MARINE BIOLOGICAL LABORATORY. Received Aucu St J 19 3.5 .. Accession No. '^5282 Given by Lea ('k.-Febiser Place Philadelphia ***flo book OP pacnphklet is to be vemoved fpom tbe Liab- Ofatopy taitbout tbe pePmlsslon of tbe Tpastees. ■y THE MICROSCOPIC ANATOMY OF VERTEBRATES BY GEORGE G. SCOTT, Ph.D. PROFESSOR OF BIOLOGY IN THE CITY COLLEGE, NEW YORK CITY AND JAMES I. KENDALL, Ph.D. INSTRUCTOR IX BIOLOGY IN THE CITY COLLEGE, NEW YORK CITY ILLUSTRATED WITH 167 ENGRAVINGS '^^ LEA & FEBIGER PHILADELPHIA 1935 Copyright LEA & FEBIGER 1935 PRINTED IN U. S. A. PEEFACE. Courses dealing with the anatomy of vertebrate animals form an important part of the curriculum of the college program of studies. Work in anatomy is almost wholly occupied with the gross anatomy revealed by dissection of representative vertebrates. Associated with the course in anatomy is work in embryology in which the emphasis is usually placed upon development in the chick and pig. In addition to embryology and gross anatomy, the student is well repaid by continuing his anatomical studies and investigating the tissue organization of the various systems. Although college textbooks in comparative anatomy and embryol- ogy of vertebrates are available, most textbooks in histology and microscopic anatomy have been prepared for medical students and the emphasis has been placed upon human material. In the present book, material has been taken from a variety of vertebrates so that it may serve as a survey of the microscopic anatomy of the verte- brates. It is a college textbook, the purpose of which is to present a working knowledge of vertebrate histology and microscopic anatomy to be covered in a semester period. It is not intended as a com- pendium of information on all aspects of so wide a field, and one so rich in interest for investigation. Therefore, the book has been kept primarily on a descriptive basis to accompany laboratory work. The possibility of wide ^•ariation and exceptions in different species make it unavoidable that students will find discrepancies between certain general descriptions and the specific example they may be studying. Since this is true, it is hoped that the student will be interested in discovering variations and will not be content with mere verification of the text. Believing that it is well for the student to acquire the habit of consulting original papers, the authors have appended a few selected references to each chapter. This list of references is in no way complete, but has been selected from the more readily available publications to serve as a starting- point. 4 PREFACE The degree of magnification of the drawings and photographs has been dehberately omitted. The statement to the effect that a certain ilhistration is 600 or 1000 times the actnal size conveys very Httle helpful information. The student will be constantly using the microscope and will have little difficulty in appraising the degree of magnification. A simple standard of measurement present in many preparations is the diameter of the erythrocyte and by it the size of other structures can be approximated when necessary. We have found it advisable to spend the first few weeks in the laboratory in a first-hand study of the varieties of the different tissue groups. The student is then given sections of organs and taught to recognize the different tissues represented in them. Then the tissue composition of organs is formally taken up. In a short time, the student is quite independent of assistance from the instructor, and this is as it should be. Each student should have for constant use during the semester a well-selected set of slides loaned to him at the beginning of the term and returnable at the end of the course. There should be available a number of reference books and a complete set of lantern slides of drawings and photomicrographs of preparations for use during the laboratory sessions. Projection of microscopic prepara- tions is a useful device for recitation purposes. Drawings are sug- gested, not as artistic representations but as pictorial descriptions of what the student actually sees. Unknown preparations are used frequently to test the student's knowledge of tissues and organs and his ability to apply it. At every step, the student is con- fronted with new problems requiring observation and the exercise of judgment in making decisions in identifying tissues or organs. Such mental exercise is valuable training. Part of the time the student will spend in learning to make preparations of the various organs studied, employing the more general methods found by experience to be most satisfactory. Mastery of technique is a worthwhile educational objective. There should be available for constant reference one or more of the text- books listed under the chapter dealing with technique, so that the student may extend his knowledge of methods as time permits. The authors hope to have the privilege of being made aware of the criticisms and suggestions which will undoubtedly arise in the minds of those who use the book. New York City. (^t. G. S. J. I. K. CONTENTS. CHAPTER I. Introduction. The Cells 14 Protoplasm 15 Cytosome 15 Nucelus 17 Cell Reproduction 18 Mitosis 18 Prophase 19 Metaphase 19 Anaphase 19 Telophase 19 Amitosis 20 Cell Metabolism 20 Tissue Culture 21 Embryology 22 Amphioxus 22 Amphibia 23 Sauropsida 24 Mammalia 25 Organogeny 26 CHAPTER II. The Epithelial Tissues, Simple Epithelia Simple Squamous Epithelium Simple Cuboidal Epithelium Simple Columnar Epithelium Stratified Epithelia .... Stratified Squamous Epithelium Stratified Cuboidal Epithelium Stratified Columnar Epithelium Transitional Epithelium Pigmentation of Epithelial Cells Surface Modifications of Epithelial Cells Growth and Regeneration of Epithelia Secretory Epithelial Cells and Glandular Organizations Exocrine Glands Unicellular Glands Secreting Areas Glandular Pockets Simple Tubular Glands Simple Alveolar Glands Compound Tubular Glands Compound Alveolar Glands Serous and Mucous Glands Endocrine Glands .... 27 30 31 32 34 34 35 35 36 37 37 39 40 41 41 42 42 42 43 43 45 45 45 45282 CONTENTS CHAPTER III. The Connective Tissues. Mesenchyme 47 Mucous Tissue 48 Reticular Connective Tissue 49 Loosely Organized Fibroelastic Tissue 50 Cell Types 50 Fibrocytes 50 Histocytes 51 Ameboid Wandering Cells .51 Mast Cells .52 Pigment Cells 52 Undifferentiated (Mesenchymal) Cells 53 Fat Cells 53 Fibers 53 Collagenous Fibers 53 Elastic Fibers 54 Function 54 Serous Membranes 55 Adipose Tissue 55 Densely Organized Collagenous and Elastic Connective Tissues ... 56 Tendons 57 Ligaments 57 Chondroid Tissues and Cartilage 58 Hyaline Cartilage 59 Elastic Cartilage 60 Fibrous Cartilage 60 Bone 61 Intramembranous Ossification 61 Endochondral Ossification 63 Microanatomy of Long Bone 66 Marrow 68 The Notochord 68 CHAPTER IV. The Blood. The Plasma 70 The Blood Cells 70 Erythrocytes 71 Leukocytes 73 Agranulocytes 74 Lymphocytes 74 Monocytes 74 Granulocytes 75 Neutrophils (Heterophils) 75 Eosinophils 75 Basophils 75 Platelets 76 Thrombocytes or Spindle Cells 76 Blood-cell Formation 76 Bone-marrow 78 Hemocytoblasts 78 Erythrocyte Formation 78 Granulocyte Formation 78 Megakaryocytes 79 The Destruction of Blood Cells 80 CONTENTS CHAPTER V. The Muscle Tissues. Smooth Muscle 81 Cardiac Muscle 84 Neurogenic and Myogenic Theories of Heart-beat 87 Skeletal Muscle 88 CHAPTER VI. The Nerve Tissue. Histogenesis of Nerve Tissue 94 The Neuron 95 The Cyton 95 Nucleus 96 Nissl Bodies 96 Neurofibrils 97 Golgi Apparatus 97 Chondriosomes (Mitochondria) 97 Cytoplasmic Processes 97 Reflex Arc 97 The Synapse 98 Types of Neurons 98 Unipolar Cells 99 Bipolar Cells 99 Multipolar Cells 100 The Nerve Fiber 101 Histology of a Peripheral Nerve 102 Receptors and Effectors 103 Sensory Endings 104 Motor Endings 105 Neurologia 106 Ependyma Cells 106 Astrocytes 106 Microgliocytes 106 Ganglia 107 Degeneration of Nerves 108 Regeneration of Nerves 108 The Brain and Spinal Cord 109 Structure of the Spinal Cord » .... 110 CHAPTER VII. The Vascular System. The Capillaries 113 The Arteries 115 Small Arteries 116 Medium-sized or Muscular Arteries 116 Large or Elastic Arteries 117 The Veins 118 The Lymph Vessels 119 CHAPTER VIIL The Lymphatic System. The Lymph Nodules 122 Agminated Lymph Nodules 122 Tonsils 122 8 CONTENTS The Lymph Nodes 124 Cortex 124 Medulla 125 The Hemolympli Glands 127 The Spleen 127 The Thymus 131 CHAPTER IX. The Integument. Introduction 134 Epidermis 134 Dermis (Corium) 134 Integument of Fishes 135 Scales 135 Integument of Amphibia 136 Integument of Reptiles 138 Integument of Birds 139 Feathers 139 Integument of Mammals 139 Skin of Palm or Sole 140 Sweat Glands 142 Hairs 142 Sebaceous Glands 144 Mammary Glands 145 Nails 146 CHAPTER X. The Respiratory System, The Respiratory System of Fishes 148 The Respiratory System of Amphibia 148 The Respiratory System of Rejitiles 150 The Respirator}^ System of Mammals 150 The Larynx 151 The Trachea 151 The Lungs 152 Bronchi and Branches 152 Vascular Supply 155 CHAPTER XI. The Digestive System. Mucous Membrane 157 The Lips 158 The Oral Cavity 159 Teeth 159 Tongue 159 Salivary Glands 164 Parotid Gland 164 Submaxillary Glands 165 Sublingual Glands 166 Pharynx 166 CONTENTS 9 The Alimentary Canal Ifi6 The Esophagus 167 The Amphibian Esophagus 1(57 The Reptilian Esophagus 167 The Mammalian Esophagus 168 The Stomach 169 The Fish Stomach 169 The Stomach of Amphibia 171 The Stomach of Reptiles 171 The Mammalian Stomach 171 The Small Intestine 173 The Small Intestine of Fishes 174 The Small Intestine of Amphibia 174 The Reptilian Small Intestine 175 The Small Intestine of Mammals 175 The Large Intestine 178 The Large Intestine of Elasmobranchs 178 The Large Intestine of Amphibia 179 The Large Intestine of Reptiles 179 The Mammalian Large Intestine 179 The Rectum and Anus of Mammals 181 The Cloaca 181 The Pancreas 181 The Liver 184 Lower Vertebrates 184 Mammalian Liver 185 The Gall-bladder 190 CHAPTER XII. The Excretory System. The Pronephios 193 The Mesonephros 193 Kidney of the Frog 194 The Metanephros 196 Mammalian Ividney 197 The Nephridial Tubule 199 Blood Supply of the Kidney 201 The Ureters '. 202 Mesonephric Duct 202 Metanephric Duct * .... 203 The Bladder 204 The Urethra 207 CHAPTER XIII. The Female Reproductive System. Ovaries 209 Fishes 210 Amphibians 211 Reptiles 212 Mammals 213 Maturing Primary Follicles 216 0\nilation 216 Corpus Luteiun 217 Corpus Luteum Spurium 218 Corpus Luteum Verum 219 The Oviducts 219 Amphibia 220 Reptiles and Birds 221 10 CONTENTS The Oviducts- Mammals 221 The Uterus 223 The Vagina 225 The Vestibule 226 (Estius 226 Menstruation 228 Pregnancy 230 Placenta 230 Chorionic Villi 231 CHAPTER XIV. The Male Reproductive System. Development of the Testes 234 Spermatogenesis 236 Sertoli Cells 236 Spermatogenic Cells 237 Interstitial Cells 239 Sperm Ducts 239 The Testes of Fishes 240 The Testes of Amphibia 240 The Testes of Reptiles 242 The Testes of Mammals 242 Tubuli Recti 245 Rete Testes 245 Vas Efferentia 245 Epididymus 245 Vas Deferens 245 Seminal Vesicles 246 The Prostate 247 Cowper's Glands 248 Copulatory Organs 248 Lower Vertebrates 248 Penis of Mammals 249 CHAPTER XV. The Endocrine Glands. The Pituitary Glands 253 The Thyroid Glands 255 The Parathyroid Glands 258 The Adrenal Glands 259 Mammalian Adrenal 260 The Cortex 260 The Medulla 262 The Carotid Glands 262 Paraganglia 263 The Pineal Body 263 CHAPTER XVI. Technique. Fixation 265 Fixing Reagents 265 Acetic Acid 265 Picric Acid 265 Formalin 266 Alcohol 266 CONTENTS 11 Fixation — Fixing Reagents — Osmium Tetroxide 266 Chromic Acid 266 Potassium Dicliromate 266 Mercuric Chloride 266 Fixing Fluids 267 Bouin's Fluid 267 Flemming's Fluid 267 Zenker's Fluid 267 Formalin Solutions 268 Carnoy's Fluid 268 Containers 268 Handling of Material 269 Dissection 269 Foreign Matter 269 Heat 269 Records 270 Paraffin Embedding 270 Celloidin Embedding 272 Sectioning 275 Rotary Microtomes and Paraffin Sections 275 The sliding Microtome and Celloidin Sections 278 Mounting Paraffin Sections 279 Serial Sections 281 Staining of Paraffin Sections 282 Progressive Staining 283 Regressive Staining 284 The Mallory Connective-tissue Stain 285 Staining With Silver Nitrate 286 Staining Celloidin Sections 286 APPENDIX. Current References Anatomical Texts Histological Texts Prepared Slides Blood . . Muscle Tissues Nerve Tissues Bloodvessels . Lymph Organs Integument . Respiratory System Digestive System Excretory System Female Reproductive System Male Reproductive System Endocrine Glands . 