous, being isolated from one another by considerable intervals, filled in with connective tissue. The portion of the genital region occupied by the rete tubules becomes elevated to such a aegree as to be quite evident in gross dissections. Coert considers the rete tubules of the rabbit to arise from a mass of unorganized rete blastema by a process of differentiation which slowly progresses inward from the periphery. According to liim, this differentiation process is not completed until after birth. I found these tubules to be distinct and clearly limited in the rabbit embryo of 16 days. This difference between our results may have been due to a difference of technique. Coert used Kleinenberg's picro-sulphuric solution as a fixing agent, while my material was fixed in Flemming's fluid followed by Heidenhain's iron hsematoxylin stain.
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Primitive sex cells are present in the rete tubules from the first, but are uncommon, the great mass of cells being similar in character to those of the peritoneum from which they arose. This similarity applies not only to the absence of cell limits in the rete cells of the pig and rabbit, but in the former animal, to the size and staining reaction of the nuclei as w«ll. In the rabbit, these nuclei stain more deeply and are slightly smaller than are the nuclei of the peritoneum and of the germinatiA^e and follicular cells of the sex cords. As will be seen in a discussion of later stages these differences tend to disappear.
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Bennet Mills Allen 125
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The rete tubules extend in a posterior direction, their bases remaining attached to the peritoneum for a long time after the sex cords have become completely separated from it. These rete tubules penetrate quite deep into the stroma, reaching the walls of the Malpighian corpuscles, to which they are often so closely approximated as to give the appearance of arising from them. The glomeruli underlying this rete region comprise those from the 6th to the 20th, inclusive, in the pig, and from the 6th to the 12th in the rabbit.
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Since the rete remains indifferent in character long after the ovary and testis have become differentiated from one another, a common description will suffice to make clear its development in both male and female, up to a relatively late stage. Primitive sex cells begin to form anew from the syncytial cells of the rete tubules in the pig embryo of 3 cm. length. When the embryo has reached 4 cm. length, the rete tubules break away from the peritoneum along the posterior threequarters of the length of the rete region. I was unable to determine whether this process is similar to that by which the sex cords become separated from the peritoneum. In any case it takes place at a much later period as above shown.
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At this stage and a little earlier, evaginations arise from the capsules of Bowman of the Malpighian corpuscles at points close to the mass of rete tubules (Plate V, Fig. 15). Their number varies from one to three. In fact many Malpighian corpuscles give off no evaginations at all, although they arise in close proximity to the rete tubules. Similar evaginations occur in the rabbit embryo of 16 and 17 days, where they were first observed by Coert, 98. I am inclined to ascribe to these a morphological significance, yet they are of no particular functional importance, because a union of the rete tubules with the capsules of Bowman is, in very many cases, established by the former coming in direct contact with the latter.
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It is interesting to note that l^ranches from the rete tubules grow out to meet the tips of the evaginations from the Malpighian corpuscles. The cells from these two sources assume similar characters and are later indistinguishable from one another. Such later stages are very deceptive, having no doubt given rise to the incorrect view that the rete tulmles arise from the Malpighian corpuscles.
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The rete tubules 1)ianch and anastomose in their course, behaving much like the sex cords in this regard. The tubules of the anterior end of the rete mass remain in connection with the peritoneum throughout later stages while posterior to this point they are separated from it by a considerable interval and are united to form a cylindrical mass, the posterior end of which projects into the anterior end of the sex gland.
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126 Embryonic Development of Ovary and Testis of Mammals
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In the rabbit, the conditions are essentially similar yet by no means so clearly shown as in the pig. Difficulties in the study of these processes in the rabbit are caused by the compactness of the tissues, the smallness of the component cells, and the indistinctness of the limits of the rete tubules.
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2. Sexual Differentiation. — It now remains to follow the ovary and testis separately as they diverge in the process of further development. Both are homologous, in that they have originated from an indifferent rudiment in which a considerable complexity of structure has become evident before it is possible to distinguish sexual differentiation.
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Unmistakable differences between ovary and testis can be discerned in the 2.5 cm. embryo of the pig, less-marked differences being evident in the embryo of 1.8 cm. lengih. A clear distinction between ovary and testis is observable in the rabbit embryo of 14| days' age.
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Previous to the period of sex differentiation, the sex gland has taken definite form, having become constricted off from the mesonephros, to which it remains attached by the relatively narrow mesentery. A transverse section shows it to be composed of the following tissues: (1) the peritoneum, or germinal epithelium as it has been generally termed, especially in the case of the ovary; (2) the albuginea, a term usually applied to the subperitoneal connective tissue of the testis, but equally applicable to the same zone in the ovary; (3) the sex cords; (4) the interstitial stroma; (5) the distal ends of certain rete tubules that have grown from the rete region into the anterior end of the sex gland.
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The prime features of sex differentiation are shown in the 2.5 cm. pig embryo. The fundamental points are the further development of the sex cords in the testis to form the seminiferous tubules and the development of the peritoneum in the ovary to form the cords of Pfliiger. These two sets of cords having a similar origin, but one which is successive in point of time, are the structures in which the functional sex products form. On the other hand, the sex cords of the ovary cease in their growth and become the medullary cords — assuming the character of the cords of Pfliiger. In the testis the peritoneum ceases to develop and becomes flattened — finally almost disappearing in later stages.
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The albuginea layer is far thinner and more compact in the testis than in the ovary. This is due to the fact that in the former it is much more closely crowded against the peritoneum by the seminiferous tubules than by the medullary cords in the case of the ovary.
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The peritoneum, cords of Pfliiger and medullary cords of the ovary, together with the peritoneum and seminiferous tubules of the testis,
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Bennet Mills Allen 137
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contain numerous globules of fat resulting from a process of fatty degeneration in these structures. Loisel, oo and 02, found the spherules of fat to occur throughout the rudimentary sex gland of the 98-hour chick embryo, and in the 5-day embryo of the California quail, and in sparrow and guinea-pig embryos as well. According to him the primitive sex cells lose them when they become spermatogonia, while certain germinative and Sertollian cells contain fat globules up to the end of embryonic life, when they disappear, to later reappear at the time of puberty and at successive periods of sexual activity. There is thus a periodicity in their formation at least in the sparrow, upon which form the greater part of Loisel's work was done. He shows that the interstitial cells are filled with fat globules at the same time that the cells of the seminiferous tubules contain them, hence there is an interrelation between the two sets of cells. Interstitial cells are rare if not non-existent in the testes of adult birds, although they occur in great numbers in the testes of adult mammals.
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He interprets the fat spherules described above as secretion products and not as the products of degeneration. It seems to me unsafe to hazard an opinion upon the physiological aspects of this process, yet it certainly does result in the destruction, both immediate and remote, of a large number of cells. It would hardly seem that in the present state of our knowledge, Loisel is justified in his assertion that the bright plumage assumed by birds during the breeding season is due to any trophic stimulus imparted by this fatty substance. Coincident with this process of fatty degeneration in these structures, certain cells of the stroma suffer extensive modification, their cytoplasm becoming granular, acquiring a centrosome, centrosphere and definite cell limits. The nuclei of these cells also enlarge and become spherical. These interstitial cells are very numerous in the testis, but quite rare in the ovary. In Iwth sex glands, they divide by mitosis.
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Plato, 97, Coert, 98, and Limon, 02, are unanimous in agreeing that the interstitial cells arise from the stroma in both ovary and testis.
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3. Further Development of the Sex Glands. A. Testis. — The peritoneum becomes less and less important in later stages, finally forming a broken and almost vestigial covering of the sex gland. The albuginea becomes thicker and more compact, but need be given little further attention as its development is a very simple process.
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We have still to consider the development of the following elements :
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1. Seminiferous tubules.
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2. Eete tubules.
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3. Interstitial cells and stroma.
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128 Embryonic Development of Ovary and Testis of Mammals
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1. Seminiferous Tubules. — The seminiferous tubules of the 2.5 cm, pig embryo are rather distinctly limited by a membrana propria, still exterior to which is a thin layer of small connective tissue cells forming a capsular investment. The cells of the tubules are of two general classes — germinative cells and primitive sex cells, between which classes are found all intermediate forms. The germinative cells are identical with those classed above under groups 2 and 3, in fact the conditions are not altered in this stage. All classes of cells are dividing by mitosis while many of the germinative cells appear to be undergoing amitotic division.
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Conditions in every regard similar to those outlined above, hold good in the rabbit material, the corresponding period being about the 16th day of embryonic life.
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Transition forms connecting the germinative cells with the sex cells occur in the pig embryos between 2.5 cm. and 13 cm. length (see Plate IV, Fig. 14), later stages showing no transition forms. The rabbit material being more extensive, shows the condition of the cells of the seminiferous tubules up to the period of sexual maturity. Intermediate forms of cells are found to connect these two types in all stages from the 16-day embryo up to the stage 8 days after birth inclusive. Testes of the 40-day rabbit show absolutely no connecting links, the two classes of cells being there found in their purity. The germinative cells occur in a single layer with their bases attached to the membrana propria, while the primitive sex cells — spermatogonia — lie in the more axial portions of the seminiferous tubules.
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A striking feature of the seminiferous tubules is their tendency to branch and anastomose (Plate IV, Fig. 12), such tendencies manifesting themselves in the very earliest stages, when the sex cords are first laid down.
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As has been previously shown, the rete tubules are pushed into the anterior end of the sex gland. In the pig, their tips project into an axial space left between the inner tips of the radially arranged seminiferous tubules, while in the rabbit, the mesentery is broader than in the pig ; hence there is left a space at the base of the testis, for the occupancy of the rete tubules.
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2. Rete Tubules. — ^In the testis of the pig, the rete tubules remain within the hilum until a period between the 8.5 cm. and 10 cm. stages, during which time they grow rapidly down the axial space, almost reaching the distal end. It is at this period that the rete tubules begin to acquire a lumen and also to send out branches^ — tubuli recti — to the inner ends of the seminiferous tubules. As manv as four of these
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Bennet Mills Allen 129
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tiibiili recti were seen to arise from a single rete tubule, being apparently called forth wherever needed. A distinction between the tubuli recti and the seminiferous tubules can be readily drawn from several criteria. The chief difference lies in the greater diameter, lack of lumen, and far greater number of sex cells of the seminiferous tubules as contrasted with the fact that the rete tubules are narrower by half, possess a lumen, and contain very few primitive sex cells (Plate V, Fig. 18). The two structures resemble one another in the character of their component cells, the germinative cells of the seminiferous tubules being practically identical with the epithelial cells of the rete tubules, and the primitive sex cells of both structures showing an exact correspondence. This homology is also seen in the fact that both structures are limited by membrana propria and capsular connective tissue investments formed in the same manner in each.
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This process of the extension of the rete tubules takes place in the rabbit 3 days after birth. Later, the seminiferous tubules grow about the eccentrically placed mass of rete tubules in such a manner as to enclose it. Eepeated anastomosis of the rete tubules results in the union of their lumina to form a large cavernous, irregular space imperfectly divided by the walls of the component tubules. The nuclei of the rete cells still have the general characters of those belonging to the germinative cells of the seminiferous tubules; but are far more elongated by lateral compression. Primitive sex cells are found in the rete tubules of the rabbit 24 days after birth, but are not present in those of the 140day rabbit; hence it is safe to conclude that the sex cells of the rete testis are not functional.
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3. Interstitial Cells. — In the pig, the interstitial cells are found to multiply by mitosis from the time of their first appearance up to the stage of the 7.5 cm. embryo, and in the rabbit testis as late as 8 days after birth. There may be new interstitial cells formed between the period of their first appearance and sexual maturity, but this seems highly improbable, no evidences of such having been seen. They begin to degenerate in the 15 cm. pig embryo, and in the rabbit 24 days after birth. This process of degeneration first manifests itself by a shrinkage of the cytoplasm. In the process of development of these interstititial cells, their cytoplasm becomes filled with fat globules that have a tendency to run together (Plate IV, Fig. 13). At the same time, the centrosphere becomes clearer and more sharply defined from the surrounding cytoplasm. Plato, 97, does not represent the centrospheres in his figures of the interstitial cells of the cat, rabbit, steer, horse and other forms studied bv him.
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130 Embryonic Development of Ovary and Testis of Mammals
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B. Ovary. — The tissues to be considered in this organ are as follows :
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1. Cords of Pfliiger and peritoneum.
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2. Medullary cords.
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3. Eete tubules.
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4. Interstitial cells and stroma.