289 289 289 290 291 291 291 292 292 292 292 293 294 294 294 295 'LI MICROSCOPIC ANATOMY OF VERTEBIIATES. CHAPTER I. INTRODITTTOX. In courses of animal biology, or zoology, students usually begin by studying the Protozoa where a single cell constitutes an indi- vidual possessing a complete organization and carrying on all functions essential to life. This conception of cellular independence and self-sufficiency is modified upon consideration of simple multi- cellular animals, such as are found among the Co'lenterates. In such animals a large number of cells are interdependent, their functions finding expression in the activity of the individual they constitute. Cellular specialization becomes evident; certain cells become par- ticularly active in secretion or in absorption; others serve for pro- tection; still others function in contraction or conduction of stimuli. Among the higher groups of animals cellular difi'erentiation and interdependence becomes more marked, so that within the individual animal body there are organizations of cells having the same struc- tural and functional features. Such organizations of cells, struc- turally similar, and their products, if any, are calleil tissues. Five different types of tissues are recognized on the basis of the structural and functional characteristics of the cells and cellular products com- posing them. These are epithelium, connecti\'e tissue, muscle, nerve and blood. Histology concerns itself with the study of the structural charac- teristics of tissues and their interrelationships with one another. The five types of tissue do not exist independently, but are asso- ciated in the formation and function of various organs. A better appreciation of how organs function is possible after a study of their tissue composition or microscopic anatomy. Histology and micro- scopic anatomy are but continuations of anatomy which concerns itself with the internal and external structures of the animal body as determined by dissection. The close association of structure and function obliges a student of anatomy to consider various 14 INTRODUCTION aspects of function with relation to the structural features, although this study is primarily the problem of physiology. The study of histology and microscopic anatomy rests upon the structural and functional differentiations of the cells. Although the knowledge founded on studies of the protoplasmic structure and function of cells belongs to the science of cytology, it is essential that the student of histology should know some of the general structural features of the cell in order to understand the conditions met w ith in the tissue and organ structures with which his prepara- tions deal. It is also important to remember that complex multi- cellular animals usually originate from the fusion of a single egg and a sperm. The progressive steps by which such a fertilized egg develops into a mature individual is the concern of embryology. We will later follow the proliferation of cells, at first similar in structure, through their early embryonic development up to the time when cellular differentiation becomes more apparent and the three cellular layers, the ectoderm, endoderm and mesoderm, are formed. From these three layers all the later tissues and organs are derived. The study of the living cell is possible, but it is still far from being sufficiently practical to be used as a method for teaching. We must rely, therefore, almost entirely upon preparations of dead tissues and organs stained to accentuate their characteristic features. It is true that the methods used in such preparations subject the cells to a number of chemical and physical changes, but the features shown by such methods have been repeatedly checked so that in knowing the reaction of cells to these procedures w^e have relatively constant factors with which to deal. In the living cell many of the structures easily seen hi prepared material are visible only with great difficulty. Although the cellular structures and intercellu- lar material appearing in our preparations may be called artifacts to some extent, they will gi\'e reliable information regarding char- acteristic structures and reactions of tissues, providing we are acquainted beforehand with the methods of preparation used. THE CELL. Although there is great variation in the size, shape, and particular functions of different cells, there are certain general structural features that can usually be demonstrated in all. An animal cell may be defined as a small mass of protoplasm externally limited by THE CELL 15 a cell membrane and containing a spherical body, the nucleus, enclosed in its own membrane. The body of the cell, or cytosome, includes all the protoplasm outside the nucleus, both nucleus and cytosome being composed of protoplasm whose composition diifers in each case. Protoplasm. — Protoplasm is chemically a complex mixture of l)r()teins and their derivatives, carbohydrates, lipoids, and inor- ganic salts associated with a large amount of water. Physi- cally, jirotoplasm has the properties of a complex colloidal system, capable of those changes in viscosity which are due to revers- ible gels occurring in the living cell. Living cells are visible as pale, colorless, homogeneous masses in which, with proper illu- mination, various refractive structures may be observed. The cell membrane and the nuclear membrane may appear brighter because of the higher refracti\ity of their components. Likewise the chromosomes of the nucleus, together with some of the formed granular or fibrillar elements of the cytosome, may stand out. For the most part, our knowledge must depend upon examination of fixed and stained material in which the various elements of the protoplasm are precipitated as insoluble substances which are then stained with various dyes. The precipitation process also preserves the membranes; and, depending upon the reagents used, other structures of the cytosome, such as the mitochondria, or chondrio- somes, Golgi apparatus, and centrioles, may be precipitated for staining and study. (Fig, 1.) Cytosome.— The appearance of the cytoplasm in fixed materials varies with the method used in preparation, and also with the type and physiological state of the cell. It was this variability with fixatives which caused much of the early disagreement and debate among scientists as to the nature of protoplasm, since each based his conception on his prepared material. Accordingly, some investi- gators held that protoplasm was essentially granular; others believed it to be alveolar and to resemble an emulsion; still others held that it had a reticulum of slender fibrils associated with fine granules. Actually all these conditions may be observed in the same type of cell, if it be treated with various techniques. However, certain definite structures or organelles are difterentiated in the cytosome. Chondriosomes, or mitochondria, are elements demonstrated within the cytoplasm after certain techniques. They are quite universal in occurrence, though variable in shape. Usually they are granular in young or embryonic cells but filamentous or rod- 16 INTRODUCTION shaped in the older tissue cells. They are composed of phospho- lipids and protein in varying proportions, and are soluble in fat solvents, but can be preserved by formalin and potassium dichro- mate. Janus green reacts with them in the living cell, and their activities may be readily followed after staining with this dye. Various observers have associated them with the formation of fat, of fibrils, and of secretion granules. They move about in the cytoplasm, grow and divide, and transform from the granular to the rod or filamentous forms (or lylce versa) in the living cell. Secretion granules Golgi bodies- es ^-^••••••.•^•♦••. Nuclear membrune- Nucleolus- Chromatin- Cytoplasm- K^^il 'ell membrane ■Vacuole ■Mitochondria (chrondriosomes) -Central body Fig. 1. — Diagram of a typical cell. The apparatus of Golgi, a reticular structure often close to the nucleus, was first demonstrated in certain nerve cells. These bodies are formed of lipoids, which are fixed with osmium tetroxide, or they may be dissolved with fat solvents, leaving empty canaliculi in their place. Their function is unknown, but their occurrence is so general that they are considered normal intracellular structures, concerned in some unknown manner with cell function. They apparently vary in composition with different states of the cell. Close to the nucleus there is to be found another structure of almost universal occurrence in animal cells, the central a])])aratus, or centrosome. In the resting cell it is inconsi)icnous, but becomes prominent during mitotic (indirect) division of the cell. During THE CELL 17 this process, a single small j^raiuilar body, the centriole, is surroiiiuled by a clear area of cyto])lasni, the centros])here. After treatment with ])n)tein-])recii)itatins ao:ents, numerons fine ])rotoplasmic rays may be made out, extending in all directions into the cytoi)lasm from the centros])here. The so-called astrosphere thus formed is not connnonly visil)le in living material nor in material otherwise fixed. The function of this ai)paratus is not clearly understood, l)ut ajjpears to be intimately connected with mitosis. In addition to the bodies already mentioned, there are other less common })articles distributed in the cytoplasm. In nerve cells and striated muscle fibers, abundant and distinct fibrils may occupy much of the cytosome. The cells in the early dixisions of the frog embryo contain yolk granules, and glycogen can be demonstrated as a stored substance in the cytoplasm of liver cells. Under favor- able conditions, droplets of fat form in certain connective-tissue cells, (iranules of pre-enzymatic nature are recognizable in gland cells, and pigment granules occur in the cytoplasm of certain epithe- lial and connective-tissue cells. A limiting membrane is not clearly evident in all cells. Some investigators believe it to be merely a condensation of the peripheral cytoplasm which probably contains lipins. By using microdissec- tion apparatus it has been possible to indicate that this membrane possesses some degree of elasticity. It has been demonstrated to be semipermeable and appears to regulate the passage of oxygen and nutriti\e materials into the cell cytoplasm as well as the passage of carbon dioxide and nitrogenous wastes out of the cell. The exposed surface of some cells may have a thick, tough external cuticle or other structural modifications. Certain groupings of cells are not completely separated by indi\'idual cell membranes and form a syncytium as in the case of cardiac and skeletal muscle. Nucleus. — In general, the nucleus of the cell is a globular or ovoid body and is separated from the cytoplasm by a definite membrane. \Yithin the membrane, the nucleus is composed of a homogeneous watery fluid, containing special proteins, the nucleoproteins or chromatin in colloidal solution. Fixation and use of basic stains bring out a granular network of chromophilic substance that increases in density as organization of the nucleus for division advances, until the chromosomes, which are mainly solidly staining bodies composed of nucleic acid, stand out clearly. Cytogenetics has demonstrated that it is the chromatin material of the nucleus which carries the hereditary factors. Nucleus and cytoplasm diflfer 2 18 INTRODUCTION in their reactions to stains. Cytoplasm takes acid stains, there- fore staining red with eosin. The chromatin of the nucleus reacts mainly to basic stains so that when hematoxylin is em])loyed the chromatin has a color varying from lilue to purplish-hlack. In the resting nucleus the chromosomes are not apparent. Their disappearance is presumably due to the incorporation of liasic pro- teins in the nucleic acid molecule, which then goes into solution. Two types of chromatin are thus indicated in preparations of cells in the resting state; oxychromatin, which has an affinity for acid dyes; and basic chromatin with an affinity for basic dyes. Within the nucleus of the resting cell there is usually present at least one sm^iU spherical body, the nucleolus, that in living cells is highly refractive. This body has an affinity for acid dyes, but may also stain with basic dyes and resemble chromatin in aj^pear- ance. Its function is not definitely known, but there are some indications that it may be associated with the storage of nuclear wastes and of chromatin. It usually disappears during the mitotic process. The nucleus is regarded as being the regulator of cellular activities, and changes in its appearance and reactions are associated with the changing states of the cell as a whole. One of the major functions in which the nucleus is intimately involved is cellular division, or reproduction. Cell Reproduction.— The formation of new cells is most active in the embryo, yet the process takes place in many tissues of the adult vertebrate body notwithstanding the fact that there are surprisingly few cells in the active process of division to be seen in the usual microscopic preparations. Probably most of the cells of which adult tissues are composed have a longer life than one would at first suppose. We should think of a cell as a com])licated biochemico- physical system whose organization is maintained for a long time, even though the materials of the system may change. Two types of cell division have been recognized, mitosis and amitosis. Mitosis, or indirect cell division, is the normal method of cell division, and a brief presentation of this process may serve to recall the major features. Mitosis.