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1. Cords of Pfiuger, and Pei'itoneum. — The cords of Pfliiger, were seen to arise in the 2.5 cm. pig embryo as columns of cells growing into the stroma from the peritoneum. During later stages, they lengthen by centrifugal growth, cell multiplication taking place largely at their points of attachment to the peritoneum. One can find all stages in the development of the oogonia (see Plate VI, Fig. 22) from the stage when they are indistinguishable from the other cells of the peritoneum from which they originate, to that in which more mature forms of oocytes are found in the deeper-lying portions of the cords of Pfliiger. There is a gradual transition in these cells; the degree of maturity corresponding with the distance from the surface of the ovary. In the rabbit, certain small nuclei of cells without cell boundaries divide by amitosis (Plate VI, Fig. 22). These cells of the cords of Pfliiger correspond to the germinative cells of the seminiferous tubules. In the ovary, they are destined to form the granulosa cells, although it might well remain an open question whether some of these amitotically dividing cells do not also transform into sex cells.
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In certain of the later stages of the pig (13 and 15 cm. embryos), tubular cords of Pfliiger make their appearance (Text Fig. 3). They extend from the peritoneum for some distance into the medullary substance. By the development of the cells forming these peritoneal tubules they become transformed into solid cords containing primitive sex cells and other elements similar to the peritoneal cells of which these invaginations were originally composed. The latter are destined, in part at least, to form the granulosa. In the manner above described, these hollow tubules become transformed into solid cords of Pfliiger, in every way homologous with the cords laid down at an earlier period of development. In still later stages, the cords of Pfliiger are found to have widened, branched, and anastomosed to such an extent as to form an almost unbroken cortical zone, through which plates of connective tissue extend in a radial direction, marking out the original limits of the cords. Their inner ends are broken up to form nests of cells which become surrounded by layers of the invading stroma. In this manner are formed the follicles with their connective tissue theca, the oocyte and granulosa cells being derived from the cords of Pfliiger.
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Kolliker, 98, Mihalkovics, 85, Eouget, 79, Biihler, 94, hold the view
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Bennet Mills Allen 131
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that the granulosa cells are derived from the medullary cords. Xone of the above named authors subjected this question to a critical study of numerous stages in any species of animal; but studied isolated and more or less mature stages. Wini-^'arter, oo, Balfour, 78, Coert, 98, Nagel, 99, and many others hold, on the contrary, that the granulosa cells arise from the cords of Pflliger.
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2. Medullary Cords. — The medullary cords which we found to be at a standstill in development at the time of their separation from the peritoneum develop into structures in all regards similar to the cords of Pfiiiger. Although homologous with the seminiferous tubules, they are distinctly female in character.
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There is a constant degeneration of follicles in these medullary cords and in the deeper portions of the cortex as well. This results in the complete destruction of the sex cells in the former before the follicles have developed far enough to possess more than a single layer of granulosa cells. The few such young follicles found in the medullary cords and inner portions of the cords of Pfiiiger of the 13 cm. embryo, are found to have disappeared in the 15 cm. stage, leaving small clumps of more or less elongated granulosa cells enclosed in the membrana propria and connective tissue investment that was previously formed about them. These clumps remain throughout later stages, the persistent granulosa cells taking on more or less the appearance of connective tissue.
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The fate of the medullary cords is quite similar in the rabbit. It will be more explicitly dealt with in connection with the rete tubules. Suffice it to say that the sex cells never pass beyond the stage of synapsis characteristic of young oocytes in a certain early stage of development, the few simple follicles that make their appearance lieing destined to degenerate as in the pig.
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3. Rete Tubides. — The subject of the rete tubules of the ovary is one of the most interesting of the whole account. i\.s previously stated, they contain primitive sex cells during the early stages in the development of both male and female. These are present in both the extra- and intraovarian portions. In that part of the rete lying within the sex gland, they increase in number during the 4 cm. and 5 cm. stages of the pig, becoming much more numerous than in that portion lying within the mesonephros. The proximity of any given portion of the rete tissue to the sex gland appears to condition the relative number of primitive sex cells found in it. The rete tubules of the ovary are at all times devoid of a lumen, and the intra-ovarian portions take on more and more the appearance of the medullary cords and cords of Pflliger. This similarity becomes very evident in the 13 cm. stage, at which period
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132 Embi\voiiic Development of Ovai\y and Testis of Mammals
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the intra-ovarian jDortions of the rete are almost wholly composed of primitive sex cells and of smaller cells in every regard identical in kind with the follicular cells. Strands of stroma tissue serve to separate the rete tubules from one another.
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Later -stages show the intra-ovarian portion. of the rete tissue to become constricted off from the extra-ovarian (mesonephric portion). The primitive sex cells in the former continue to develop until in the 18 cm. embryo there are found t3'pical follicles (Plate VI, Fig. 20), each with its oocyte and a single layer of granulosa cells. These oogonia and oocytes are short-lived, however, being already in process of degeneration, resulting in their total destruction before the 20 cm. stage, where all traces of the sex cells have disappeared from the rete tissue, both in the ovary and in the mesonephros. The only trace of rete tissue found in the ovary at this time consists of clumps of connective tissue and elongated modified granulosa cells. These resemble the vestiges remaining after the degeneration of the medullary cords and of the inner follicles of the cords of Pfliiger.
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In the rabbit, the process is not so striking, the sex cells having disappeared from the rete tissue in the 17-day embryo, long before there has been any trace of follicle formation. The rete tissue is bunched at the anterior end of the ovary in contact with the medullary cords. Coert, 98, states that the rete extends by no means so far distally in the ovary as it does in the testis. I have also observed this fact in the rabbit, while in the pig it is very marked as already shown. The close resemblance between the cells of the rete and medullary cords makes it difficult and, in later stages, impossible to distinguish between them. The only criterion is the presence in the latter of scattered sex cells, these being absent from the rete tubules after the 17th day of embryonic life. This distinction, however, is quite unreliable.
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The subsequent history of the mass of tissue formed by the union of the rete and medullary cords is an uneventful one. After the primitive sex cells of the medullary cords degenerate in the young rabbit, 17 days after birth, the rete and medullary cords are seen as slender strands, lying in the dense stroma between the lymph spaces of the medullary portion of the ovary (Plate VII, Fig. 26). Their nuclei often become columnar through the pressure exerted upon them. These rete-medullary cord rudiments have now reached a period of quiescence in Avhich they are remarkably persistent, remaining until after the animal has passed the stage of puberty. In both pig and rabbit, they persist as vestigial structures, playing absolutely no further role in the development of the sex gland.
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Bennet Mills Allen 133
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■i. Interstitial Cells and Stroma. — The first generation of interstitial cells in the pig ovary appears in the 2.5 cm. emhryo. They divide by mitosis, but are on the whole short-lived, disappearing in the stage of 4 cm. The stroma consists of fibrous connective tissue filling in all the space between the remaining structures, and forming a very important element of the ovary. No interstitial cells are found in the rabbit ovary until the stage of 45 days after birth, when a few cells are to be found, which can unmistakably be assigned to this class. Their presence is associated with the degeneration of certain follicles in which a theca interna has developed from the stroma investment. Such a theca interna is not formed until the follicle has acquired about three layers of granulosa cells and the rudiment of a follicular cavity. Their development can be best understood in the ovary of the 85-day rabbit. Fully-formed follicles at this stage are seen to be surrounded by a connective tissue investment which consists of an inner layer of modified cells — theca interna — and an outer layer of ordinary connective tissue cells. All transition forms between these two kinds exist (Plate VII, Fig. 26), showing that the cells of the theca interna have originated from the general stroma by a process of transformation in which the cell body becomes cylindrical instead of fibrillar, the cytoplasm becomes clearer, the nucleus larger and spherical, and a centrosome makes its appearance. In realit}^, the theca interna is separated from the follicle by a thin layer (follicular capsule) of attenuated connective tissue cells which send fibres in a diagonal direction through the theca interna to the theca externa. This arrangement has been previously described l)y a number of authors, Paladino, 87, Clarke, 98, and Rabl, 98.
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Very many of these follicles are degenerating at this stage. As soon as the granulosa cells have begun to degenerate by chromatolysis, those of the theca interna begin to enlarge slightly. A few cells from the innermost fibrous layer (follicular capsule) and from the theca interna break through the basement membrane and enter the cavity of the follicle. The theca interna derivatives undergo fatty degeneration, eventually disappearing together with the granulosa cells. The only elements that ultimately persist in the mass of degenerating cells enclosed by the follicular capsule are the thin connective tissue cells that have become separated from the capsule. These remain unaltered after all the cells that have migrated from the theca interna have disappeared by fatty degeneration and the granulosa cells by chromatolysis.
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During the degeneration of the cells enclosed by. the follicular capsule, the cells forming the latter join to form a thick densely-staining membrane which contracts and thiq]vens as degeneration proceeds. It
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134 Embryonic Development of Ovary and Testis of Mammals
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persists after all the granulosa and inner theca cells and even the ovum have entirely disappeared. It shows a more or less fibrous structure in these later stages, finall}' disappearing without leaving a trace. The above described closing-in of the follicular capsule stretches the connective tissue strands joining the capsule with the theca externa. In this manner, the cells of the intermediate theca interna are arranged in radiating columns. These develop into the interstitial cells to be described later. This view has already been advanced by Schottlaender, 91, Clarke, 98, Plato, 97, Limon, 02, and a number of others.
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At a stage immediately succeeding the disappearance of granulosa cells and ovum, the nuclei of the still undeveloped interstitial cells become amoeboid and then undergo a rapid process of amitotic division (Plate VII, Fig. 27). This is not described in any of the literature, although Dr. Frank E. Lillie and Dr. C. M. Child of this laboratory both inform me that they have noted the same phenomenon.
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Van der Stricht, 01, finds that in the formation of the corpora lutea of the bat (Vespertilio) the cells of the theca interna having taken on the form of lutein cells, divide by mitosis for a short period, after which division ceases entirely. Sobotta, 96, also has found scattered mitotic figures in the theca interna of the rabbit after discharge of the ovum, although he does not ascribe to this layer the formation of the lutein cells. Eabl, 98, finds them to divide before the beginning of atresia, not after that process has set in.
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After this process of amitotic division is completed the interstitial cells rapidly increase in size and finally develop into the mature form in which the cytoplasmic body is voluminous, clearly bounded, and stuffed full of fat globules; the nucleus is enlarged and roimded; and a welldefined centrosphere and centrosome have appeared. These interstitial cells are similar to the lutein cells of the corpora lutea in all regards save size. Even this criterion is not a safe one by which to distinguish the two sets of elements. Certain interstitial cells of the ovary of a 6-months' old virgin, having become separated from the mass and lying free in the loose stroma, are found to have enlarged to the dimensions of the smaller lutein cells of the corpora lutea.
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Although the work of Sobotta, 96, Honore, 00, and others has led them to assert that the lutein cells of the corpora lutea originate solely from the granulosa cells of the discharged follicle, there are a large number of workers who hold the view that they originate solely from the cells of the theca interna. Among such authors may be mentioned Clarke, 98, Van Beneden, 80, and Kolliker, 98. Van der Stricht, 01, and Schulin, 81, consider them to arise from both sources. This question
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Bennet Mills Allen 135
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does not jDroperly come within the limits of this work, yet I cannot refrain from pointing out the very close resemblance betw^een the interstitial and lutein cells (Plate VII, Figs. 29 and 30). It seems quite improbable that two groups of cells, almost identical as these are, could have arisen from such diverse elements as the connective tissue cells of the theca interna, on the one hand, and the granulosa cells on the other.
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Successive generations of lutein and interstitial cells push the earlierformed groups of cells toward the center of the ovary, where they undergo hyalin degeneration. De Sinety, 77, finds in the human subject that the number of atretic follicles is greater during pregnancy than at other times. I cannot substantiate this; but am inclined to consider pregnancy to make no difference in their number in the rabbit. This view I can support by a number of ovaries, taken from immature rabbits, mature virgins, immature virgins, pregnant animals and one taken from a rabbit that had borne young but had been isolated for three months.
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4. Primitive Sex Cells. — The primitive sex cells occur from the earliest stages studied (pig, 6 mm. length; rabbit, 13-day embryo) on through all later stages of development. Similar cells have been found in the earliest stages of the Elasmobranchs by Beard, 00, 02, 03, Eabl, 98, Woods, 02, and in the Teleost by Eigenmann, 91. I have myself found the large yolk-filled primitive sex cells of these authors in turtle embryos (Trionyx) and may say that I am now at work upon this subject. It is too early to give results, but it may be stated with certainty that these cells occur in the embryo of 3 mm. length and are seen to be apparently migrating from the entoderm through the splanchnopleuric mesoderm to the point where the latter joins the somatopleuric mesoderm. It is here that the sex glands are to form. This observation corresponds with that of Beard, 03.
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The nuclei of such cells are larger than those of the entoderm and mesoderm among which they lie, but resemble them at this stage in the fact that they show very little chromatin material as contrasted with the very pronounced chromatin network which they show in later stages (1.5 cm. embryo). In this stage the large yolk spherules are found to be breaking up into small granules which remain in one or two clumps in the cytoplasm. These cells at this stage show a very marked resemblance to the primitive sex cells of the pig and rabbit. In fact I have no hesitation in identifying them as their homologues. In the pig and rabbit they are found not only in the genital ridge, but outside of it as well, in the earlier stages. So far as my own work goes, I have found them in the mesentery of the alimentary canal. Eigenmann, 91, found
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136 Embryonic Development of Ovary and Testis of Mammals
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them even in the brain region of the young Teleost (Micrometrus). There seems to be no doubt of their sexual character, because they are present in such great numbers in the sex glands, and have all the characteristics of those cells (spermatogonia and oogonia) from which the sex products eventually form.
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It has been shown in the pig and rabbit, however, that these sex cells, appearing in the indifferent stages, do not contribute to the formation of functional sex products in the ovary. The same is probably true of the testis, it being at least certain that the great bulk of the spermatogonia are formed from the germinative cells — cells derived originally from the peritoneum and maintaining, first, their indifferent character at least so far as our technique is able to show. Schonfeld, oi, and Loisel, oo, have shown, the former in the case of mammals, the latter in birds, that spermatogonia and Sertoli i an cells arise during adult life from these germinative cells (indifferent cells of Schonfeld). What interpretation shall we then put upon the so-called primitive sex cells (primitive ova, XJreier, Urkeimzellen, etc.) ? I consider them to be spermatogonia in the testis and oogonia in the ovary. They have almost reached that degree of specialization at which we might call them oocytes and spermatocytes. The fact that they are still found in process of mitotic divisions excludes them from these latter classes of cells. They should more properly be termed spermatogonia and oogonia of the second order, in accordance with the use of these terms by Loisel in the case of birds and by many authors who have written upon the subject of the sex cells of invertebrates.
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These primitive sex cells found in the early stages of embryonic life have, then, undergone a process of precocious development; but for some reason, this process is not carried beyond a certain point. The stimuli or favorable conditions that brought about the formation of these secondary spermatogonia in regions outside of the sex gland, may be later present only in the sex gland itself, or indeed, only in certain parts of it. The influence exerted by the sex gland in bringing about the development of the sex cells is beautifully illustrated by the process above described by which egg follicles and spermatogonia develop in large numbers in the rete tubules lying within the sex gland, while they deN-elop sparingly in those parts lying within the mesonephros. In such cases as we have seen, the degree of development and number of sex cells forming in any given region of the rete tissue is dependent upon the proximity of that region to the sex gland. It must be clearly understood that I do not deny the possibility of an early specialization and segregation of the sex cells as claimed by Nussbaum, 80, Eabl, 96,
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Bennet Mills Allen 137
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Beard, oo, 02, 03, Eigenmanu, 91, Woods, 02, and others; but I do assert that the proof of such an assertion has not yet been furnished. The development of the sex cells must be followed from the earliest embryonic stages to the period of sexual maturit}^, before one can prove that the cells under consideration are the only ones that give rise to the sex products, or that they give rise to them at all.
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5. MoRPHOLOC4iCAL Eelationships. — The morphological significance of the sex gland structures may be expressed in terms of the biogenetic law somewhat as follows : The genital ridge represents a primitive condition in which the sex gland extended along the entire length of the mesonephros. In this sex gland, the gonads appeared in the form of tubules or vesicles opening into the body-cavity in the case of both ovary and testis. It was impossible, in this piece of work, to determine whether there is any segmental arrangement of the sex cords in the forms studied. Such, however, would most probably be the condition in the primitive t^-pe.
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The evaginations from the Malpighian corpuscles no doubt represent the " segmental strange," " genital kanale," etc., of those authors who have worked upon the development of the sex glands in the Anamnia. In the Amphibia, for instance, they have a truly segmental arrangement, according to Hoffman, 87, Spengel, 76, Semon, 92, while Hoffman, 89, Braun, 77, Mihalkovics, 85, Semon, 87, Weldon, 85, show a like segmental arrangement to occur in the Eeptilia. Much might be said in this connection, as the literature upon the subject is quite rich in suggestive facts.
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According to Shreiner, there are 2 to 3 glomeruli in each somite of the pig. My own work has shown that a connection takes place between the rete tissue and those glomeruli with which it comes in contact, there may be as many as three evaginations called forth from a single glomerulus or there may be none at all. For these reasons we cannot assert a strict homology between these evaginations and the " segmental strange " of the Anamnia, yet the former probably represent a modification of the same process, as that by which the formation of the latter are formed.
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Returning to the subject of the genital ridge as a whole, it would seem fair to conclude that the sex cords which in the ancestral forms lay posterior to the present limits of the sex gland, have disappeared. The rete tubtiles would represent sex cords anterior to the sex gland that had retained more or less of their ancestral condition, but have become modified to meet the requirements of their function as efferent ducts for the male sex products. The union of the rete tubules with the sex 11
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138 Embryonic Development of Ovary and Testis of Mammals
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cords is more intimate in the male than in the female, owing to the fact that the connection is not, and probably never was, of functional importance in the latter.
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One is struck with the fact that there is a complete, or almost com})lete, separation of the medullary cords of the ovary from the peritoneum at almost the same time that the seminiferous tubules of the testis break away from it. This separation takes place essentially in the same manner in both sexes. Coert, 98, considers the medullary cords to represent a system of ducts which served in phylogenetically earlier periods, to carry the female sex products to the Wolffian duct. Waldeyer, 70, considers them to represent vestigial seminiferous tubules arising from the mesonephros. Paladino, 87, and Harz, 83, confound them in part with the long rows of interstitial cells of the adult animal. One might assume that there was one stage in the jjhylogenetic history of the sex glands in which both medullary cords and seminiferous tubules furnished sex products that were conducted to the rete efferentia through the rete tubules. If one hold this view, he must grant that the formation of the cords of Pfl tiger or second generation of ovarian cords represents a return to the primitive condition in which the female sex products are again discharged into the body-cavity from the surface of the sex gland. This view would hardly seem to be a reasonable one, hence I, at least, would prefer to consider the cords of Pfliiger to be mere interrupted continuations of the medullary cords. Winiwarter, 00, holds a view very similar to the last, his well-known diagram practically expressing my own conception of the process. There is no exact correspondence in number between the cords of Pfliiger and medullary cords, the former being much more numerous that the latter.
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The medullary cords never assume the characteristics of seminiferous tubules.
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The assumption that the separation of the medullary cords from the peritoneum is not and never was phylogenetically of functional importance, leads to many interesting questions pertaining to the influence of heredity in the transmission of sexual characters. Is it possible that developmental processes of functional importance to the testis alone must be transmitted to the female or vice versa, simply because the germ cells which have united to produce the embryo transmit tendencies to the formation of both male and female, both of which assert their power, though one be ever so feeble? In this connection it is of great interest to note that Laulaine, 86, has found that the peritoneum of the 7 to 8 day chick testis thickens after the separation as though it were about to develop along the line of the ovary. This is merely temporary,
 +
 +
 +
 +
Beunet Mills Allen 139
 +
 +
as it again thins out and loecomes relatively unimportant in later stages. In any case, there is a remarkably close correspondence between the general processes of development of the testis and ovary.
 +
 +
I wish to express my deep obligations to my professor. Dr. Frank K. Lillie, for constant guidance and valuable assistance throughout the course of this work.
 +
 +
VI. SUMMARY AND CONCLUSIONS.
 +
 +
1. The sex glands and rete originate from the genital ridge composed of the thickened mesonephric peritoneum and underlying tissue, proliferated from it.
 +
 +
2. The testis is composed of (A) the seminiferous tubules, (B) stroma.
 +
 +
A. The seminiferous tubules are formed as solid invaginations of the peritoneum, which later became separated from it, and undergo subsequent growth by the activity of the component cells. These are of two kinds, (1) primitive sex cells, spermatogonia of the second order; and (2) germinative cells. Intermediate forms connect these two kinds and may be interpreted as the primitive cells from which both varieties origirate. They occur' up to a certain stage in development, and may possibly recur periodically in adult stages.
 +
 +
B. The stroma consists of (1) loose connective tissue, (2) the albuginea — formed from the cells comprising the proximal portions of the seminiferous tubules together with possible additions of other connective tissue from the stroma, (3) interstitial cells formed from the stroma. These are formed contemporaneously with the appearance of fatty degeneration in both peritoneum and seminiferous tubules.
 +
 +
3. The ovary is made up of homologous groups of structures.
 +
 +
A. The medullary cords and cords of Pfliiger are both derived by invagination of the peritoneum, the former being in all regards homologous with the seminiferous tubules. The cords of Pfliiger are invaginations of the peritoneum, formed after the medullary cords have become separated from it. Both medullary cords and cords of Pfliiger contain oogonia and follicle cells. Follicles formed in the medullary cords are never functional and cease to form in later stages. They degenerate together with other young follicles of the inner ends of the cords of Pfliiger. Both medullary cords and cords of Pfliiger contain (1) primitive sex cells (oogonia) ; (2) follicular cells — probably homologous with the germinative cells of the seminiferous tubules; while intermediate forms of cells are found in the peripheral part of the ovary.
 +
 +
B. The stroma consists of (1) loose connective tissue, from which are derived the theca interna and theca externa of tlie follicles; (2) a zone
 +
 +
 +
 +
140 Embryonic Development of Ovary and Testis of Mammals
 +
 +
homologous with the albuginea, but of a loose consistency, which renders it indistinguishable from the remainder of the stroma; (3) interstitial cells homologous with those in the testis. In the pig ovary, these interstitial cells appear very sparingly, at the same time that they appear in the testis, but very soon disappear. They develop in later stages in both pig and rabbit ovary. Details of this phenomenon were studied only in the rabbit, in which animal the cells in question were first found 45 days after birth. They are formed from the cells of the theca interna in response to conditions created by the degeneration of the follicles about which they lie. Many striking points of similarity link these interstitial cells with the lutein cells of the corpora lutea. Both finally sufi'er hyalin degeneration in the interior of the ovary.
 +
 +
4. Eete tubules are formed in connection with both testis and ovary,
 +
 +
A. The tubules forming the rete ovarii and rete testis originate in the region of the genital ridge anterior to their respective sex glands. They are homologous with the sex cords which they also reseml^le in the fact that they contain primitive sex cells. In the testis they form a core surrounded more or less completely by the seminiferous tubules, and extend almost the entire length of the -organ. They project but a short distance from the hilum into the ovary. In both sexes they are connected with certain glomeruli of the anterior part of the mesonephros, by means of more or less vestigial evaginations from the capsules of Bowman.
 +
 +
B. Each cord of the rete testis acquires a lumen and sends numerous side branches (tubuli recti) to the seminiferous tubules, from which they differ only in diameter and in the relative number of primitive sex cells contained in them. This similarity is in later stages confined to tliose portions, of the rete tubules that lie within the testis. These finally undergo modification in form and lose their sex cells by degeneration.
 +
 +
C. Homologous relations exist between the -rete ovarii and the medullary cords. The rete ovarii never acquire a lumen, remaining solid, like the medullary cords which they greatly resemble. So great is this resemblance in the case of the rabbit, that the two structures cannot be distinguished from one another in post-embryonic stages. The slender strands of these indistinguishable elements persist in a quiescent state in the ovary of the adult. In the pig, numerous oogonia appear in the intra-ovarian rete tubules, many of them even developing into young follicles, all of Avhich degenerate before birth, leaving masses of follicular cells similar in all regards to the remains of the medullary cords.
 +
 +
 +
 +
Bennet Mills Allen 141
 +
 +
Conclusions.
 +
 +
1. The sex cords — medullary cords and seminiferous tubules — are homologous structures formed as tubular invaginations of the peritoneum.
 +
 +
2. The cords of Pfiiiger are formed from the same source and in the same manner as the sex cords, but at a slightly later period of time.
 +
 +
3. The rete cords are formed at the same time as the sex cords and in the same manner.
 +
 +
4. The connective tissue of the sex-gland — stroma and albuginea — is derived from the peritoneum.
 +
 +
5. The interstitial cells of both ovary and testis are formed from connective tissue in reference to a process of degeneration occurring in the sex gland.
 +
 +
6. The primitive sex cells seen in the earliest stages are precociously developed oogonia or spermatogonia of the second order, similar cells developing during later stages, from apparently undifferentiated peritoneal cells.
 +
 +
7. The sex glands exert a specific influence, causing follicles to form in the intra-ovarian rete of the pig, and bringing about the development of spermatogonia in the intra-testicular rete of both pig and rabbit. Such sex elements are not functional, because of the fact that they suffer early degeneration.