— The mitotic process is usually ari)itrarily (li\idc(j into four stages: namely, prophase, metaphase, anaphase, and telophase. These imperceptibly grade into one another, since they are but easily distinguishable steps in a continuous sequence of phenomena. THE CELL 19 7-'ro;;//fl.s'r. This phase of nuclear actixity is preceded and accompanied by activity of the two centrioles in the center of the cytocentruni or attraction s])here. When activity })egins, the centrioles ni()\'e a])art until they occui)y a position op})osite each other 90 degrees from their resting position, with the nucleus between them. Astral rays a])pear in the cytoplasm, radiating out from each centriole. In pre])arations, these rays often resemble fibers. Some of them form a fibrillar apparatus between the two centrioles, which is known as the achromatic s])indle, or, together with the centrioles, the amphiaster. Simultaneously the nuclear membrane and nucleolus disappear, and nucleoplasm mingles with cytoplasm. The distributed chromatin granules of the resting nucleus are api)arently organized into a long, coiled, tangled thread, which shortens through condensation of its constituents and finally forms a specific numlier of pieces, or chromosomes. The num})er of chromosomes forming at this stage is usually constant for all somatic cells of each species. The chromosomes move so that they lie on the sijindle in an imaginary })lane midway between the two cen- trioles at right angles to the axis. Longitudinal splits in the chromo- somes now become api)arent as grooves, though they may actually have split much earlier in the prophase stage, and some of the astral rays (fi})ers) ai)i)ear to attach to chromosomes. Metaphase.^Dwv'm^ this second phase longitudinal splitting is completed and each chromosome is found to be equally divided into two longitudinal halves. The two halves of each chromosome separate as they move toward their respective centrioles, a feature marking the beginning of the anaphase. Anaphase. — In this third stage, the chromosomes continue toward the centrioles. In late anaphase a groove api)ears in the cell mem- brane corresponding with the location of the equatorial ])lane, which is marked by a granular line. This groove deepens as the chromo- somes pass to opposite poles. The chromosomes assemble at opposite poles and initiate the telophase stage. Telophase. — hi the final phase the equatorial constriction, or groove, continues so that eventually 2 daughter cells are formed, each with its own cell membrane. Two daughter nuclei are organ- ized in the same way as was the original resting nucleus, though they are smaller in size. In this process, a nuclear membrane forms about each daughter nucleus, and the chromatin becomes flistributed in it as it^was in the resting nucleus of the parent cell. A nucleolus 20 INTRODUCTION usually appears in each nucleus at the end of the telophase. Also, by this time, the centriole has divided into a diplosome, or 2 cen- trioles, near each daughter nucleus. If the daughter cells chance to be part of a very young embryo, the next cell division will occur very quickly. But if the cells are part of an adult organism, some time may elapse before they divide. Amitosis. — Now and then cells appear to divide directly by constriction without the elaborate preparations occurring in mitosis. This process has been named amitosis. In such cases the nucleus first separates into two parts by constriction, after which the cyto- plasm likewise divides. It has been observed in pathological tissues and in the case of some old cells in tissue cultures. In most of the so-called cases of amitosis, the technique may have been at fault or the cells only appear to divide amitotically, due to some abnormal condition interfering with the mitotic process. Cell Metabolism.— The living cell is a dynamic system in which chemical and physical changes are constantly taking place. All such transformations of matter and energy occurring in li^'ing proto- plasm are designated by the term metabolism. In this term are included the anabolic processes involved in building tissues up as well as the katabolic or breakdown processes. Nutritive sub- stances taken into the cell are subjected to the action of enzymes which facilitate the breaking down of the complex compounds into their simpler units. These units are then utilized in building up the elements of protoplasm which serve for repair and growth. The energy liberated by oxidation reactions is utilized for the chemical and physical changes involved in movement, in secretion and excretion, and for the continuation of those metabolic changes constantly involved in the maintenance of life. All cells regardless of the degree of their functional specialization must still carry on a basic general metabolism necessary to maintain their own life. Thus a muscle cell, in addition to the basic metab- olism maintaining its life, has special metabolic processes endowing it with the property of contraction. Cytomorphosis designates the structin-al changes undergone by a cell in its passage from the embryonic state, through its differenti- ated phase to senescence and death. Death or necrosis of cells is followed by cellular disintegration, at which time the enzymes active in the living cell in the synthesis of ])r()teins from amino-acids begin breaking down the proteins into amino-acids which are then diffused from the cell. During this process of autolysis the chromatin of the nucleus contracts into a dense irregular chromatic mass. THE CELL 21 Tissue Culture. One of the aehieveiueuts of modem research has been the deveIo})meiit of methods whereby small fragments of tissues com])osed of various cells can be isolated and grown a])art from the organism in especially prepared media. If proper conditions of warmth, aeration, and asepsis are maintained in the culture media, the cells of such tissue cultures may be kei)t alive, grow, and divide mitotically over a considerable period and a number of generations. It has been ])ossible by these methods to isolate certain cell types, observe their growth, and determine whether they maintain certain characteristics or transform to other types. It also opens a method of attack upon the jihysical and chemical properties of the various cells maintained under culture conditions. One of the outstanding contril)utions has been the study of growth and regeneration of nerve cells. Still other observers have studied the behavior of other ty])es of cells under diflPerent physical and chemical experimental conditions. Another method of attacking the study of living cells utilizes a micromanipulating ap])aratus in conjunction with the microscope. By means of this apparatus those proficient in the technique have been able to dissect cells or inject minute quantities of chemicals into them. As a result of these microdissections and microchemical studies much has been learned about the physical and chemical nature of living cells. REFERENCES. Bensley, R. R., and Gersch, I. 193.3. Studies on cell structure by the freezing-drying; method, Anat. Rec, 57, 369. Bensley, R. R., and Hoerr, N. L. 1934. Studies on cell structure by the freezing-drying method: V. The chemical basis of the organization of the cell, Anat. Rec, 60, 251. Studies on cell structure by the freezing-drying method: VI. The preparation and properties of mitochondria, Anat. Rec, 60, 449. Cameron, Gladys. 1935. Essentials of Tissue Culture Technique. Farrar and Rinehart, Inc., New York. Chambers, R. 1927. Microdissection studies: The visible structure of cell protoplasm and death changes, Am. .Jour. Physiol., vol. 43. Cowdry, E. V. 1924. General Cytology, Chicago, 111., Univ. Chicago Press. Gray, J. 1931. E.xperimental Cytology, Cambridge, Cambridge Univ. Press. HoBER, R. 1930. The present conception of the structure of the plasma membrane, Biol. Bull., 58, 1-17. Lewls, F. T. 1925. A further study of the polyhedral shapes of cells, Parts I, II, III, Proc Am. Acad. Arts and Sci., Boston, 61, 1. 1928. The effect of cell division on the shajje and size of he.xag- onal cells, Anat. Rec, vol. 33. Schrader, F. 1934. On the reality of spindle fibers, Biol. Bull., 67, 519. Wilson, E. B. 1928. The Cell. New York, The Macmillan Company. See Appendix for general text references. 22 INTRODUCTION EMBRYOLOGY. A vertebrate normally begins life as a single cell, the zygote, which is formed by fusion of a sperm cell and an ovum in the process of fertilization. Almost immediately the zygote begins dividing by mitosis to form an increasing number of cells of decreasing size. Each cell so formed in these early divisions is called a blastomere, and the process itself is termed cleavage. The amount of yolk originally stored in the ovum varies in different classes of verte- brates, and since the presence of yolk modifies cleavage and later developmental stages by hindering mitotic activity where it is abundant, the morphology of later stages varies. Nevertheless, homologous stages in development can be recognized. Amphioxus.— The relatively simple development of Amphioxus is usually studied as exemplifying clearly the early stages, since there Ectoderm Endoderm Archenteron — Blastopore 8 CELL STAGE 32 CELL STAGE BLASTULA GASTRULA Fig. 2. — Diagram of early stages in development of Amphioxus. (Modified from Arey's Developmental Anatomy, W. B. Saunders Company.) is only a very small amoimt of yolk stored in the ovum. The yolk present is localized toward one end of the cell, known as the vegetal pole, and at the opposite end, the animal pole, protoplasm is more concentrated. However, the yolk is so small in amount that mitotic activity is little affected and nearly equal blastomeres are formed by cleavage. As mitosis continues, the cells become more numer- ous and smaller, those toward the vegetal pole becoming slightly larger than those at the animal pole. An the cells continue to divide, they maintain a peripheral ])osition and enclose a central cavity. (Fig. 2.) Thus a hollow sphere, the bJastiiJa, is formed, with its wall composed of a single layer of very small cells enclosing a central segmentation cavity filled with liquid. Mitotic acti\'ity continues following blastula formation, and the slightly larger vegetal cells are folded into the segmentation cavity. Invagination of these cells continues to form an inner layer adjoining the outer cell layer and practically obliterating the segmentation ca\'ity, thus EMBRYOLOay 23 forniini!; a j^astruhi. The ()i)t'ninti; at tlic ])()iiit where the invagina- tion of \egetal cells occurred is called the hld.sioporc. The outer layer of cells conijHJses the ectoderm ; the iiuier layer is the endoderm; and the cavity within the endoderm layer of cells is the archenterun, or primitive gut, which oi)ens to the exterior by the blastopore. A third germ layer, the mesoderm, makes its initial appearance from a series of ])aired i)ouch-like evaginations along each side of the dorso-lateral wall of the endoderm of the primitive gut. These evaginations, the coelomic pouches, later separate from the endo- derm. The ctrlomic ])()uches on either side enlarge and grow ven- trally around the gut, the transverse walls of adjacent pouches fuse and are ()l)literated so that a connnon cavity, the coelome, is formed. The outer wall of the mesoderm adjacent to ectoderm is known as somatic mesoderm and together with the ectoderm is called the somat()i)leure, which gives rise to the bodv wall. The inner hiver Ectoderm- i Primitive gut --Wp^Qs^.^'^SSyt^ „ , Endoderm 8 CELL STAGE BLASTULA GASTRULA Fig. 3. — Diagram of early stages in development of the frog. of mesoderm adjacent to the endoderm of the gut wall is known as splanchnic mesoderm and together with the endoderm composes splanchnopleure, which gives rise to the gut wall. Amphibia. — In this group, as exemplified by the frog, the eggs contain considerable yolk. The nucleus and bulk of the cyto])lasm are restricted to the animal pole and the abundant yolk is concen- trated toward the \'egetal pole. Cleavage is complete but the cells are of unequal size, those toward the vegetal ])ole being distinctly larger than those of the animal i)ole. The segmentation cavity of the blastula is reduced in size with yolk-laden cells on its floor. (Fig. 3.) Gastrulation is atypical, invagination of vegetal cells being limited, and the gastrula appears to be formed as a result of overgrowth and infolding of the more actively dividing cells of the animal pole in the region of the blastopore. The gastrula is similar to that of Ami)hioxus, excei)t for the larger yolk-laden cells below the floor of the archenteron. 