 +
 +
BIBLIOGRAPHY.
 +
 +
Allex, B. M., 03. — The Embryonic Development of the Ovary and Testis
 +
 +
of the Mammalia (preliminary account). Biological Bulletin, Vol. V,
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 +
1903. Balfour, F. M., 78.— On the Structure and Development of the Vertebrate
 +
 +
Ovary. Quart. Jour. Micro. Science, Vol. XVIII, 1878. Balfour, F. M., 74. — On the Development of the Elasmobranch Fishes, Urino genital System. Complete Works, Vol. I, 1885. Beard, J., 03. — The Germ-cells of Pristiurus. Anat. Anz., Bd. XXI, 1903. Beard, J., 03.— The Numerical Law of Germ-cells. Anat. Anz., Bd. XXI, 1903. Beard, J., 00. — The Morphological Continuity of the Germ-cells in Raja batis.
 +
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Anat. Anz., Bd. XVIII, 1900. Beard, J., 02. — Heredity and the Epicycle of the Germ-cells. Biol. Centralbl.,
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 +
Bd. 1902. Benda, C, 89. — Die Entwickelung des Saugethierhodens. Verh. d. Anat.
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 +
Gesellsch. 3 Versam., 1 Sitz., 1889. Benda, C, 87, — Untersuchungen iiber den Bau, etc., etc., und Folgerungen fiir
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 +
die Spermatogenesis der Wirbelthieren. Arch. f. mikr. Anat., Bd.
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XXX, 1887. Beneden, E. van, 80. — Contribution a la connaissance de I'ovaire des mam mifgres. Arch, de Biol., T. I, 1889. BoRX, E., 94. — Die Entwickelung der Geschlectsdriisen. Anat. Hefte, Ergbn.
 +
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4, 2, 1894.
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142 Embryonic Development of Ovar}' and Testis of Mammals
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BouiN, M., oo. — Histogenese de la glande femelle chez Rana temporaria. Arch.
 +
 +
de Biol., T. XVII, 1900. Braun, 77. — Das Urogenitalsystem der einheimischen Reptllien. Arbeiten
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a. d. Zool. Zoot. Institiit in Wiirzburg, Bd. IV, 1877. BUHLER, A., 94. — Beitrage zur Kenntniss der Eibildung beim Kaninchen und
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der Markstrange des Eierstockes beim Fuchs und Menschen. Ztschr.
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f. Wissensch. Zool., Bd. LVIII, 1894. Child, C. M., 97. — Centrosome and Sphere in Cells of the Ovarian Stroma
 +
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of Mammals. Zool. Bull., Vol. I, 1897. Clarke, J. G., 98. — Ursprung Wachsthum und Ende des Corpus luteum.
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Arch. f. Anat. u. Entwickelungsgsch., 1898. CoERT, H. J., 98. — Over de ontwikkeling en den bouw van de geschlachtsklier
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bij de Zoogdieren. Leiden, 1898. EiGENMANN, C. H., 91. — On the Precocious Segregation of the Sex-cells of
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Micrometrus aggregatus. Jour, of Morphol., Vol. V, 1891. Flemming, W., 85. — Ueber die Bildung von Richtungsfiguren, etc. Arch. f.
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Anat. u. Physiol., 1885. Harz, W., 83. — Beitrage ziir Histologic des Ovarium der Saugethiere. Arch. f.
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mikr. Anat, Bd. XXII, 1883. Henneguy, L. F., 94. — Recherches sur I'atresia des follicules de de Graaf.
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Jour, de I'Anat. et de la Physiol., T. XXX, 1894. Herbst, C, 01. — Formative Reize in der tierischen Ontogenese. Leipz., 1901. Hertwig, O., 96. — Lehrbuch der Entwickelungsgeschichte des Menschen und
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der Wirbelthiere, 1896. His, W., 65. — Beobachtungen tiber den Bau des Saugethier-Eierstocks. Arch.
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f. mikr. Anat., Bd. I, 1865. Hoffman, C. K., 87. — Zur Entwickelungsgeschichte der Urogenitalorgane bei
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den Anamnia. Ztschr. f. wiss. Zool., Vol. XLIV, 1887. Hoffman, C. K., 89. — Entwickelungsgeschichte der Urogenitalorgane bei den
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Anamnia. Ztschr. f. wiss. Zool., Bd. XLVIII, 1889. HoNOR]6, C, 00. — Recherches sur I'ovaire du lapin. — II. Recherches sur la
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 +
formation des Corps jaunes. Arch, de Biol., T. XVI, 1900. JuNGERSEN, 89. — Beitrage zur Kenntniss der Entwickelung der Geschlechts organe bei den Knochenfischen. Arbeit, aus dem Zool. Zoot. Inst.
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 +
in Wiirzberg, Bd. IX, 1889. Kolliker, a., 98. — Ueber die Markkanale und Markstrange in den Eierstocken
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 +
junger Hiindinnen. Verh. d. Anat. Gesellsch., Kiel, 1898. Kolliker, A., 98. — Ueber die Entwickelung der Graafschen Follikel der
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 +
Sagethiere. Wiirzb. Sitzber., Juni, 1898. Kolliker, A., 98. — Ueber Corpora lutea atretica bei Saugethieren. Verh. d.
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 +
Anat. Gesellsch., Kiel, 1898. Laulanie, F., 86. — Sur le mode d'evolution et la valeur de I'epithelium ger minatif dans le testicule embryonnaire du Poulet. C. R. Soc. Biologie,
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 +
Paris, T. Ill, 1886. LAULAN16, F., 86. — Recherches sur les connexions embryogeniques des cordons
 +
 +
medullaires de I'ovaire avec les tubes seminif^res. C. R. Soc. Biologie, Paris, T. Ill, 1886. Laulanie, F., 88. — Sur I'origine commune et le role variable de I'epithelium
 +
 +
germinatif et des cordons sexuels dans I'ovaire. C. R. Soc. Biologie,
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Paris, T. V, 1888.
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Bennet Mills Allen 143
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LiMO>', M., 02. — Etude histologique et histogenique de la glande interstitielle
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 +
de I'ovaire. Arch. d'Anat. micro.. T. V, fasc. II, 1902. LoisEL, G., 00. — Etudes sur la spermatogenese chez le moineau, domestique.
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 +
J. de I'Anat. et de la Physiol., T. XXXVI, 1900. LoisEL, G., 02. — Sur I'origine embryonnaire et revolution de la secretion
 +
 +
interne du testicule. C. R. Soc. de Biologie, T. LIV, fasc. 26, 1902. LoisEL, G., 02. — Sur I'origine du testicule et sur sa nature glandulaire. C. R.
 +
 +
Soc. de Biologie, T. LIV, fasc. V, 1902. LoisEL, G., 02. — Sur le lieu d'origine, la nature et le role de la secretion interne
 +
 +
du testicule. C. R. Soc. de Biologie, T. LIV, No. 27, 1902. MacCallum, J. B., 02. — Notes on the Wolffian Body of Higher Mammals.
 +
 +
Amer. Jour, of Anat., Vol. I, No. 3, 1902. MacLeod, 81. — Contribution a I'etude de la Structure de I'ovaire des mam miferes. Arch, de Biol., T. I, 1880, also Arch, de Biol., T. II, 1881. Mihalkovics, 85. — Entwickelung des Ham und Geschlechtsapparates der Am nioten: I der Excretionsapparat, II die Geschlechtsorgane, III die
 +
 +
Geschlechtsdriisen. Separatabdruck aus der Internat. Monatsschr. f.
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Anat. u. Histol., 1885, Bd. II, H. 9. MiNOT, C. S., 94. — Gegen das Gonotom. Anat. Anz., Bd. IX. MoLLER, F., gg. — Ueber das Urogenitalsystem einiger Schildkroten. Ztschr. f.
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wiss. Zool., Bd. LXV, 1899. Nagel, W., 89. — Ueber die Entwickelung des Urogenitalsystem des Menschen.
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Arch. f. mikr. Anat., Bd. XXXIV, 1889. Nagel, W., 89. — Ueber das Vorkommen von Primordialeiern ausserhalb der
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 +
Keimdriisenanlage beim Menschen. Anat. Anz., Bd. IV, 1889. NussBAUM, M., 80. — Zur Differenzierung des Geschlechts im Thierreich. Arch.
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 +
f. mikr. Anat., Bd. XVIII, 1880. NussBAUM, M., 01. — Zur Entwickelung des Geschlechts beim Huhn. Verhandl.
 +
 +
d. Anat. Gesellsch.. Bonn, 1901. Paladino, G., 87. — Ulteriori richerche sulla distruzione e rinnovamento continue del parenchima ovarico nei mammiferi. Naples, 1887. Paladino, G., 94. — La destruction et le renouvellement continuel du paren chyme ovarique des Mammiferes. Arch. ital. de Biol., T. XXIV, 1894. Pflugek, E., 63. — Ueber die Eierstocke der Saugethiere und des Menschen.
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Leipzig, 1863. Plato, J., 97. — Zur Kenntniss der Anatomic und Physiologie der Geschlechtsorgane. Arch. f. mikr. Anat., Bd. XLVIII, 1897. Prenant, a., 89. — Contribution a I'histogenese du tube seminifere. Internat.
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Monatsschr. f. Anat. u. Physiol., T. 1889. Prenant, A., 90. — Remarque a propos de la constitution de la glande genitale
 +
 +
indifferente et de la histogenese du tube seminifere. Compt. rend, de la Soc. de Biol., 9e Serie, T. II, 1890. Rabl, C, 96. — Ueber die Entwickelung des Urogenitalsystem der Setachiers. Morphol. Jahrb., Bd. XXIV. Theorie des Mesoderms. VI, Ueber die erste Entwickelung der Keimdriise. Morphl. Jahrb., Bd. XXIV, 1896. Rabl, H., 98. — Beitrage zur Histologie des Eierstockes des Menschen und der Saugethiere nebst Bemerkungen iiber die Bildung von Hyalin und Pigment. Anat. Hefte, 1 Abth., Bd. LVII, 1898.
 +
 +
 +
 +
144 Embryonic Development of Ovary and Testis of Mammals
 +
 +
RouGET, 79. — Recherches sur le developpement des oeufs et de I'ovaire chez les
 +
 +
mammiferes apres la naissance. Compt. rend, de I'Acad. des Sci. a
 +
 +
Paris, T. LXXXVIII, No. 3, 1879. RouGET, 79. — Evolution comparee des glandes genitales male et femelle chez
 +
 +
les embryons de mammiferes. Comp. rend, d I'Acad. des Sci. a Paris,
 +
 +
T. LXXXVIII, 1879. ScHMiEGELOw, E., 82, — Studien iiber die Entwickelung des Hodens und Neben hodens. Arch. f. Anat. u. Physiol., 1882. ScHONFELD, H., oi. — La spermatogenese chez le taureau et chez les mammiferes en general. Arch, de Biol., T. XIX, 1901. ScHOTTLAENDER, J., 91, — Bcitrag zur Kenntniss der Follikelatresie nebst einige
 +
 +
Bemerkungen iiber den unveranderten Follikeln in den Eierstock
 +
 +
Saugethiere. Arch. f. mikr. Anat, Bd. XXXVII, 1891. ScHOTTLAENDER, J., 93. — Ueber den Graafschen Follikel, seine Entstehung
 +
 +
beim Menschen und seine Schichsale bei Mensch und Saugethiere.
 +
 +
Arch. f. mikr. Anat., Bd. XLI, 1893. ScHULiN, K., 8i. — Zur Morphologie des Ovariums. Arch. f. mikr. Anat., Bd.
 +
 +
XIX, 1881. Semon, R., 87. — Die indifferente Anlage der Keimdriisen beim Hiihnchen und
 +
 +
ihre Differenzierung zum Hoden. Jena, Ztschr. f. Naturwiss., Bd.
 +
 +
XXI, 1887. Semon, R., 92. — Der Bauplan des Urogenitalsystems der Wirbelthiere. Jen aische Ztschr., Bd. XXVI, 1892. Semper, C, 75. — Das Urogenitalsystem der Plagiostomen. Arb. aus d. Zool.
 +
 +
Zoot. Inst, in Wiirzburg, Bd. II, 1875. SiNETY, 77, — De I'ovaire pendant la grossesse. Comp. rend, de I'Acad. d. Sci.
 +
 +
a Paris, 1877. SoBOTTA, J., 96. — Ueber die Bildung des Corpus luteum bei der Maus. Arch. f.
 +
 +
mikr. Anat, Bd. XLVII, 1896. SoBOTTA, J., 98. — Noch einmal Frage der Bildung des Corpus luteum. Arch. f.
 +
 +
mikr. Anat, Bd. LIII, 1898. Spengel, J. W., 76. — Das Urogenitalsystem der Amphibien. ' Arb. aus dem
 +
 +
Zool. Zoot. Inst, in Wiirzburg, Bd. Ill, 1876. Van dee Steicht, O., 01. — La ponte ovarique et I'histogenese du corps jaune.
 +
 +
Bull. Acad. Roy. Med. de Belgique, IV Ser., Vol. XV, 1901. Waldeyer, W., 70. — Eierestock und Ei. Leipzig, 1870. Waldeyer, W., 02. — Die Geschlechtszellen. Handb. d. vergl. u. exper. Ent wick. d. Wirbelthiere, 9 u. 10 Lief., 1902. Weldon, W., 85. — On the Suprarenal Bodies of the Vertebrata. Quart. Jour.
 +
 +
Micro. Sci., Vol. XXV, 1885. Winiwarter, H., 00. — Recherches sur I'ovogenese et I'organogenese de I'ovaire
 +
 +
des mammiferes (Lapin et Homme). Extr. d. Arch, de Biol., T. ' XVII, 1900. Woods, F. W., 02. — Origin and Migration of the Germ-cells in Acanthias.
 +
 +
Am. Jour, of Anat., Vol. I, No. 3, 1902.
 +
 +
 +
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Bennet Mills Allen 145
 +
 +
EXPLANATION OF PLATES.
 +
 +
All figures except No. 1, were outlined witti the aid of a camera lucida.
 +
 +
al., albuginea. m. r., mesenteric ridge.
 +
 +
c, blood corpuscle. nil., nucleus.
 +
 +
c. B., capsule of Bowman. oc, oocyte.
 +
 +
cen., centrosphere. p., peritoneum.
 +
 +
c. P., cord of Pfliiger. p. s., primitive sex-cell.
 +
 +
c. p., epithelial plate. r., rete.
 +
 +
ev., evagination of capsule of r c, rete cord.
 +
 +
Bowman. r. t., rete tubule.
 +
 +
f. c, follicular capsule. s. c, sex cord,
 +
 +
gr., glomerulus. st., stroma.
 +
 +
ger., germinative cell. s. tu., seminiferous tubule.
 +
 +
gr., granulosa cell. s. g., sex gland.
 +
 +
i. c, interstitial cell. t., testis.
 +
 +
m., mesonephros. t. r., tubulus rectus.
 +
 +
m. f., mesenteric fundament. th. i., theca interna.
 +
 +
m. p., membrana propria. th. e., theca externa.
 +
 +
PLATE I.
 +
 +
Fig. 1. Reconstruction of the anterior end of the left mesonephros. Pig embryo, length 12.5 cm.
 +
 +
Fig. 2. Fundament of the rete ridge. Pig embryo, length 0.7 cm. Transverse section, x 893.
 +
 +
Fig. 3a. Fundament of sex gland. Pig embryo, length 0.7 cm. Transverse section, x §93.
 +
 +
Fig. 3b. Same as 3a, showing primitive sex cell in portion of peritoneum. X893.
 +
 +
Fig. 4. Fundament of sex gland. Pig embryo, length 1.0 cm. Transverse section, x 893.
 +
 +
PLATE IL
 +
 +
Fig. 5. Fundament of sex gland. Pig embryo, length 1.25 cm. Transverse section, x ^08.
 +
 +
Fig. 6. Fundament of rete. Pig embryo, length 1.4 cm. Transverse section. X893.
 +
 +
Fig. 7. Fundament of sex gland and mesentery. Pig embryo, length 1.4 cm. Transverse section, x 893.
 +
 +
PLATE in.
 +
 +
Fig. 8. Fundament of sex gland (peripheral portion). Pig embryo, length
 +
 +
1.6 cm. Transverse section, x 608.
 +
 +
Fig. 9. Fundament of sex gland (peripheral portion). Pig embryo, length
 +
 +
1.7 cm. Transverse section, x 893.
 +
 +
Fig. 10. Medullary cord of ovary. Pig embryo, length 5 cm. x 893.
 +
 +
 +
 +
146 Embryonic Development of Ovary and Testis of Mammals
 +
 +
PLATE IV.
 +
 +
Fig. 11. Rete cord in rete ridge. Pig embryo, length 1.8 cm. x 893.
 +
 +
Fig. 12. Seminiferous tubules and stroma of testis. Pig embryo, length 1.8 cm. X S93.
 +
 +
Fig. 13. Seminiferous tubule and stroma of testis (transverse section). Pig embryo, length 3 cm. x 893.
 +
 +
Fig. 14. Seminiferous tubule of testis (longitudinal section). Pig embryo, length 7.5 cm. x 893.
 +
 +
PLATE V.
 +
 +
Fig. 15. Capsule of Bowman and rete cords. Pig embryo, length 4 cm. X 893.
 +
 +
Fig. 16. Capsule of Bowman and rete cords. Pig embryo, length 7.5 cm.
 +
 +
Fig. 17. Intra-ovarian portions of rete cords. Pig embryo, length 8.5 cm. X893.
 +
 +
Fig. 18. Tubulus rectus and seminiferous tubule. Pig embryo, length 13 cm. X 893.
 +
 +
PLATE VL
 +
 +
Fig. 19. Seminiferous tubule. Pig embryo, length 15 cm. x 893.
 +
 +
Fig. 20. Follicle in intra-ovarian rete. Pig embryo, length 15 cm. X 893.
 +
 +
Fig. 21. Rete tubule of testis. Pig embryo, length 13 cm. x 893.
 +
 +
Fig. 22. Portion of cortex of ovary. Rabbit embryo, 23 days. X 893.
 +
 +
Fig. 23. Seminiferous tubule (longitudinal section). Rabbit embryo, 21
 +
 +
days. X 893.
 +
 +
Fig. 24. Seminiferous tubule (transverse section). Rabbit, 24 days after
 +
 +
birth. X 893.
 +
 +
PLATE Vn.
 +
 +
Fig. 25. Cord of Pfliiger, with lumen. Pig embryo, length 15 cm. x 893.
 +
 +
Fig. 26. Tissues of a rabbit ovary. 78 days after birth, x 893.
 +
 +
Fig. 27. Interstitial cells in process of amitotic division. Rabbit ovary, 93 days after birth, x 893.
 +
 +
Fig. 28. Interstitial cell of ovary. Virgin rabbit, 6 months old. x 893.
 +
 +
Fig. 29. Interstitial cell of ovary. Rabbit in 14i/^ day of first pregnancy. X893.
 +
 +
Fig. 30. Lutein cell of corpus luteum, same animal as for Fig. 29. X 893.
 +
 +
 +
 +
OVARY AND TESTIS OF MAMMALS BENNET MILLS ALLEN
 +
 +
 +
 +
PLATE I
 +
 +
 +
 +
 +
AMERICAN JOURNAL OF AN ATOMY--VOL. Ill
 +
 +
 +
 +
OVARY AND TESTIS OF MAMMALS BENNET MILLS ALLEN
 +
 +
 +
 +
PLATE II
 +
 +
 +
 +
 +
AMERICAN JOURNAL OF ANATOM Y--VOL. Ill
 +
 +
 +
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OVARY AND TESTIS OF MAMMALS BENNET MILLS ALLEN
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PLATE III
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AMERICAN JOURNAL OF AN ATOM Y--VOL. II
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OVARY AND TESTIS OF MAMMALS BENNET MILLS ALLEN
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PLATE IV
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AMERICAN JOURNAL OF ANATOMY--VOL III.
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OVARY AND TESTIS OF MAMMALS BENNET MILLS ALLEN
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PLATE V
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AMERICAN JOURNAL OF AN ATOM Y--VOL. Ill
 +
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OVARY AND TESTIS OF MAMMALS BENNET MILLS ALLEN
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PLATE VI
 +
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AMERICAN JOURNAL OF ANATOMY—VOL. Ill
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 +
OVARY AND TESTIS OF MAMMALS BENNET MILLS ALLEN
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PLATE VII
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AMERICAN JOURNAL OF ANATOMY— VOL. Ill 12
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ON THE STRUCTUEE OF THE HUMAN UMBILICAL VESICLE.
 +
 +
BY
 +
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ARTHUR W. MEYER,
 +
 +
From the Anatomical Laboratory of the Johns Hopkins University.
 +
 +
With 5 Text Figures.
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In the history of embryology the discovery and interpretation of the yolk sac must always remain one of the most interesting chapters. The, to us, naive speculations as to its significance, at a time when " anatomists feared to make a thorough examination of ova and preferred rather to preserve them in alcohol," lend a peculiar interest to the study of the early literature on this subject. Many of the embryologies and anatomies of that time give much attention to the yolk sac, and it is not uncommon to find several chapters devoted to the discussion.
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The credit for the first description of the human yolk sac seems to lie between Hoboken and Noortwyck. Wrisberg, however, gave the first accurate description of it, in full cognizance of the fact that what he described was a yolk sac comparable to the yolk sac of birds. The latter is referred to by Wrisberg as the " vesicula erythroides " of von Pockel, imconscious of the fact that von Pockel really described the allantois and not the yolk sac, as he believed. It is possible that Noortwyck was the first to recognize the yolk sac of the human embryo. Hoboken did not recognize it, and, according to Mayer, this was left for the great Albinius who first pictured a human embryo with the umbilical vesicle in situ. It was this fact which caused Zinius, in his monograph, to refer to the yolk sac as " de vesicula embryonis Albiniana." Neither this designation, nor that of " vesicula alba " of Hunter, found favor, however, for both were soon displaced by the term " vesicula umbilicalis " first used by Blumenbach.
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Up to 1835 the greatest diversity of opinion existed regarding the functions of the yolk sac, and many interesting theories were advanced. Oken, while recognizing the meaning of the organ and demonstrating its occurrence in several of the mammalia, promulgated the idea that the intestine arose in the vesicle itself. Kieser, in 1810, claimed to have proven that the intestine develops in the yolk sac, and that it is then slowly taken into the abdomen. Van Euysch and Ossiander, on the contrary, took it for an hydatid and a pathological formation respec American Journal of Anatomy. — Vol. III.
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156
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The Structure of Human Umbilical 'Wsicle
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tively. Mayer's exhaustive monograph, which appeared in 1835, removed many of these misconceptions; though regarding its functions he says, " ueber den eigentlichen Zweck des Nabelblaschens schweben wir in ganzlicher Ungewissheit." It is interesting to note, however, that most of the early investigators ascribed nutritive and hgematogenous functions to it.
 +
 +
The following study is based mainly upon eighteen normal human umbilical vesicles in the collection of Dr. Mall, to whom I am greatly indebted for the unrestricted use of his extensive collection of human embryos, and for many helpful suggestions. Besides these normal specimens, a number of pathological ones, and some taken from placenta at birth, were examined. They were all stained in alum-carmine" and imbedded in paraffin. An endeavor was made to set the imbedded vesicle so that its long diameter, which usually lay in the same direction as the remnant of the umbilical stalk, was at right angles to the microtome knife. In all cases in which this was not possible, account was taken of the fact in the study of the sections.
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As the following table shows the size of the vesicles, not including those taken from placenta at birth, varies from one to six millimeters in embryos from 11 to 110 days old:
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TABLE OF EMBRYOS AND ATTACHED UMBILICAL VESICLES.
 +
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The numbered emhryos in the first colunui refer to the cabinet of Dr. Mall.
 +
 +
Length of Embryo Diam. of Vesicle Approx. Presence of
 +
 +
in millimetei'S. in millimeters. age in days. tubules.
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Embryos of the Second Week.
 +
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Peters 0.19 0.19 .. None
 +
 +
vonSpee 0.37 l.OSbyl.O 13 None
 +
 +
No. II 0.80 1.00 by 1.5 13 Several
 +
 +
Keibel 1-00 1.00 .. Many
 +
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V. Spee-Gfe 1..54 1.8 by 1.5 13 Many
 +
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Embryos of the Third Week.
 +
 +
No.12 3.1 1.5bylbyl 13 Several
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 +
Janosik 3.0 3.5 15 No mention
 +
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No. 76 4.5 3.0 19 Many
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 +
No.80 4.5 4.0 19 Some
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Embryos of the Fourth Week.
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No. 18 7 26 None
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No.2 7 7by4.5by4.5 26 Some
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Embryos of the Fifth Week.
 +
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No.187 9 6by4.5by4.5 30 Many
 +
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No. 163 9 5.2 by 3.7 30 Some
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No. 113 — 30 Many
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No.187 10 4 33 Macerated
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Embryos of the Fifth and Sixth Weeks.