24 INTRODUCTION The dorsal wall of the archenteron consists of relatively few layers of small endoderm cells, while the floor consists of many layers of large yolk-laden cells. Certain cells along the dorsal mid-line differentiate into a rod-like structure, the notochord, and other cells proliferate off on either side of the notochordal region to form mesoderm masses separated from ectoderm and endoderm. Eventu- ally the right and left sheets meet along the mid-ventral line and fuse. Before this fusion occurs, the mesoderm mass begins to split into two sheets on each side a short distance away from the noto- chord. This continues so that eventually, as in Amphioxus, there will be an outer somatic sheet associated with ectoderm and an inner splanchnic sheet associated with endoderm. The space between them is the coelome, or body cavity. Blastoderm Segmentation cavity Ectoderm Blastocwle A nterior Archenteron Invaginated endoderm Blastopore Neural fold Neural groove Mesodermal somite ~Somafopleure ~S plan ch nopleu re Endoderm- Fig. 4. — Diagram of early stages in development of reptiles and birds. (Modified from Arey's Developmental Anatomy, W. B. Saunders Company.) Ectoderm- Somatic mesoderm-,;^ Splancfinic mesoderm '^ Sauropsida. — In reptiles and birds, where the egg contains a large amount of yolk, the nucleus with a small amount of cytoplasm is located at the animal pole. This restricted protoplasmic region divides into cells, but the larger yolk portion takes no part in cleavage. Cleavage is therefore partial and results in a small disc of cells on top of the spherical yolk mass. This disc of cells, called a blastoderm, continues to divide by a series of vertical and horizontal planes, so that in the late cleavage stage there are a few layers in the central ])art of the disc, but only a single lay(>r of cells EMBRYOLOGY 25 at the margin. A small space between the blastoderm and the yolk represents the segmentation cavity. Additional marginal cells continne to form in such a manner that they eventually surround the yolk, but never take part in the formation of the embryo itself. Gastrulation is accomplished by an ingrowth of cells along the ])osterior margin of the blastoderm. (Fig. 4.) Following endoderm formation, a ])rimitive streak appears on the blastoderm as a linear thickening in which a shallow depression, the primitive groove, appears. This groove is believed to be homologous with the blasto- pore of Amphioxus. Mesoderm arises from lateral proliferations of cells of the primitive streak which form plates that grow laterally between the ectoderm and endoderm on either side of the mid-line. The plates split distally, thus forming the somatopleure and the splanchopleure with ectoderm and endoderm, respectively. The coelome is the space between somatic and splanchnic mesoderm. Mammalia.— Although the eggs of mammals possess a minimvun of yolk, the development of the embryo does not follow the simple plan outlined for the egg of Amphioxus. Development of the mammal is complex and possibly modified because of evolution from an ancestral condition in which yolk was abundant in the egg, a condition still present in Monotremes. Yolk has ])ractically dis- appeared from most mammalian eggs and the zygote developing within the parental body secures nutritional supplies from maternal tissues. Such modifications change the developmental processes by which the embryo becomes established. Cleavage is complete and the early stages superficially resemble those of Amphioxus. Later clea^'age results in the formation of an inner cell mass surrounded by a single layer of cells, called the trophectoderm, which is in contact with the wall of the uterus of the mother. The trophectoderm grows more rapidly than the inner cell mass which is left attached only at one point on the trophectoderm wall. The cavity between the two regions is filled with fluid and is homolo- gous with the segmentation cavity of lower forms. This stage is recognized as a specialized blastula and is called a bJastocj/st. The embryo jjroper develops from the inner cell mass which is homolo- gous with the disc-like blastoderm of the reptiles and birds. Cells derived from the lower surface of the inner cell mass form endoderm and the remainder of the mass becomes ectoderm. Eventually a primitive streak appears on the blastocyst and is morphologically similar to that in Sauropsida. A primitive groove also appears which is homologous with the blastopore of lower 26 INTRODUCTION forms. Mesoderm forms on either side of the primitive streak and grows into the space between the ectoderm and the endoderm, as in preceding groups. The mesoderm mass spKts into two sheets, a somatic layer associated with ectoderm to form somatopleure and a splanchnic layer associated with endoderm to form splanchnopleure. Organogeny.— The development of similar organs differs in details in different classes of vertebrates and a complete account belongs to the field of comparative embryology. Later, as each tissue and system of organs is considered, reference to early embryology will be made wherever it is considered essential to an understanding of the microanatomy of the organ in question. The following table summarizes the structures usually considered as developing from ectoderm, mesoderm and endoderm: From Ectoderm. I From Mesoderm. From Endoderm. Epidermis of skin, hairs, Cartilage, bone, peri- Epithelium of digestive nails, sweat and sebaceous c ho ndrium, periosteum, ten- tract from pharynx to rec- glands, claws, superficial ' dons, ligaments; other con- ' turn; glands of this tract. portion of feathers, arrec- tor pili muscles in skin; | cells; almost all muscle tis lens of eye; part of cornea retina; conjunctiva; lacri mal gland; most of pituit- ary body; epithelium lining neys; gonaducts and uri- mouth and nasal cavities; nary ducts: cortex of adren- sensory cells of many sense als; pleura?; pericardium; nective tissues; microglia | inckiding jjancreas and liver; epithelium of larynx, sue; blood cells; marrow; trachea, and lungs; gland- blood vessels and lymph I ular epithelium of thyroid vessels; gonads and kid- , and parathyroid glands; epithelium lining the Eus- tachian tube and middle ear. organs; pineal gland; me- dulla of adrenal gland; peritoneum; lymph organs; scales in fishes and bony enamel of teeth; all nerve plates in reptiles; major cells and most neuroglia ' part of feathers. ! cells. In the past it has been a common ])ractice to emphasize the origin of the tissues and organs with regard to the germ layers, but embryo- logical studies show considerable \-ariations with regard to such origins. Until the embryologists have settled the debatable points we are not justified in placing nuich emphasis on origins. REFERENCES. Arey, Ij. B. 1934. Developmental Anatomy, Philadelpliia, W. B. Saunders Company. Adelmann, H. B. 1932. The development of the prechordal plate and meso- derm of Amblystoma punctatum, Jour. Morph., 54, 1-53. DE Beer, G. R. 1934. An Introduction to Exp3rimental Embryology, Oxford, England, The Clarenden Press. Keith, A. 1933. Human Embryology and Morpliology, 5th ed., Baltimore, William Wood & Co. Richards, A. 1931. Outline of Comparative Embryology, New York, John Wiley & Sons, Inc. Shumway, W. 1935. Vertebrate Embryology, 3rd Ed., New York, J. Wiley & Sons. CHAPTER II. THE EPTTHELIAL TISSUES. The various types of epithelial tissues are composed of one or more layers of cells lyino; so close to one another that there is practicall}' no intercellular material. Membranes of this tissue cover the free surfaces of the body and line not only the ducts con- necting with the surface, but also cavities within the body not connected with the outer surface. One of the chief general func- tions of these tissues is ])rotection, such as preventing the loss of body fluids or the invasion of foreign material. Most types of epithelia have some secretory activity, but those lining numerous glands are ])rimarily secretory. Epithelia are intimately concerned in processes involved in respiration, in the assimilation of nutritive materials, and in the elimination of wastes. Classification. — It must be admitted that any classification of the various types of epithelia will be artificial, and the justification of any scheme will lie in the ease and completeness it oft'ers for dealing with the types in question. A classification of e])ithelia is not possible on the basis of function alone, for the exact function of many types is not yet clear, and others may have more than a single function. Embryological origin likewise fails, since often the same type is derix'ed from ectoderm, endoderm, and mesoderm. The most satisfactory scheme uses the form and arrangement of the cells. Even on this basis, allowances must })e made for intergrading forms. Two large groups are separated depending upon the presence of one or more than one layer of cells in the tissue. Tissues belonging to the first group have a single layer of cells and are called simple epitheha. In the second group are the stratified epithelia, where more than a single layer of cells compose the tissue. Various types within these two groups are then separated with reference to the form and arrangement of the cells composing each. SIMPLE EPITHELIA. This group is usually subdivided into three types, namely, squamous, cuboidal, and columnar. (Fig. 5.) These terms are convenient for indicating types commonly appearing in histological 28 THE EPITHELIAL TISSUES preparations, but between them there exist numerous intermediate forms. Those cells having the form resembling scales or irregular discs are called squamous. In other cells the three dimensions are approximately equal and these are called cuboidal epithelial cells, although they are rarely perfect cubes. The columnar type has elongate prismatic or roughly hexagonal cells that resemble irregular columns. It is unfortunate that in the study of histological preparations the field presented by the microscope is primarily two-dimensional. The third dimensional aspect of the objects under study is depen- dent upon the focus, and in the case of very thin sections it is neces- sary to study a series of consecutive sections and develop the ability to form a com])osite picture incorporating this aspect. As an example of limitation imposed by studying the two dimensions presented by histological preparations under the microscope, con- sider the possible sections through a cell having the form of a SQUAMOUS CUBOIDAL COLUMNAR Fig. 5. — Diagram of types of simple epithelial cells. hexagonal column with a central bean-shaped nucleus. (Fig. 6.) If a series of cuts pass through such a cell parallel with the long axis, thin sections will be obtained which appear as rectangles, and not all sections will have the nucleus represented. P\irthermore, if the first cut through the region of the nucleus takes only a small portion of its convex surface, such a section will appear to have a spherical nucleus, wdiile sections through the body of the nucleus will have an ovoid form. Xo one section so obtained gives any clear evidence of the third dimensional aspect or the picture of the cell as a whole. If another series of thin sections is obtained using cuts at an angle of 45 degrees with the long axis, then there will be a variation from trapezoids without nuclear portions to rectangles containing roughly spherical nuclear portions. As the angle of cutting approaches a right angle with the long axis, the hexagonal structure of the cell appears in the sections, although distorted. Sections from cuts made at right angles to the longi- tidinal axis will show hexagons, some of which will have no miclear portion, while others will have a spherical nucleus centrally situated. Therefore, in order to visualize the complete structure of such a SIMPLE E PIT HE LI A 29 cell it would he necessary to have at least se\eral sections at right angles to and parallel with the long axis. In studying tissues and organs, an attem])t should be made to \isuali/.e the third dimensional aspect and consider the relationship of the cells with each other. In the example just considered the cell was a regular, independent unit, hut as a part of tissues all cells are modified by pressures of adjoining cells and internal pressures, so that the connnon form is an irregular })rism which in sections presents polygonal figures. In I Fig. 6. — Diagrams of the appearance of a variety of sections of a typical columnar epithelial cell. general, those cells which appear as squares in sections passing through their central region at right angles to their base are spoken of as cuboidal, but usually they are iso-prismatic in form. Likewise, cells appearing as rectangles in sections passing at right angles to their base are spoken of as columnar cells, but are long prismatic structures when the third dimensional aspect is considered. The squamous cells appear as fusiform structures in sections resulting from cutting at right angles to their basal or attached surface. The three types indicated show great variation as a result of irregularities imposed by adjacent cells and the location of the tissue so that inter- grading types are common. 30 THE EPITHELIAL TISSUES Simple Squamous Epithelium.