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No. 175 13 3.7by3 36 Many
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No. 167 14.5 5.5 by 5.3 38 Many
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No. 5 18.5 4.7by4.5 43 Macerated
 +
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Embryo over Six Weeks.
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No. 33 20 5 by 3 by 3 43 Some
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No.l45» 33 4.5bv4by2.5 57 Few
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No. 176 38 4.8 by 3.6 by 3.5 61 None
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No. 184 50 5 by 4 by 3.9 70 None
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No. 171 60 6.5 by 4.3 77 None
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 +
No. X 110 4.5 by 4 110 None
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Artliiir AV. Meyer 157
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 +
As all these measurements were made after preservation in alcohol, shrinkage must be borne in mind, although it is of no practical importance since estimations of age were not based upon them.
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The vesicles are usually pyriform in shape, somewhat flattened in one diameter, and slightly roughened by protrusion and ridges below which blood islands and blood vessels usually lie. A few specimens are smooth, inflated, translucent sacs without any outward sign of blood islands or blood vessels. Others are collapsed irregularly folded and filled with calcareous-like material. There is never any regularity in the folding of the vesicle, however. Usually the folds were present while the vesicle still lay between amnion and chorion ; while in some cases they were produced during the hardening and imbedding. In several of the inflated vesicles the blood vessels are plainly visible throughout their entire length and can be seen entering the umbilical stalk.
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The umbilical stalk is present in the detached vesicles, as a short (5-15 mm.) stump only. It is thread-like, about .75 mm. in diameter, and never appears twisted. In cross sections the cavity of the vesicle can be traced up to the stalk, and after ending blindly a strand of characteristic entodermal cells can be traced for some distance towards the abdominal end; after which the lumen of the stalk reappears at varying intervals. This lumen, which never contains anything but a slight amount of amorphous material, is often completely occluded by the bounding entoderm.
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The stalk itself is composed of three layers in the greater part of its extent. On the exterior there is a thin layer of ccelomic epithelium (mesothelium) which continues indefinitely downward over the vesicle itself. In most vesicles it stops at the upper border, but in three specimens it forms a complete outer layer. The entodermal cells which bound the lumen have all the characteristics of those lining the vesicle itself, except for a slight decrease in size. Between these two layers mesoderm is found. Nearer the body of the embryo the latter usually predominates, while it is scarcely represented at all near the upper border of the vesicle.
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Besides these three layers the blood vessels form a conspicuous part of the umbilical stalk. They are not constant in number in various parts of the stalk. Sometimes three arteries and two veins are found, while in other cases one vein and two arteries are present. They can generally be distinguished by the character of their walls. The wall of the vein is formed by a single layer of very flat cells, while that of the arteries usually has an additional outer layer, composed of somewhat flattened
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158 The Structure of Human Umbilical Vesicle
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entodermal cells. This difference in structure, which is evident with the low power of the microscope, is found to disappear soon after the upper border of the vesicle is reached. In the structure of the walls of the blood vessels of the yolk sac itself there is never any difference as far as I am able to ascertain. The position of the vessels in both stalk and vesicle is usually well out towards the periphery, and in some cases only the ccelomic epithelium covers them.
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For the microscopic structure of the youngest umbilical vesicles reference to the literature is necessary. Peters, in his monograph, gives the size of both embryo and vesicle as 0.19 mm. Unfortunately, the preservation of the umbilical vesicle of Peter's ovum was not such as to prompt a detailed description of it. We are told, however, that it is composed of entoderm and mesoderm, and in the accompanying plate (Peters, Taf. Ill, Fig. 33) some contents containing globules and cells are represented. In this plate the lower half of the vesicle shows no clear demarcation between mesoderm and entoderm; while in the upper half a fairly clear line of division between the two is indicated. The character of the mesodermal and entodermal cells is not given in the monograph, except that the latter are spoken of as " unscheinbaren Entodermzellen." Blood islands and blood vessels are not represented.
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In an embryo of 0.37 mm. described by Graf Spee a marked advance in the structure of the umbilical vesicle exists. In this case the entoderm, which is one-layered, is composed of cubical cells, while the mesoderm is made up of irregular masses of cells with protrusions on the distal half of the vesicle, below which blood islands are found between entoderm and mesoderm. The' latter is thus pushed out while the entoderm in these places is said to be more wavy, its cells of greater variety and stained more intensely.
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The distal part of Graf Spee's embryo Gle (an embryo 1.54 mm. long) is said to be full of gaps — " ausserst liickenreich." Some of these gaps have an epithelial lining of flat cells of the nature of embryonic endothelium. Blood Anlagen are found in the wall of the vesicle only. In the proximal third of the latter the entoderm and mesoderm are thin and membranous, while in the distal two-thirds they are of varying thickness. The protrusions on the surface are said to be due to collections of cells between . entoderm and mesoderm. It is of interest to note in this connection that Keibel states that the umbilical vesicle of an embryo 1.0 mm. long, described by him, is in every particular like that of embryo Gle of Graf Spee.
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In the later article of Graf Spee, already referred to, he says that in embryos of three or four weeks the entoderm forms true glandular struc
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Arthur W. Meyer 259
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tures with small necks and large distal ends, which in embryos of nine weeks are branched and are found in all parts of the mesoderm
 +
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and fat oft '^'rW ' '^*^™^°^' P^^^^^^' ^^e origin and fate of these glandular structures, and to throw some light m^n
 +
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their possibe function. So far as I have been able to learn, Graf Spee
 +
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was the first to mention and to describe them, and no one else seems to
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have suggested any explanation of their presence. These glandular
 +
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structures which for the sake of brevity I shall call tubules, are present
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m the walls of nearly all vesicles taken from embryos less than two
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months old m the collection of Dr. Mall. The vesicles of A^s. n and
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U of this collection, embryos 0.8 mm. and 2.1 mm. respectively, are
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Identical in structure. Both are in a state of good preservation, and
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their structure m cross section as represented in Fig. 1.
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bloodvessel
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FIG. ]. Umbilical „siole ot ,„ embrjo 2.1 mm. long ,N„. 12,. x 35,
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narrow'll,','f '" " 'T/T"' *" ^"^P'^' »^"°'J'-^' ^bules with
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1 / , f , - " '" **'" mesoderm close to the entoderm. These socalled glandular stn^ctnres do not branch and can be traced throng from
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fo.fn;r;h:tsLL':';tr;Tot '""^^ ^'^'"^ °' '-- *"^-- '^
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Here thf. ,„„„1 f * , ' ' ™'"'^° ™ millimeters long.
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Here the number of tubules is considerably greater and a direct connec.on be ween many of them and the entoderm exists (Fig. 2). In many a es the tubules end as evaginations of the entoderm "and arc thr^n direct communication with the cavity of the vesicle. Others are "nd" r^ly connected with the entoderm by bands of entoderma Icel ^ht s 111 others he isolated m the mesoderm. As showu in Fig. 3 all transi ions are found from a slight evagination of the entoderm t los d ubules lj.ng detached from the entoderm in the mesoderm. Altl gh they can be traced through a series of fifteen to twenty-five sections Z are never seen to branch. On the other hand the branching described by
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160
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The Structure of Human Umbilical Vesicle
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Graf Spee is well seen in a vesicle taken from an embryo thirteen millimeters long. In such a vesicle (Fig. 4) we find an almost complete canalization of the mesoderm while the entoderm is but little changed. The
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■ 7— mesoderm
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-?^^^lubule
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Fig. 2. Umbilical vesicle of an embryo 7 mm. long (No. 2). X 35.
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tubules are much larger and longer and are formed by a layer of flat cells which often approach the cubical type. Contact of tubules is common but definite branching is infrequent. The lumina are wide and contain
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Fig. 3. Tubules from the vesicles of an embryo 7 mm. long (No. 2). X 35 ; (a) Simple evagination of entoderm — first stage; (5) Same, second stage; (c) Isolated tubule.
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confused masses of amorphous material similar to that found in the cavity of many of the younger vesicles. They never seem to open directly into the cavity of the vesicle, although often the entoderm only separates their lumina from it. They are of many sizes, shapes and lengths, and lie irregularly distributed in the mesoderm. When not in contact they
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Arthur W. Meyer
 +
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161
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often have irregiihir masses of entoderm between them or are separated by mesoderm. Their abundance gives a striking appearance to sections of the vesicle which is well expressed by Graf Spee as " ausserst liickenreich." It is worthy of note that the lumina of the tubules have greatly increased in diameter while the thickness of the bounding endotheliimi has, absolutely as well as relatively, decreased. In many cases the shape of the individual cells also has changed from cubical or pyramidal to a membranous-like layer of greatly flattened cells.
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In older vesicles these tubules occur but rarely. This is usually the case in vesicles of the ninth and tenth weeks, although one vesicle taken
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Fig. 4. Umbilical vesicle from an embryo 13 mm. long (No. 175). X 2.5.
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from a normal embryo of the fifth week has already reached the stage of those three or four weeks older. Generally these older vesicles have a very different structure than those of four or five weeks and contain masses of calcareous matter.
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It seems then that these tubules make their appearance during the second week, reach their greatest development by the fourth or fifth week and then gradually disappear by the eighth or ninth week. These stages are well represented in embryos Nos. 11 and 12; 113 and 175; and 145, 176 and 184 respectively. This conclusion is at variance with the observation of Graf Spee on embryo Gle, but as the widest variations as to the presence, structure and size of these tubules exist the contradiction does not seem surprising. As a rule the only constant characteristic was their direction. This was almost invariably in the direction of the
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162 The Structure of Human Umbilical Vesicle
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long diameter of the vesicle, for only occasionally was a tubule cut at other than a slight angle to its long diameter. Even when such was the case it could generally be accounted for by the fact that the plane of the microtome knife was not at right angles to the long diameter of the vesicle.
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In spite of the large amount of material at my disposal, I am unable to reach any satisfactory conclusion as to the meaning of these tubules. Their presence is not at all a constant one. Vesicles of the same age and size often present wide divergencies of structure which are hard to reconcile. I feel justified, however, in suggesting an explanation of the manner of formation, which an examination of the material at my disposal will, I think, corroborate. Two methods of formation can be distinguished: (1) evagination of the entoderm and (2) development from irregular extensions of entoderm into the mesoderm. That the first step in the formations of many tubules is a slight evagination of the entoderm, as Graf Spee has stated, is very evident. I have found all transitions between such a stage and perfect tubules lying isolated in the mesoderm. This isolation can be readily brought about by a gradual deepening of the original evaginations accompanied by constriction and consequent fusion. This process seems to be further indicated by the occurrence of tubules which communicate with the cavity of the vesicle by their ends only, while others are closed at both ends and lie isolated in the mesoderm close to the entoderm. It seems highly probable to me that an active proliferation of the mesoderm might play a part in this separation of the tubules and their further removal to the periphery of the mesoderm.
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Even if correct, however, this explanation cannot account for those tubules in whose lumina masses of unmistakable mesoderm are found. This is the case in No. 33, an embryo twenty millimeters long. In this specimen there are striking evidences of the formation of tubules by proliferation from irregular extensions of entodermal cells. Such inclusions of mesoderm might evidently result from tubule formation by invagination of the entoderm, but it is hard to find any satisfactory evidences of such a process of inclusion. That another method than that of evagination of the entoderm must have been followed, however, in the case of No. 32, is clearly indicated not only by the masses of mesoderm contained in the tubules, but especially by the fact that strands of mesoderm are found in various stages of inclusion by the entoderm. That an active proliferation of the entoderm into the mesoderm does occur, is further indicated by those specimens in which almost
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Arthur W. Meyer 163
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the entire wall of the vesicle is composed of entoderm (Fig. 5), for in young specimens the entoderm is composed of a single well-defined layer of cells (Fig. 1).
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In embryos of the seventh to tenth week the entoderm and sometimes the tubules can be found in various degrees of degeneration. This is true of Nos. 145, 176 and IS-t, embryos of 33, 38 and 50 mm. long, respectively. As the mesoderm is generally increased in thickness in these specimens it seems as though the degeneration of the entoderm is accompanied by a proliferation of the mesoderm. The latter at this time takes on the characteristics of a streaked fibrous connective tissue, and becomes compacted. The degeneration of the entoderm is apparent not only in the inner layer which lines the cavity of the vesicle, but is seen especially well in the groups of entodermal cells which lie scattered throughout the mesoderm. In many cases the entodermal cells are represented by granular detritns without any remnants of nuclei, while in
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3 — mesoderm ^bloodvessel
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Fig. 5. Umbilical vesicle from an embryo 7 mm. long (No. 18). X 25.