— The term squamous literally means scaly, and epithelia of this type are composed of flattened, plate- like cells whose cytoplasm is so scanty that the nucleus, which is Fig. 7. — A photograph of a surface view of simple squamous cells forming the top- most layer of a frog's epidermis. Xji9^\ ^# Fig. 8. — Photograph of a section through the kidney of a frog. Simple squamous epithelial cells are shown lining the space surrounding the glomerulus. Cuboidal cells form the walls of the surrounding sections of uriniferous tubules. The arterial vessels are shown entering the glomerulus at the top. centrally placed, causes a slight bulge. (Fig. 7.) In some cases the lateral faces or edges adjoining other cells are regular, but more often the boundaries are irregular and interlock with irregularities in adjacent cells. In sections, which may pass at right or even SIMPLE EPITHELIA 31 uhlique aii ; ^'V^^Jf -'-^ z> ^ * * I ^ Wk^' .,^^ "^i^^f^^Sfm^^^^m ^V% HlViP^C Fig. 14. — Photograph of stratified squamous epithelium from esophagus of monkey. the epidermis become spherically distended by accumulation of secretions which are later liberated on the skin surface. Stratified Cuboidal Epithelium.— The basal cells are cuboidal or low colunniar, the intermediate region of several cell layers has irregularly polygonal cells, and the surface cells are roughly cuboidal with a rounded outer margin. This type is commonly found form- ing the epidermis of urodeles, where scattered cells of the inter- mediate layers are secretory and become distended with their secretion ])r()ducts until the,\' appear almost spherical. (Fig. 90.) Stratified Columnar Epithelium.— The basal layers and intermediate cell layers are similar to those of the preceding stratified types, but the superficial cells are elongated prisms that appear col- umnar in sections. It is found in mammals lining, the vas deferens and some of the larger excretory ducts of certain glands. 36 THE EPITHELIAL TISSUES In some cases, as in the vas deferens, the cohnnnar cells may be ciliated. Among the lower vertebrates this type seems to be more commonly distributed, although many cases so distinguished may prove to be a pseudostratified arrangement such as we have already mentioned. It appears in the alimentary tract of many lower forms and in the excretory ducts. In some regions, as in the esophagus of many amphibians (Fig. 15), some of the columnar superficial cells are ciliated, and others are transformed into goblet cells filled with mucin. Fig. 15.- -Stratified ciliated columnar epithelium from the frog's esophagus, the long superficial cells and several layers of small basal cells. Note Transitional Epithelium. — In this type the basal layer of cells is cuboidal or low columnar in appearance in sections, while at the surface of the membrane are very large cells convex on their free boundary and with conca\'ities on their lower surface into which underlying polyhedral cells fit. Binucleated cells commonly occur in this tissue. Transitional epithelium lines the pelvis of the kidney, the ureters, the bladder and part of the in-ethra of mammals, in which group it appears to be well de^ eloped. Its cells seem to be capable of considerable displacement; they slide by one another under tension so that in a distended membrane of this type there appear to be only two or three layers of cells. The surface cells stretch most and the underlying cells are drawn out into a single or double layer beneath them; as the tension on the membrane is released the cells slide back, until the stratified appearance of a number of layers is resumed. INIembranes of this type show grada- tions to those reseml)ling a stratified cuboidal or stratified cohnnnar, conditions connnonlv occurring in tlic urethra. (Fig. b>0.) SURFACE MODH'K'ATIOXS OF EI'ITIIKIAAL CELLS 37 PIGMENTATION IN EPITHELIAL CELLS. In various locations epithelial cells normally contain pigment granules, and this constant characteristic has lead to speaking of such epithelia as being of the pigmented type. However, this does not indicate the morphology of the cells, which may be rightly classified as cuboidal or other types. To follow the scheme of morphology in classification, such cells should be classified on the basis of their form and arrangement and then the presence of the pigment should be noted for the gi\en type in certain locations. An example of this tissue occurs in the outer layer of the retina, where hexagonal cells adjacent to the inner surface of the choroid coat are heavily pigmented. Pigmentation granules also occur in the lower layers of cells in stratified squamous epithelium forming the epidermis of various mammals and in the liver cells of many lower forms. SURFACE MODIFICATIONS OF EPITHELIAL CELLS. The free boundaries of epitlielial cells may be modified in various ways. The superficial layer of protoj^lasm in some cases is con- densed into a firmer portion continuous with subjacent less dense protoplasm. In sections such a surface condensation appears as a bright line and may extend about the entire cell, although limited in some cases to the exposed surface. Occasionally this denser layer presents perpendicular striations, which are considered as fine, hair- like processes of protoplasm held together by intervening less differentiated protoplasm. Such an arrangement, is known as a striated border. The surface of other cells has a brush border in which densely packed, non-motile, hair-like, protoplasmic structures project slightly beyond the surface. The elements of such a border are associated with small granular swellings at their base and are thought to be active in absorption. The peak of such dift'erentia- tion is founfl in the ciliated boundaries of various types of epithelial cells where the fine, hair-like, protoplasmic processes are much longer than those of the brush border. These processes, the cilia, of which there may be as many as a hundred from a single cell, often possess the power of movement. Less commonly cells have single, whip-like, protoplasmic processes, called flagella, which are usually motile. According to some authors, cilia possess a protoplasmic shell enclosing a hollow core, which being rhythmically filled and emptied with more fluid protoplasm imparts to these structures their charac- 38 THE EPITHELIAL TISSUES teristic mo^'ement. According to other investigators, cilia have a contractile band along each of two opposite surfaces and the move- ment is effected by the alternate contraction of these bands. In either case the movement appears as a sharp initial bending and a slow recovery. The motion of one row of cilia apparently initiates similar activity in adjacent rows, so that a series of waves pass along the field of cilia. Since the l)eat is in one direction, particles caught on the tips of the cilia are usually propelled in the direction of the beating. Ciliated epithelia lining the respiratory passages tend to mo^'e dust particles and material in the lumen out into the mouth or nasal sinuses. Ciliary action is independent of nerve action, as may be demonstrated by removing the ciliated membrane from the roof of the frog's mouth and studying it in a saline solution hours after the frog itself is dead. Cilia possessing the power of movement are called kinocilia, to distinguish them from static cilia or stereocilia of such cells as those lining the epididymis. Another structural featiu-e associated with the free boimdary of some epithelia is a cuticle. The superficial protoplasm of the cell is not modified in any marked manner and is not continuous with this modified liorder which is formed by the cell during development. The cuticle may become impregnated with \'arioiis salts and be firm and hard. The enamel of the teeth is an example of such a special- ization. This type of modification plays a much wider role in the invertebrates where cuticles are more complex and form the skeleton of many forms. The boundaries of epithelial cells in contact with adjacent cells are so delicate that they do not always show in preparations. Some believe a cementing substance is present between adjacent cells but it has also been indicated that the adherence of the cells to each other is due to the adhesion of adjacent cell membranes. Proto])lasmic bridges may extend from cell to cell. (Fig. 16.) These ha^'e been interpreted by some as solid strands between vacuoles in the intercellular spaces or as products of technique and therefore artefacts. In silver nitrate technique silver salts are deposited about the cells so that in face \'iew a network of poly- hedral outlines define the cells as described in the case of mesothe- lium. The cells composing an epithelial membrane are ca})able of gliding by one another when the membrane is stretched. The basal surface of simple epithelia and the basal siu'face of the deepest layer of the stratified types may possess a cuticle. In addition, most epithelial cells rest upon a distinct basement mem- (rROWTH AND REGENERATION OF EPITHELIA 39 brane, which is a homogeneous hiyer of nou-ceUular substance derived ])resumably from the su})jacent connective tissue with which it is most intimately associates 1. The l)asement menil)rane does not ap])ear to be ])resent in all cases. It is absent in the follicles of the thyroid gland where cuboidal cells compose the epithelial mem- branes. One of the characteristics of epithelial tissues is the fact that they rest upon connective tissue. E\'en in those cases where the epithelial membrane is much folded, connective tissue fills in the Fig. 16. — Photograph of a section through the stratified squamous epithelium of the toe of Necturus, showing intercelhilar strands or intercellular bridges. spaces between the folds. This subjacent region of connective tissue carries a network of blood and lymph vessels which come into close contact with the base of every epithelial membrane, but do not extend in among its cells. There is a plexus of nerve fibers in the connecti\'e tissue below the basement membrane and some of these fibers pass along the base of the cells while others extend between the cells. GROWTH AND REGENERATION OF EPITHELIA. As one might ex])ect from the exposure of various epithelia to mechanical and physiological wear, there is a varying quantitative loss of cells that must be replaced. The continual wearing awa>' of the superficial layers is characteristic of stratified squamous 40 THE EPITHELIAL TISSUES epithelia. This is especially pronounced in the case of the epidermis of the skin, which protects underlying tissues and still retains a sensitiveness to external stimuli. If the upper superficial scaly layer of dead cells continue to accumulate to considerable thicknesses, there is an increased protection with an accompanying loss of general sensitivity. This actually occurs in the callosities localized at points of constant friction and pressure. The maintenance of a protective layer of optimum thickness despite the wearing away of the superficial layers is made possible through the continual replace- ment of cells from the basal layers, where they are being produced through normal mitotic activity. A lesion in this type of epithelium is quickly repaired through the activity of these basal cell lasers. P^pithelia of other types in other locations are also subject to con- tact with substances causing gradual or abrupt destruction and loss of cells. In the alimentary tract, dead cells of the columnar type are replaced by mitotic activity of less differentiated cells intercalated among them. In many places mitotic activity is rarely observed under normal conditions. In healing of surfaces from which the epithelimn has been lost through injury the newly formed cells of the bordering region appar- ently migrate by a sliding movement to form a covering layer prior to the regeneration of the tissue characteristic of the injured region. Experiments have also shown changes in type in the case of the pseudostratified ciliated epithelium of the trachea of the cat, which, after repeated treatments with a formalin solution, became a stratified squamous membrane. In the embryo cat the esophagus is lined with a ciliated columnar epithelium, but in the adult this region has a lining of stratified squamous, the type commonly found in places subjected to external wear. SECRETORY EPITHELIAL CELLS AND GLANDULAR ORGANIZATIONS. Probably all epithelial cells to some extent elaborate active substances through biochemical processes of their protoplasm; these processes being collectively spoken of as secretion. Although protection and absorption are the major tasks of some epithelial cells, there are others primarily concerned with this process of secretion. In those cells active in secretion there occurs an accu- mulation of very small secretion granules in the cytoplasm. The origin of these granules is debated, but they appear first in the basal SECRETOh'Y AX J) EI'ITIIELIAL CELLS 41 region, then usually pass toward the surface of the cells \\here they are discharged to become part of the active secretion. There is considerable variation in the method by which secretion is carried to completion in different types of glandular cells. The use of the term glandular to indicate such secretory cells places the emphasis u])on function, but the form of most of these cells will fit into the classification of types already