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other cases the cell outlines are faintly seen, and the nuclei are well preserved. Large amounts of cellular detritus can be found in the cavity of such a vesicle, and it does not seem unlikely that the cell remnants found among the calcareous contents of vesicles taken from placentae at birth have this origin. This cellular detritus is especially well seen in Nos. 187 and 176, the cavities of which vesicles are almost completely filled with granular debris containing many large cells having the characteristics of entodermal cells. In older vesicles, those from embryos of sixty and one hundred millimeters, for example, we find, on the contrary, a condition almost identical with that found in full-term vesicles, except that the walls of the latter are more compacted and look still more as though composed of mature fibrous connective tissue.
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The signs of degeneration in these older vesicles are not limited to the entoderm, however, for many of the blood vessels show marked degeneration of their walls and of the nucleated red blood cells contained within. The vessels are often pigmented and without a proper lining. The pigment reminds one strongly of blood pigment and looks very much indeed like hsematoidin. The entire absence of vessels in the old
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164 The Structure of Human Umbilical Vesicle
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vesicles and their extreme vascularity in the early stages alone seem sufficient to indicate a gradual degeneration.
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 +
The walls of these vesicles, as already stated, vary greatly in thickness and in the character of the cells composing them (Figs. 2, -i, 5). Usually the greatest thickness is found at the distal end. Both entoderm and mesoderm are present in all vesicles except that of No. 187, below eight weeks of age. In these specimens the ccelomic epithelium in addition extends over the entire surface of the vesicle. This envelope is invariably composed of a single layer of very much flattened cells with elongated nuclei.
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The mesoderm also presents great variations in thickness, though not in the character of its cells. These cells, though cuboidal or cylindrical in a few instances, not infrequently look like embryonic connective-tissue cells in the young vesicles, while in those of ten weeks and older it has the characteristics of fibrous connective tissue, as already noted. In these specimens it is denser, and stained more deeply near the cavity of the vesicle. The tubules and blood vessels invariably lie in the mesoderm, but are frequently surrounded by extensions or by groups of entodermal cells. In younger vesicles the blood vessels and blood islands usually cause an elevation of the mesoderm above the points where they lie.
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 +
The entoderm is composed of a single layer of cuboidal, pyramidal, and exceptionally in a small area, of cylindrical cells in vesicles of two to four weeks, but is absent in those over seven weeks of age. In a few specimens no distinct demarcation between entoderm and mesoderm can be found, though usually they are clearly defined in all the younger vesicles (Fig. 1).
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A series of six imibilical vesicles taken from placentae at birth were found almost identical in structure with tlxe vesicles of JSTos. 184, 171 and X. The walls of these vesicles are composed of a dense, wavy layer of fibrous connective tissue of varying thickness, which blends more or less with amnion and chorion. The cavity contains an irregular mass of calcareous matter among which cell remnants are plainly visible. Even those vesicles which are inflated sacs contain a small amount of calcareous matter, while those which are compressed and irregularly folded contain a firm mass of calcareous substance, which completely fills the cavity of the vesicle. Eemnants of the early blood vessels or of tubules are never found nor can any recognizable remnants of the entoderm be detected. Unless, as previously suggested, the cells lying among the calcareous matter have this origin. The striking similarity between the structure
 +
 +
 +
 +
Arthur W. Meyer 1G5
 +
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»
 +
 +
of these vesicles and those from embryos of the third month plainly shows that the condition of the vesicle as found at birth is reached early in the life of the embryo.
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 +
The occasional large size of the umbilical vesicle in full-term placentae which contained a normal foetus, is very remarkal)le. I have seen vesicles that measure fifteen by ten millimeters. Such occurrences are hard to reconcile with the supposition that the umbilical vesicle reaches it? greatest development in the fourth week. oSTor is it easy to see how mechanical forces can produce these large inflated vesicles. The only suggestion that occurs to me without further study of full-term vesicles, is that hypertrophy takes place at the time when the transformation of the wall of the original vesicle into fibrous connective tissue occurs.
 +
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BIBLIOGRAPHY.
 +
 +
VON Baer. — Entwickelungsgeschichte der Thiere. Konigsberg, 1837. BiscHOFF. — Entwickelungsgeschichte des Menschen iind der Saugethiere, Bd.
 +
 +
VII, Leipzig, 1842. Chiariuge. — Archives Italiennes de Biologie, Vol. XII, 1889. Haller. — Grundriss der Physiologic. Berlin, 1788. His. — Anatomic Menschlicher Embryonen, Bd. I, II. Hunter. — Anatomia uteri humani gravidi tabulis illustrata. Birmingham,
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 +
1774.
 +
 +
On anatomical description of the human gravid uterus and its con tents. London, 1794. Van Heukelom. — Archiv f. Anatomic und Physiologic, 1898. Janosik. — Archiv f. Mikroskopische Anatomic, Vol. XXX, 1887. Keibel. — Archiv f. Anatomie und Physiologie, 1890. KiESER, D. G.— Der Ursprung des Darmkanals und der Vesicula umbilicalis
 +
 +
dargestellt im Menschlichen Embryo. Gottingen, 1810. KoLLiKER.-^Grundriss der Entwickelungsgeschichte, Leipzig, 1880. KoLLMANN. — Entwickelungsgeschichte des Menschen, 1898.
 +
 +
Archiv f. Anatomie und Physiologie, 1879.
 +
 +
Mall.— Journal of Morphology, Vol. XII, 1897. New York, 1902.
 +
 +
The Johns Hopkins Hospital Reports, Vol. IX, 1900.
 +
 +
Journal of Morphology, Vol. V, 1891.
 +
 +
Meckel. — Beitrage zur vergleichende Anatomie, Bd. I, Heft I, 1808. Mayer.— Acta Leopoldina, Vol. XVII, 1834. MiNOT. — Embryology, New York, 1892.
 +
 +
Laboratory text-book of embryology, 1903.
 +
 +
Muller. — Archiv f. Anatomie, 1834.
 +
 +
Peters. — Die Einbettung des Menschlichen Eies, 1899.
 +
 +
ScHULTZE. — Das Nabelblaschen ein Constantes Gebilde in der Nachgeburt des Ausgetragenen Kindes, Leipzig, 1861.
 +
 +
ScnuLTZE. 0. — Grundriss der Entwickelungsgeschichte des Menschen, Leipzig, 1897.
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166 The Structure of Human Umbilical Vesicle
 +
 +
VON Spee. — Archiv f. Anatomie und Physiologie, 1889, 1896.
 +
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Anatomischer Anzeiger, Vol. XII, 1896.
 +
 +
Miinch. Med. Wochenschrift, No. 33, 1896.
 +
 +
ToiJRNEUx, F.— C. R. Soc. Biol., Paris, Ser. 9, T. I., 1899. Velpeau. — Embryologie et Ovologie Humaine, 1833.
 +
 +
Annales des Sciences Natural, 1827.
 +
 +
Williams. — Obstetrics, New York, 1902.
 +
 +
 +
 +
THE EMBRYONIC DEVELOPMENT OF THE INTERSTITIAL CELLS OF LEYDIG.
 +
 +
BY R. H. WHITEHEAD, M. D.,
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Professor of Anatomy, Medical Department, University of North Carolina. From the Hull Laboratory of Anatomy, University of Chicago.
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With 10 Text Figures.
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The interstitial cells of Leydig furnish such a striking feature in the testis of mammalian embryos that one is surprised to find that their development has received very little study. Doubtless this is due to the fact that embryologists in their investigations of the development of the testis have had their attention focused upon the much more important subject of spermatogenesis.
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These cells have been known for a long time. Leydig discovered them in 1850, and stated that they were a constant constituent of the mammalian testis. He regarded them as connective-tissue cells, and classed them with fat and pigment cells. This view was adopted by Koelliker in 1854. Boll, 6g, observed an intimate relation between them and the blood vessels, and believed that Leydig's cells composed the walls of capillaries. Von Ebner, 71, studied them in several mammals, and concluded that they were " a peculiar form of connective tissue." F. Hofmeister, 72, seems to have been the first to approach the problem of the nature of these cells by a study of embryonic material. Examining the testis of human embryos at four and seven months, he found that Leydig's cells constituted about two-thirds of the bulk of the gland in the embryo of four months, and only about one-tenth in that of a boy about eight years old; at puberty they were greatly increased in number, and contained much fat and pigment. He too regarded the interstitial cells as connective tissue, and thought that he could detect transition forms between them and the fixed connective-tissue cells. Waldeyer, 74, classed them with his plasma cells, but later regarded them as perithelial. Harvey, 75, noticed, as others had done, their resemblance to nerve cells, and advanced the view that they were derived from the sympathetic nervous system. This view, however, has been discredited by all writers on the subject.
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American Journal of Anatomy. — Vol. III.
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168 The Development of the Interstitial Cells of Leydig
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Hansemann ' regards it as certain that Leydig's interstitial suhstance belongs to the connective-tissne gronp, because he believed he could demonstrate an intracellular substance with Van Gieson's stain. He made the interesting observation that in the hibernating marmot no Lej^dig's cells were present, and that evidences of spermatogenesis were also lacking during that period: whereas in the waking animal the cells were present in such large numbers as to produce a picture resembling large-celled sarcoma. These observations, together with those of others upon fat and pigment contained in the cells, lead him to conclude that Leydig's cells constitute a distinct organ.
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In 1896, Fr. Eeinke ' made the discovery of crystalloids in Leydig's cells. He found that these bodies were absent before puberty, present in large number during active sexual life, and again absent in old age.
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Von Bardeleben,^ from whose article the references to the earlier literature were taken, studied Leydig's cells in the teste's of criminals, the organs being removed immediately after execution. He was impressed by the epithelial appearance of the cells, and noted that the cell-margins were not smooth, but rather serrated — the expression of intercellular bridges connecting adjacent cells. He found no intercellular substance, properly speaking, and no mitotic figures, though frequently he saw evidences of direct division. He thinks that Leydig's cells are almost identical in appearance with the Sertoli cells of the seminal tubules, and believes that they are in fact youthful forms of Sertoli cells. They are capable, he says, of passing through the walls of the tubules, there to become Sertoli cells and take the place of such as are worn out in the performance of their function. In the last analysis, according to him, Leydig's cells are epithelial in nature, and are derived from the germinal epithelium.
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J. Plato * describes minute canals in the walls of the seminal tubules through which, he thinks, fat and pigment from the interstitial cells stream into the Sertoli cells, to be used as pabulum in spermatogenesis. To support this hypothesis he undertook a study of the development of
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Ueber die grossen Zwischenzellen des Hodens. Arch. f. Anat. u. Physiol.,
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Leipzig, 1895, Physiol. Abth., p. 176.
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^ Beitraege zur Histologie des Menschen. Arch. f. mikr. Anat., Bonn, 1896, Bd. XLVII, p. 34.
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' Beitraege zur Histologie des Hodens und zur Spermatogenese beim Menschen. Arch. f. Anat. u. Physiol., Anat. Abth., Supplement-Band, Leipzig, 1897, p. 193.
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Die interstitiellen Zellen des Hodens und ihre physiologische Bedeutung.
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Arch. f. mikr. Anat., Bonn, 1897, Bd. XLVIII, p. 280.
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E. H. Whitehead 169
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Leydig's cells in cat embryos/ using unstained sections of material fixed in Hermann's fluid. He begins his observations with the embryo of seven weeks, which, we may note, is quite a late stage. Here he finds Leydig's cells in all stages of transition to fixed connective-tissue cells, the transition 23roceeding from the neighborhood of the blood vessels towards the seminal tubules. He could find but one Leydig's cell containing a mitotic figure. Fat is present only in minute droplets. " In the embryo at term the Leydig's cells are in close apposition with the walls of the tubules, and their nuclei are eccentric in position; drops of fat are present in the portion of the cell-body which lies opposite the nucleus. The subalbugineal layer of Leydig's cells is quite thick. In the newborn cat the subalbugineal layer of cells has almost vanished, owing to the increase in length of the tubules. Fat is wanting in m^py of the cells, which present, therefore, a spongy appearance. He concludes that Leydig's cells are developed from the connective tissue which accompanies the blood vessels of the testis, somewhat after the manner of typical fat cells, and regards them as trophic nurse-cells ("trophische Huelfzellen "), whose function is to pass their specific inclusions into the seminal tubules.
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M. V. Lenhossek ^ confirms, in the main, the observations of Eeinke as to the crystalloids. He is inclined to regard the interstitial cells as epithelial. He thinks that the presence of crystalloids in them and the absence of connective-tissue cells elsewhere in the body similar to them are decided evidence against the opinion which classes them with the connective tissues. He advances the theory that they are unused remains of the germinal epithelium, and that their function is to store up pabulum, which they give over on demand to the seminal tubules.