studied. Groups and organizations of actively secreting epithelial cells are called glands, and two broad divisions are sei)arated on the basis of whether the secretion formed is liberated into a hmien from the free boundary of the cells or whether it passes in the opposite direction to enter the vascular system. Those organizations of cells secreting by the first method, that is by liberating their secretion into ducts which carry it to the surface of the epithelial membrane from which the gland developed, are called exocrine glands. The endocrine glands are those glandular tissues lacking excretory duct systems and whose secretions pass into the vascular system. Exocrine Glands. ^The secretory process is completed in se\eral ways in different types of exocrine glands. In merocrine glands, granules form in the cytoplasm of the cells and accumulate toward the free boundary to be discharged as the active secretion; the process is then repeated. Most glandular secretions, such as those of the digestive glands, are derived in this manner. In a holocrine gland, as exemplified by a sebaceous gland in the skin of a mammal, the major portion of the active cell develops into a secretion mass which, when dislodged to form the secretion, is accompanied by the death and disintegration of the cell. A new cell then forms from the underlying layers of the stratified epithelium forming such glands and the process is repeated. Another type of secretion is termed apocrine, and in this case a secretion mass forms in the apical portion of the cell; this apical region becomes greatly enlarged and is finally cut off from the basal nucleated portion. This t}'pe of secretion occurs in the mammary gland. The cells whose apical portions have been cut off do not disintegrate following the libera- tion of the secretion but develop a new apical portion and repeat the process. A convenient classification of the exocrine glands may be built upon their organization regardless of the method of secretion. Unicellular Glands. ^In the skin of fishes and amphibians there are scattered single cells that secrete a fluid substance. Also, among the columnar cells lining the gut regions are the numerous goblet cells, already mentioned under columnar epithelia. Mucin, 42 THE EPITHELIAL TISSUES which is elaborated in the cytoplasm of these cells, collects toward the free end, water is absorbed, and the volume of the secretion is increased so that the apical end becomes much distended. The distention progresses until there remains so thin a sheath of cyto- plasm about the secretion that a rupture occurs and there is a discharge of the mucus. Such goblet cells apparently may undergo a number of repetitions of this apocrine type of secretion before disintegrating. Secreting Areas.— As a further step beyond the unicellular condi- tion, there are certain areas among cells forming an epithelial membrane where a small aggregation of cells function as a special secreting group, while the surrounding cells, although similar in form, function chiefly in protection and absorption. Such areas occur among the columnar cells lining the uterus, in the epithelial membrane lining the trachea, and in the thin membrane of cuboidal or low columnar cells forming the choroid plexes in the brain. Glandular Pockets.— Occuvrmg, at intervals in the epithelial mem- branes lining the various ducts there are shallow pockets lined by secretory cells. In the trachea, cloaca, and urethra, for example, are small pockets usually lined with mucous secreting cells. Those cells near the mouth of the pocket are cuboidal or short columnar, while toward the bottom of the pocket are broader and longer columnar cells. Simple Tubular Glands. — As the name implies, these glands result from tubular invaginations of epithelial membranes during develop- ment. The oviducts of the frog and other egg-laying forms are usually lined with tubular glands whose secretions are added to the descending eggs. The epithelial wall of the large intestine of mam- mals possesses a multitude of simple tubular glands closely adjoining each other, their walls composed of columnar cells, of which the majority are of the goblet type. The cavity in these tubes is called the lumen of the gland and may vary considerably in length. The secretions of the cells pass into the lumen and out to the surface of the epithelial membrane from which the gland forms. A modification of these straight tubular glands can be seen in the type represented by the sweat glands in mammalian integument. These glands have an excretory superficial portion which is long and spirals from the epidermis through the underlying connective tissue to the deeper, much-coiled portion. The deeper portion is com- posed of actively secreting cells and may be called the secreting end-piece of the gland. The mucosa of the fundus of the stomach SECRETORY AM) EPITHELIAL CELLS 43 in mammals is composed of branched tubular o;lan(ls with a single short excretory duct portion and two or more longer, slightly twisted, tubular ])ortions composed of secretory cells. (Figs. 17 to 19.) Simple Alveolar (llaiuLs. —These glandular invaginations develop a spherical termination instead of the tubular type just described. In simple alveolar glands the enlarged si)herical terminal jiortion is called an alveolus or acinus. These are connnon in the skin of certain lower vertebrates but do not occur among the mammals. An example of this type may be found in the skin of the frog where a saccular base lies below the stratified squamous epithelium of n^ gH Fig. 17 Fig. Fig. 19 Fig. 17. — Simple tubular gland. Fig. 18.— Coiled tubular (sweat) gland. Fig. 19.— Simple branched tubular gland (stomach). the skin epidermis and connects with the surface by a passageway through the strata of epithelial cells. This simple type of gland may also be modified b\- having two or more alveoli attached to the end of a single excretory duct, as in the case of the sebaceous glands where alveoli are connected with the lateral walls of a common excre- tory duct. (Figs. 20 and 21.) Compound Tuhiilar (Hands.— The formation of compound glands may be visualized by assuming that, instead of stopping with the simple tubular invagination of embryonic development, the early invagination has gone on developing branches from its deeper portions. These secondary branches give rise to tertiary ones, and so on. (Fig. 22.) The outermost end-portions of the smallest 44 THE EPITHELIAL TISSUES branches alone possess the secretory cells and the other portions form the excretory duct system. The mucous glands of the mouth cavity, the prostate, and the kidney are examples of such tubular glands. Fig. 20 Fio. 21 Fig. 20. — Simple alveolar gland (frog skin). Fig. 21. — Simple branched alveolar gland (sebaceous). Compound glands are invested with loosely arranged connective tissue extending l)et\veen the excretory ducts and secretory end- pieces. The large masses thus separated by connective tissue are known as lobes and the smaller subdivisions are called lobules. Fig. 22 Fig. 23 Fig. 24 Fig. 22. — Compound tubular gland. Fig. 23. — Compound tubular-alveolar gland (submaxillary). Fig. 24. — Compound alveolar gland (lung). The main excretory- ducts accompanied by blood and lymph vessels, and ner\'es are carried in the connective tissue between the lobules. Immediately surrounding the basement membrane of the gland cells of the secreting end-pieces is a capillary network and also a plexus of nerve fibers. SECRETORY AND EPITHELIAL CELLS 45 The liver in lower forms is a ('oiii])ouii(l tuhiilar j>;laii(l in form, hut in the higher vertebrates this form may he lost during development, as will be seen when studying this organ. Compound Alveolar Glands. Thv manner in wiiicli these glands develop is similar to that in the case of tubular glands, but the secreting end-pieces are expanded into alveoli. (Fig. 24.) The lung develops as this type of gland but is not usually considered as a gland, though it might be considered as secreting carbon dioxide and absorbing oxygen. The mammary gland is another example illustrating the structure of a compound alveolar gland. The salivary glands and the pancreas, considered as tubulo-alveolar, have the outward appearance of compound alveolar glands, but the lumens of the secreting end-pieces are tubular in form. (Fig. 28.) SeroNs and Mucous Glands. — On the basis of secretory products there are some glands whose secretions are of the watery or serous type; others secrete a thicker, viscous, mucilagin- ous substance, called mucus. The cells secreting these two ty^jes of material have certain distinctive features. (Fig. 25.) The serous cells usually ha^•e a clouded cytoplasm with a rounded luicleus located toward the center of the cell ; the mucous cells are generally larger, haxe a clearer cytoplasm, aufl have an oval, flattened nucleus located well tow^ard the base of the cell. The secreting end-pieces of the serous glands have a small lumen when compared with the relatively wide lumen in the end-pieces of mucous glands. The ])ancreas presents an example of serous secretion. The salivary glands, which var\' somewhat in this respect, may present either serous or mucous or a combination of both, as will be observed in studying these glands later. Endocrine Glands.^ These glandular organizations are very richly supplied with blood vessels, which their secretions enter and are circulated by means of the blood stream. The thyroid is an example of this ty])e of gland; it begins its development as do other glands, feq fv~ Si ^fLVver of cuboidal epithelium. The other endocrine glands are best studied in the chapters dealing with them. REFERENCES. BowEN, R. H. 1929. The cytology of glandular secretion, Quart. Rev. Biol, 4, 299. Bralter, a. 1926. The regeneration of transitional epithelium, Anat. Rec, 33, 137. Chambers, R., and Renyi, G. S. 1925. The structure of the cells in tissues as revealed by microdissection: I. The physical relationships of cells in epithelia. Am. Jour. Anat., 35, 385. Florey, H., Carleton, H. M., and Wells, A. G. 1932. Study of alterations in epithelia, Brit. Jour. Exp. Pathol., 13, 269. Gaebler, O. H. 1921. Bladder epithehum in contraction and distention, Anat. Rec, 20, 129. Thuringer, J. M. 1924 1928. Mitotic activity in stratified epithelium, Anat. Rec, 28, 31; 40, 1. See Appendix for general text references. CHAPTER III. TIIK rDXXF/TIVF. TISSFES. In contrast to epithelial tissues, where intercellular material is ab- sent or insignificant in amount, in connective tissues the emphasis is placed upon the nature of the intercellular material. The cells of con- nective tissues usually form a small part of the tissue mass and are partially or completely separated from each other by the various types of intercellular material which they form. This emphasis upon the non-cellular, intercellular products of connective tissues is directly associated with the role they play. In general, connective tissues form the supporting structures of the body, from the heavy framework of the bony skeleton to the finer networks supporting the capillaries. In addition to the formation of intercellular products some of the cellular components of the tissues are active in storage and phagocytosis. The various t^-pes of connective tissues trace their origin back to the mesoderm of early stages of embryonic development. Classification.— As one might expect from the nature of connective tissues, classification of the various t>-pes is based both upon the cellular constitution and the nature and arrangement of the inter- cellular deposits. There are intergrading forms and also some variation in the general characteristics considered as distinguishing the t\"pes. so that here, as in the case of epithelia, there may be difficulty in classification. Two t^-pes, mesenchyme and mucous tissue, are mainly embryonic. In the adult vertebrate, the following t^npes are distinguished: reticular tissue, loosely organized fibro- elastic tissue, adipose tissue, densely organized fibrous and elastic tissue, cartilage, and bone. These X\"pes include modifications or subdivisions which may be best considered in order when describing the features of each tx-pe. MESENCHYME. As soon as the three germ layers are well established in the embryonic development, cells begin moving away from the meso- derm to occupy spaces between it and the ectoderm and endo- derm. These early, wandering cells compose mesenchyme tissue. 48 THE CONNECTIVE TISSUES They have an irregular, stellate form with branching cytoplasmic processes, and have the appearance of a syncytium. The nucleus is relatively large and mitotic figures are often evident. An appar- ently homogeneous, liquid, intercellular substance fills in the spaces between adjoining cells. (Fig. 2().) From such mesenchyme, or embryonic connective-tissue cells, are derived the various dift'er- entiated cells that form the cellular elements of the connective-tissue types of the adult. They also give rise to cells that in turn differ- entiate into endothelium, blood, and smooth muscle cells. Fig. 26. — Diagram of mesenchyme cells. MUCOUS TISSUE. This is a special type of connective tissue found in embryos, and reacts to stains for mucus. In the fresh condition, as seen in the case of an umbilical cord, it presents a jelly-like appearance due to the abundant gelatinous intercellular material. Within this gelatin- ous matrix are fine fibers and scattered cells with long branching processes. The ends of the branches of one cell are in contact with other cell branches, so that a network of cells and fibers extends throughout the mucoid ground substance. In the case of the umbilical cord this tissue occurs between the blood vessels and ducts RETICULAR. CONNECTIVE TISSUE 4!) passing from the ])la('enta to the fetus. A siniihirl\- api)eariiig tissue may l)e fouud iu other refjions of the embryo ])rece(Iing the chffer- entiation of the fihroelastic conneeti\-e tissue. RETICULAR CONNECTIVE TISSUE. This widely (Hstrihuted tissue is not easily demonstrated in routine ])reparations. It ean l)e demonstrated assoeiated with secretory epithelial organizations of glands and in the framework of lymph organs. The cells resemble mesenchxine cells but are larger, with a large, oval nucleus and long branching cytoi)lasmic processes making contact, if not continuing with adjoining cells. (Fig. 27.) Associated with the cells are branching fibers which have an affinity for siher salts, so that they are called argyrophil Fig. 27. — Diagram of reticular tissue. fibers. By the use of silver salts which l)lacken them, these fibers can be demonstrated as branching and anastomosing to form deli- cate networks in all regions of the body. They are continuous with the collagenous fibers of the loose fibroelastic connective tissue which do not react in the same maimer to the sih'er salts. Embryo- logically the argyrophil fibers appear before the collagenous type, so that reticular tissue may be considered as the finer, less differ- entiated continuations of the loose fil^roelastic connective tissue, an intermediate condition l)etween it and mesenchyme. The wide distribution of reticular tissue in close association with and transition 4 50 THE CONNECTIVE TISSUES to the loose fibroelastic type often make it difficult to differentiate. The appearance of the reticular cells may be studied in routine preparations of lymph glands of mammals. In the central portion of such a gland there is a diffuse arrangement of tissue and a net- work of reticular cells may be observed forming a support for the lymphocytes. LOOSELY ORGANIZED FIBROELASTIC TISSUE. This is probably the most widely distributed type of connective tissue and also contains the component elements to be found in other types. It is well exemplified by subcutaneous tissue W'hich is easily accessible for study. When the skin is removed from a freshly killed mammal, a moist, white, filmy tissue is seen lining the under- surface of the skin. It tears easily as the skin is removed; part remains attached to the skin and part adheres to the underlying tissues of the body. It has been called areolar tissue because open spaces may be seen among the fibrous intercellular elements when spread out on a slide. In the normal state of the tissue, however, these spaces are filled with a tissue juice, or ground substance, so that it is advisable to use the longer, more descriptive term instead of the term areolar which is misleading. Study of this tissue clearly reveals cells and fibers, but the ground substance or tissue juice noted by the feeling of moisture in the fresh tissue, is difficult to demonstrate in preparations. Cell Types. — Cells are not easily seen in fresh tissue mounts, nor is it easy to demonstrate all the types in any one preparation. They are often studied by means of intravitam staining, a process in which small amounts of harmless dyes, such as neutral red, are introduced subcutaneously in a living animal, and, later, a small amount of the subcutaneous tissue is carefully removed for study. In such cases, certain cells take up the dyes in a characteristic fashion. Fibrocytes. ^Verived directly from mesenchyme, these cells become larger, elongated, and flattened. They have long cyto- plasmic processes and in edge views or sections they appear spindle- or rod-shaped. The cytoplasm is clear and stains only faintly with acid dyes, thus making the cell outline indistinct in preparations. The nucleus is relatively large and oval and appears lightly stained. A nucleolus is usually present. In some ])re])arations furrows appear on the surface of the cells presumably as a result of their close association with fibers. Early investigators concluded that LOOSELY ORGANIZED FIBROELASTIC TISSUE 51 these cells produce the material toriniii^f the fibers, and the terms fibroblast and fibrocytes were applied to them. The long proc- esses appear to be in contact with similar processes of neighbor- ing cells, if not actually fusing with them, but the cells a])i)ear to act as independent units rather than as a syncytium. It is not believed that the fibrocytes transform into the other types of cells found in this tissue. (Fig. 28.) Histiocytes.— This type of cell is almost as numerous as the fibro- cyte. It is smaller and has a few blunt cytoplasmic processes. Fig. 28. — Photograph of a spread of subcutaneous tissue of the cat showing histiocytes and nuclei of fibroblasts. The cytoplasm stains with acid dyes, and the cellular outline is visible. The nucleus is relatively small, oval or bean^shaped, with distinct chromatin masse which cause it to stain darkly and stand out clearly in preparations. A nucleolus is not usually present. Ordinarily these cells are at rest, but imder inflammatory conditions they migrate in the tissue spaces by ameboid motion. This type of movement is not exliibited by the fibrocytes. The histiocytes are capable of phagocytic actiAity and engulf bacteria, colloidal dyes, or other particulate matter. I )uring active phagocytosis they are known as macrophages and their numbers increase at points of infection and inflammation. The terms clasmatocyte and resting wandering cell, are also used as names for these cells. Amehoid Wandcriuy (VV/.s'.— These cells resemble certain of the non-granular leukocA'tes of the blood so closeh' that some investi- 52 THE CONNECTIVE TISSUES gators have concluded that many are derived from lymphocytes and monocytes escaping into the tissue from the capillaries, and others are derived from mesenchymal cells in the tissue. The nucleus is relatively large and the cytoplasm stains with basic dyes. They vary in size and shape, so that gradations from those re- sembling the leukocytes to those resembling typical histiocytes may be found, this being especially true in inflamed tissue. Mast Cells.— This type of cell occurs in most vertebrates but varies in distribution. It is characterized as having cytoplasmic granules that stain selectively with basic aniline dyes. The cyto- plasm of these cells is completely filled with granules, which in some animals are water-soluble. In mammals the cells are usuallv Fig. 29. — Photograph of chromatophores in the connective tissue of a section of cleared unstained king of the bullfrog. irregularly rounded, but in certain amphibians long cytoplasmic pro- cesses are often present. When neutral red is used as a vital stain, the cytoplasm appears filled with dark red granules, which methylene blue stains purple. Pigment Celfe.— Elongated cells with irregular cytoplasmic pro- cesses containing pigment granules occur commonly in loose fibro- elastic connective tissue in certain regions and are known as chroma- tophores. (Fig. 29.) In mammals such pigment cells occur below the epidermis and in the choroid coat of the eye, but in lower verte- brates similar cells are much more widely distributed and occur in many internal organs. In fish and amphibians the ])igment cells in the skin take part in the color changes exhibited by many forms. Such changes are considered as due to alteration in tiie distribution LOOSELY ORCAMZEI) Fl Bh'OELASTlC TISSUE 53 of the jjranules. rignuMit cells containing the black melanin graiuiles are called melanophores; those with yellow-red granules are called xanth()])h()res; and those with brown-red granules are designated erythrophores. No single species necessarily otters examples of all three types of cells. In some fishes guanine crystals contained in the pigment cells are responsible for the silvery color of the scales. Undifferentiated {Mesenchymal) Cells.— Msiny mesenchyme cells remain undift'erentiated and scattered throughout this type of con- nective tissue, usually near the blood vessels. Such cells are smaller than the fibrocytes but not otherwise sharply distinguishable from them in appearance. However, these cells are capable of dift'erenti- ating into various types of connective tissue. Fat Cells. — Certain cells of this tissue are characterized by a storage of fat droplets until the cell becomes distended to a spherical shape, with its nucleus and thin layer of cytoplasm pushed to the periphery. (Fig. 30.) Such cells occur singly or in groups along Fig. .30. — Diagram showing afrumiilation of fat inside of a fibroblast and the develop- ment of this into a fat cell. capillaries, and organizations of them constitute adipose tissue, which we will consider separately. Fibers. The fibrous elements which give the name to this tissue are divided into two tyi)es, a white or collagenous and a yellow or elastic fiber. Collagenous Fibers. (Fig. 31.)— These fibers are almost trans- parent in the living tissue and are called collagenous because they yield a gelatin when boiled in water. Fixed and stained spreads show a network of fibers extending in all directions. Study shows that the strands are bundles of microscopic fibrils, the larger strands containing more fibrils than the smaller ones. These fibrils are presumably held together in bundles by a cement substance which dissolves when fresh material is soaked in lime water. The fibrils do not branch but their organization into bundles is such that the bundles branch and form networks. In dilute alkalies like potassium hydroxide, and such acids as acetic, 54 THE CONNECTIVE TISSUES the fibrils become swollen. They are digested by acid solutions of pepsin. In preparing slides to show them, fixing solutions contain- ing acetic acid should be avoided since it causes swelling and distortion of the collagenous material. They are stained by eosin and other acid dyes but stand out sharply with aniline blue. Their jiroperties appear to show some variation in dift'erent regions of the same animal as well as in difi^erent animals. They do not stretch, but through their arrangement in networks adapt themselves to possible expansions of the tissue by changes in the meshes. Elastic i^iter^.— Homogeneous threads or small bands of varying size occur as distinct, refractive, and non-fibrillar elements. They are composed of a compound called elastin, and branch to form a network. These fibers are decidedly elastic; under tension they stretch but resume their original form when re- leased. When they are broken the ends curl up. Grouped in large numbers, these fibers ap- pear yellow in color. They are not affected by boiling in weak acids, or alkalies, but are slowly digested in solutions of pepsin and trjqjsin. Specific stains, such as resorcin-fuchsin and orcein make them stand out as distinct branching threads in preparations. Through combination with one another these fibers form bands or membranes in the walls of large arteries. Function.— Loose fibroelastic connecti^•e tissue forms a strong elastic material which is light and flexible and adapted to holding together other tissues which are especially concerned with secretion, absorption, movement, storage, and conduction. It is the chief tissue for the support of the blood vessels and the capillary networks supplying nutrition and oxygen to the tissues involved in secretion and excretion. Furthermore, before reaching other cells both food and oxygen must pass from the cai)illaries into the groimd substance or tissue juice of this tissue. Likewise, such wastes of metabolism as urea, carbon dioxide, and water pass through the tissue juice on their way to the capillaries prior to elimination. Thus, the element Fig. 31. — Diagram of loose fibroelastic connective tissue. 