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H. Beissner,' in an article intended mainly as a refutation of the opinions of Plato, calls attention to the work of M. iSTussbaum in 1880. The latter held that the nests and strands of Leydig's cells were invested by a membrane similar to the wall of the seminal tubules, so that one might compare them with the Pflueger's tubules of the ovary. He suggested that they were groups of germinal epithelium which had stopped developing at an early stage — a suggestion somewhat like that of v. Lenhossek.
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^ Zur Kenntniss der Anatomie und Physiologie der Geschlechtsorgane. Arch. f. mikr. Anat., Bonn, 1897, Bd. I, p. 640.
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^ Beitraege zur Kenntniss der Zwischenzellen des Hodens. Arch, f . Anat. u. Physiol., Leipzig, 1897, Anat. Abth., p. 65.
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^ Die Zwischenzellen des Hodens und ihre Bedeutung. Arch, f . mikr. Anat., Bonn, 1898, Bd. LI, p. 794. 13
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170 The Development of the Interstitial Cells of Leydig
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Among recent text-books of histology, Boehm and Davidoff state that Leydig's cells " are probably remains of the Wolffian body " ; Szymonowicz says that we mnst assume that they are connective tissue.
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Thus it appears that there are two principal views as to the histological nature of Leydig's cells. According to the one, they belong to the connective tissues (Leydig, Koelliker, v. Ebner, Hofmeister, Hansemann, Plato) ; according to the other, they are epithelial cells derived from the germinal epithelium (Nussbaum, v. Bardeleben, v. Lenhossek). It also appears that these views are, in the main, deductions from the study of adult conditions. It is worthy of note that the two investigators who have made a special study of the subject in mammalian embryos, Hofmeister and Plato, both conclude that Leydig's cells are derived from the interstitial tissue of the primitive testis. Their investigations, however, are incomplete, in that they were not made upon a series of embryos extending into the early stages, but upon a few isolated examples in the later stages of development.
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As my work upon this subject was nearing its completion, there appeared a preliminary account of a study of the embryology of the ovary and testis by Bennet M. Allen,' carried out upon pig and rabbit embryos, in which the following statements are made concerning the interstitial cells : " The connective-tissue elements of ovary and testis are derived from the peritoneum. In early stages they are not distinguishable from the cells which make up the sex-cords, except that the latter are marked off from the stroma by their membrana propria . . . the albuginea is largely formed by the actual transformation of the basal parts of the sex-cords into connective-tissue elements. The interstitial cells are characterized by a large nucleus, distinct cell-boundaries, a centrosome and centrosphere, and very granular cytoplasm. They first appear in the stroma of both ovary and testis of the pig of 2.5 cm. length. They are far more numerous in the testis than in the ovary. Their appearance is coincident with that of a large number of fatty globules in the peritoneum and sex-cords. In the testis they persist for a long time. . . . In both organs they divide by mitosis. This process soon ceases in the ovary, while in the testis, on the other hand, division figures are found in the interstitial cells at a stage as late as the 7.5 cm. embryo. In the testis of the 15 cm. embryo they (the interstitial cells) have begun to degenerate. This process manifests itself in a shrinkage of the cyto " The Embryonic Development of the Ovary and Testis of the Mammalia. Biological Bulletin of the Marine Biological Laboratory, Woods Holl, Mass., Vol. V, No. 1.
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E. H. Whitehead 171
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plasm." " In the ovary of the 85 day rabbit they are very common, their origin from the theca interna of atretic follicles being clearly shown. This, taken in connection with the additional fact that they make their appearance in the 3.5 cm. pig embryo coincident with the fatty degeneration of the germinative cells of the seminiferous tubules and their ovarian homologues, together with that of many cells of the germinal epithelium, would lead us to conclude that cell-degeneration offers the stimulus or condition that brings about the formation of the interstitial cells."
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The observations about to be described were made upon pig embryos. The material was fixed principally with Zenker's fluid, and stained with haematoxylin and Congo-red, iron-hgematoxylin and Congo-red, and by Mallory's method for connective tissue. A series was fixed in Flemming's fluid, and studied either stained with iron-hsematoxylin or unstained. Another series was used for frozen sections and staining with Sudan III. Also a few other methods were employed for special purposes.
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It should be remarked at the outset that the theory that Leydig's cells are derived from the epithelium of the Wolffian body cannot obtain in the pig; for in this animal Leydig's cells appear before connection has been made between the epithelial constituents of the testis and Wolffian body. Furthermore, in the case of the pig, at least, the tubules of the rete testis grow into the Wolffian body and establish connection with the Bowman's capsules of the glomeruli, and not vice versa. In this connection see also J. B. MacCallum : Notes on the Wolffian Body of Higher Mammals; Amer. Jour. Anat., Bait., Vol. I, No. 3, p. 245; and the article of Allen previously referred to.
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As I find myself in accord with the conclusion of Allen, that the interstitial tissue of the testis is derived from the peritoneum, meaning thereby the mesothelium of the genital ridge, I may omit the account of my study of the earlier stages, and proceed to the description of the intertubular tissue of the testis in the pig of 23 or 33 mm., a stage immediately preceding the appearance of Leydig's cells.
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In the pig of this length the testis may readily be identified, as the rudiments of the tunica albuginea and the mediastinum are fairly distinct, the primitive seminal tubules are well defined, and their basement membrane is formed. The intertubular spaces in the more central portions of the gland are, on the whole, larger than those near the periphery. In the latter situation they consist mainly of capillaries derived from vessels of the albuginea, whereas in the former case they are as wide as, or even wider, than the tubules, owing to the presence in considerable
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173 The Development of the Interstitial Cells of Leydig
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quantity of a loose cellular tissue. The constitution of this tissue is shown very well by Mallory's stain. In sections thus stained (Fig. 1) it is seen to be composed of a mixture of cells and fibrils. The cells often have little or no cytoplasm, some appearing to be mere naked nuclei; but others show a collection of cj'toplasm at one pole. The nuclei are spherical or ovoid, except when closely packed together, in which case they incline to the spindle-shape. They contain much nuclear sap in which is a network of chromatin; and usually there is a
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Pig. 1. Pig 22 mm. Shows the structure of the intertubular tissue. Mallory's connective-tissue stain. X 800.
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quite distinct nl^cleolus. Mitotic figures are present here and there. The cells or nuclei are imbedded in a network of fibrils which take the aniline-blue of the stain. It seems clear that this tissue is a young connective-tissue syncytium in the sense of Mali/ In all essentials it is quite similar in structure to the deeper layers of the albuginea, with which it is continuous, and to the mesenchyme in general.
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Leydig's cells were first definitely encountered in embryos 24 mm. long. In the sections they appear scattered about in the intertubular
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® On the Development of the Connective Tissues from the Connective-Tissue Syncytium. Amer. Jour. Anat., Bait., 1902, Vol. I, No. 3.
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E. H. Whitehead
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173
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spaces, sometimes singly, sometimes in small groups, without any very regular order as regards the other constituents of the testis, except that they are most numerous in the more central intertubular spaces; at this time there are very few, or none at all, immediately under the albuginea. Some of them show mitotic figures. They frequently arrange themselves along the basement membrane of the tubules. In size and shape they vary greatly (Fig. 2); some are spindle-shaped with the nucleus near the center of the spindle; some are oval with the nucleus in the larger end, while at the opposite end the cytoplasm tapers to a process; some are irregularly oval or spindle-shaped, while others are polygonal with eccentric nuclei. The difference -in size is due principally to varying amounts of cytoplasm. Their nuclei are quite similar to those of the cells which compose the intertubular tissue of the pig of 22 mm. and stil compose the larger part of it in the pig of 24 mm. The nuclei of the Leydig's cells are perhaps larger and more spherical, and may stain more deeply, but in general they are indistinguishable from those of the other cells in the intertubular spaces. The cytoplasm is very granular, and stains well with acid dyes, so that the cells stand out very distinctly. They are markedly branched. In sections stained with hsematoxylin and eosin the cell-margins may appear quite smooth; if Congo-red be employed as the cytoplasmic stain, some notion of the branching may be obtained, but Mallory's method for connective tissue shows the branches best (Fig. 2). The branches vary much as to size. It is difficult, frequently, to determine whether they merely interlace with one another or are in actual continuity; in some places, however, the latter relation seems clear enough to justify the conclusion that, at first, Leydig's cells form a syncytium. Figures two and three are taken from rather marked examples of this condition. In addition to thus forming syncytium, some of the processes seem to be continuous with the exoplasmic network of the fixed connective-tissue cells. Thus practically the only difference between the young " interstitial substance " of Leydig and the interLiibular tissue of the preceding stage is the greater amount of cytoplasm possessed by the former; even the syncytial arrangement is retained for rt short time. Hence the conclusion is drawn that Leydig's cells are derived from the cells of the intertubular tissue, which, as we have seen.
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Fig. 2. Pi<7 2-J mm. A group of young Leydig's cells. Mallory's connective-tissue stain.
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X 800.
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174 The Development of the Interstitial Cells of Leydig
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is a mesench}anal structure differing in no essential from the mesenchyme in general.
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During the next succeeding stages a number of interesting changes may be noted. The Leydig's cells undergo rapid increase both in number and size, so that they soon come to be the predominating constituent of the intertubular spaces. The fixed connective-tissue nuclei, on the other hand, become smaller and relatively much less numerous. The increase in the number of the Leydig's cells is due, in large measure, to karyokinesis, as mitoses are fairly abundant; but doubtless it is also due, in part, to the continued conversion of mesenchyme cells into Leydig's cells. These cells now begin to assume a fairly typical form; the majority of them are polygonal, and the nucleus, spherical in shape and eccentric in position, contains much chromatin and a large nucleolus. Various other shapes, however, are observed which seem to be due to mechanical conditions. Occasionally they are arranged alongside the tubules, so that the latter in cross section appear surrounded by a sheath
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of Leydig's cells outside of the basement membrane. Soon after they are first seen in the more central intertubular spaces they begin to make their appearance under the albuginea, where they rapidly increase, particularly large masses being found along the points of attachment of the septa. During this time also the branches begin to disappear, and soon there is no evidence of a syncytial arrangement. This change seems to occur last in the subalbugineal cells; quite a marked branching can sometimes l)e made out in this situation in even as late a stage as the embryo of 3.5 cm. At the stage of 3.5 cm. (Fig. 3) the Leydig's cells present the greatest size to which they attain in the early embryo, and are very striking objects in preparations made by Mallory's method, which can be used so as to give a fair differential stain. They are very granular, and the
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Fig. 3. Pig 3.5 cm. A group of Leydig's cells from just beneath the albuginea. A delicate reticulum is forming-. C.B., centrosphere B.. ; Mallory's connective-tissue stain. X 800 .
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R. H. Whitehead
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175
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cytoplasmic network is much looser at the periphery of the cell than it is around the nucleus; the meshes of the net seem to have been distended, and the coarse granules are very apparent at the nodal points. As will be seen later, the same appearance, but in a much exaggerated degree, is found in the last stages of the embryonic development of these cells. During this period also the fixed connective-tissue cells begin to build a delicate reticulum (Fig. 3).
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Following the stage shown in the embryo of 3.5 cm. there is a progressive decrease in the size of the Leydig's cells, the process affecting
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Fig. 4. Pig 5.5 cm. A group of tubules and an intertubular space. A quite perfect reticulum for the Leydig's cells has been formed. C.B., centrosphere B. ; Mallory's connective-tissue stain. X 800.
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both the cell-body and the nucleus, though the change is more marked in the former (Figs. 4 and 5). There is much condensation of the cytoplasmic network, together with actual disappearance of cytoplasm. This process reaches its acme in the pig of 14 cm (Fig. 5), where many of the cells are reduced to their primitive condition of almost naked nuclei. This change was noted by Allen (loc. cit,), but the term " degeneration " employed by him scarcely seems appropriate ; atrophy would doubtless be a more appropriate term. This atrophy, we shall see, is merely temporary. Very few intertubular spaces can be found which are as wide and contain as many and as large Leydig's cells as the one represented in the figure; they are very scanty also beneath the
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176 The Development of the Interstitial Cells of Leydig
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albuginea. Between the tubnli recti, on the other hand, in which sitnation the intertnbnlar spaces are much wider, they are larger and fairly nnmerous. A possible explanation of the atrophy of Leydig's cells is
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Fig. 5. Pig Ik cm. The tubules are much larger, the spaces and the Leydig's cells much smaller than in preceding stages. Mallory's connective-tissue stain. X 800.
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suggested by a study of the growth of the seminal tubules. During the time the Leydig's cells are atrophying the tubules are growing rapidly,
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especially in length, and become markedly convoluted, thus reducing the width of the intertubular spaces,- especially of those situated beneath the albuginea (Fig. fi). This, taken in connection with the fact that the cells of the subalbugineal region
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