1, bundles of collagen- ous fibers; 2, elastic fibers. Tissue spaces are indicated between the fibers. ADIPOSE TISSUE 55 of the tissue not usually denioustrated in preparations i)lays an extremely vital part in metabolism. This type of tissue plays an important role in repair following injuries and infeetions and is closely linked to serological reactions which are occupying those interested in innnunology. SEROUS MEMBRANES. The peritoneum, pleunie, and pericardium are thin layers of fibroelastic connective tissue covered with mesothelium. The cellu- lar elements are more numerous in these membranes than in the loose fibroelastic connective tissue of other regions. The mesenteries are composed of similar thin membranes of connective tissue with mesothelium on both surfaces. The cells of serous membranes produce a watery, somewhat viscous fluid, which lubricates the surfaces of organs as they move over one another during their physiological activities. Histiocytes as well as white blood cells, especially neutrophils, increase in number in the fluid ground sub- stance of these membranes in inflammation. Fat cells may accu- mulate in certain regions to form considerable masses. ADIPOSE TISSUE. This is a modification of the loose fibroelastic type in which many of the cells become active in storage of fat. (Fig. 32.) The fat- storing cells are considered by some to be directly derived from fibrocytes but others believe them to be more directly derived from undifl'erentiated mesenchyme cells, called adipoblasts. How- ever derived, these cells occur in large or scattered masses among the other elements of loosely organized fibroelastic tissue. During transformation of fibrocytes or adipoblasts into typical fat cells, droplets of fat accumulate within the cytoplasm. The cell becomes distended into a spherical mass with a protoplasmic periphery. When a number of fat cells are formed close together, the spherical form of each is modificfl at points of contact, so that such cells appear as polyhedral bodies resembling a mass of small soap bubbles. In such groups the dift'erent portions of the cell membranes may be seen by changing focus. In a number of loca- tions, fat cells accumulate in small or large masses, as will be observed in sections of the skin, the mesenteries, the supporting tissues of the thymus, kidney, and intestinal tract. Under condi- 5G THE CONNECTIVE TISSUES tions of starvation the fat stored in these cehs is hberated by met- aboHsm and used as a source of energy. The cells then resume a condition in which only scattered droplets appear in the cytoplasm, but with favorable conditions the storage may be repeated. Fat accumulated during the summer season in hibernating animals is metabolized during the winter dormant season in maintaining the life of these forms. In ordinary routine techniques preparing tissue for examination, the fat is dissolved out, leaving the cells as mere empty shells in the final preparation. Fat cells may be handled so that the fat is fixed, as when using osmium tetroxide, and carried through to the final preparation. Other solutions, such as Sudan ITT, stain fat specifically and may also be used to indicate it. Fig. 32.— Adipose tissue of the fat body of Amblystoma. DENSELY ORGANIZED COLLAGENOUS AND ELASTIC CONNECTIVE TISSUES. Under this heading there are irregularly arranged tissues resem- bling the loose fibroelastic type but having their intercellular material present in far greater (juantity. There are other types in which the intercellular fibrous materials, either elastic or collagenous, are arranged ])aralleling each other. Dense irregularly arranged fibroelastic tissue with the collagenous fibers ])red()miuating and with ])roportionately few cells, which are mainly fibroblasts, forms the dermis of the skin in mammals, i)art COLLAGENOUS AND ELASTIC CONNECTIVE TISSUES 'u of the wall of thr ' the same as in intramembranous ossification. In the center of the cartilaginous piece ossification activity is preceded by enlargement of the cartilage cells and their arrangement into rows accompanied by a calcification of the hyaline matrix separating them. Two things happen simultaneously. From the inner cellular region of the perichondrium, bud-like vascular tufts burrow into the cartilage and develop a primary marrow tissue. Within the center of the cartilage a number of cartilage cells and some of the matrix disintegrate and are resorbed, and into the spaces thus formed the tufts of primary marrow tissue extend. These invaginations of tissue carry in osteoblasts and other elements of bone-marrow, which continue to extend into the progressively forming cavities. At first, the osteoblasts deposit bony matrix on calcified cartilage spicules not resorbed in the first dissolution process and then at many other points form spicules of bone in the same manner as in intramembranous bone formation. (Fig. 35.) The dissolution of cartilage progresses toward either end of the cartilage piece and marrow tissue deposits bony spicules until the major portion of the cartilage region has been replaced by spongy bone. Associated with the destruction of the cartilage are chondroclasts, large multi- nucleated cells. With the initiation of ossification within the cartilaginous piece, there also occurs a change along the periphery where the inner cells of the perichondrium become osteogenic, and this outer fibrous sheath surrounding the developing bone becomes the periosteum. In the same manner as in the plates of membranous bone, osteo- blasts from the periosteum then deposit jieripheral or periosteal lamella? about the spongy bone de\eloping internally. In this type of bone there is also a resorption of the early bony matrix in the course of growth and moulding of the mature bone. During these disintegration processes, tubular channels become hollowed out and the osteogenic tissue projecting into them deposits 64 THE CONNECTIVE TISSUES secondary })ony matrix in the form of concentric lamella^, one within the other, surrouncHng a central cyhnch'ical cavity containing some osteogenic tissue, blood vessels, and nerve fibers. Such a series of lamellae with the ca\'ity just described is called an Haversian sys- tem; the lamellie are Haversian lamelhe and the central cavity is Fig. 35. — Phc)tograi)h of a portion of the diaphysis .shown in Fig. .Hf). The arrange- ment of the cartilage cells, the resorption of the matrix between the adjacent cells and the extension of marrow tissue are shown in studying from top toward the bot- tom. In the central and lower portion bony spicules have been deposited by the osteoblasts; at the right (dark portion) the periosteum is depositing layers of bony matrix in the same manner as in the case of membranous bone. A ijrojection of vas- cular tissue (light area beginning in middle of right margin) is shown extending from the perichondrium through the periosteal bone into the central marrow tissue. the Haversian canal. These systems run ])rimarily lengthwise of the bone, but there are lateral connections between adjacent Haversian canals. Many small canals without lamelhe extend fi'om the surface of the bone into Ilavei'sian canals and similar canals extend from the central marrow ca\'it\' outward connecting with Ilaxersian BONE {):^ canals. These are called \ Olkiiianirs canals and carry N'ascular tissue into the Haversian canals. In many ])hices hiindles of collagenous fibers from the ])eri()steum extend into the periosteal l)one to form Sharper's fil)ers which tightly attach the periosteum to the underh ing hone. Fig. 36. — Photograph of a longitudinal section of the developing rabbit femur. Part of its diaphysis (below) and an epiphysis (above), show endochondral bone formation. In both, the cartilage is resorbed and replaced by spongy bone, in the epiphysis the final bone is irregular and spongy, but in the diaphysis Haversian systems are formed. Growth of the intervening hyaline cartilage region and pro- gressive resorption of it and replacement by bone make possible growth in length of the bone. In the epiphysis there is a network of broad bony pieces and large marrow cavities; in the diai^hysis there are more numerous thinner bony spicules separated by narrow cavities. The majority of the bony strands here are deposited paralleling the length of the bone along lines indicated by the paralleled columns of cartilage cells which are resorbed as the marrow tissue extends. Fig. 35 shows the right portion of the section in greater detail. Growth in thickness of a long bone is made possible liy resorption; this increases the central marrow cavity, and new bone is added as periosteal lamelht^. Resorption extends into ]M-imary Haversian systems, carrying along osteogenic tissue into the marrow ca\ities 5 66 THE CONNECTIVE TISSUES so formed, and new Haversian systems are formed. The central marrow cavity becomes lined with endosteum which resembles periosteum and forms endosteal lamelhe. Growth in length of long bones is brought about by the continued formation of more cartilage at the extremities of the shaft and its progressive replacement by bone. (Fig. 36.) Between birth and maturity ossification centers appear in the terminal cartilage pieces or epiphyses and form spongy bone similar to that formed in the vertebrae. In both of these locations Haversian systems are not formed. Each bony epiphysis is separated for some time from the end of the shaft or diaphysis adjacent to it by a plate of cartilage, but at maturity even these plates change to bone so that one con- tinuous bone is formed and growth in length is at an end. The ends of bones retain a disc of cartilage which acts as a pad in the region of joints. In the case of endochondral bone formation in the small lower vertebrates, the process of bone deposition appears to be primarily peripheral, little spongy bone being formed in the course of cartilage resorption of the shaft. Epiphyval region Epiphysial region Shaft Spongy bone Marroir cavity Hard bone Fig. 37. — Diagram of a long bone. Microanatomy of Long Bones. (Fig. 37.)— Let us take a long bone such as the femur and di\ide a consideration of it into the following parts periosteum, epiphyses, diaphysis, and the marrow. Periosteum covers the bone except at the end-surfaces exposed to contact with adjacent bones at the joints where ])erichondrium and a pad of cartilage occur. Periosteum is a dense, fibrous con- nective tissue externally but grades into material of looser texture toward the b(me. It su])ports arteries, veins, lymphatics, and nerves, which are carried into the Haversian canals of the bone by Volkmann's canals. The bundles of collagenous fibers, Sharpey's BONE 07 fibers, extciuling from the periosteum into the bony matrix ht)l(i the two tightly together. Each epii)hysis is larger than the shaft in diameter and has a very thin jjeripheral layer of hard, compact bone enclosing a mass of spongy bone between whose spicules is red marrow. Among the constituents of red marrow are: a small anioimt of loose fibro- elastic connective tissue and some fat cells; reticular tissue; erythro- cytes; and a variety of cells in different phases of differentiation from primitiAc blood cells to both red and white blood cells; blood vessels; lymphatics; and nerve fibers. *, * jv ^^ * vS®^'''™*'' Haversian ^^^^^;^>^ InfersLHial-%_:'l 1^^^^ •^^^J^^'^-J^f' huiicllw Fig. 38. — Diagram of cross-section of shaft of a long bone. The diaphysis has four systems of lamella^. (Fig. 38.) Imme- diately })elow the periosteum are several periosteal lamella^ encircling the shaft, these are called the external circumferential lamelhv. Internally, adjacent to the marrow cavity, a few similarly disposed endosteal lamellse occur and are described as internal circumferential lamella?. Between these outer and inner lamelUe are arranged sys- tems of Haversian lamellse. Portions of Haversian lamell* left over from earlier Haversian systems are called interstitial lamella^ and occur between complete later Ha^'ersian systems. Attention has already been called to the lacuntt^ and canaliculi occupied by the G8 THE CONNECriVE TISSUES osteocytes in livino; bone. (Fig- 39.) These cells secure their nutrition from the Haversian canal which carries the vascular tissue. There is one main nutrient foramen near the middle of the shaft which carries the medullary artery from the periosteum into the central marrow cavity and at the epiphyses there are several smaller foramina. Within the matrix of the bone there are numerous collagenous fibers extending in different directions in adjacent lamellte, thereby increasing the strength of the shaft. In the central marrow cavity the same marrow elements are present as in the epiphyses but there is an increasing accumulation of fat cells in this tissue with advancing age and loss of blood-forming activity. Fig. .39. — Photograph of a portion of a cross-sention of an Haversian system showing the lacuntp and canaUculi occupied by the osteocytes in Uving bone. The alternating dark and Hght lamelhe indicate differences in their fibrous organization. Marrow. — In young animals, medullary spaces in bone are filled with red marrow. This is composed of very loose fibroelastic ct)n- nective tissue cells, reticular fibers and cells, nerve fibers, a few fat cells, medullary sinusoids, a great variety of primiti^-e blood cells and stages in blood cell formation, mature blood cells, and mega- karyocytes. Since it is an hemapoietic center it is more fully ex- plained under blood. With increasing age the marrow in the medullary cavity of the shaft changes in character. The hemapoietic proi)erty is lost. A great predominance of fat cells develops and since it has a light yellow color it is known as yellow marrow. The marrow in the epiphyses remains red. REFERENCES 69 THE NOTOCHORD. A rod of cells, the notochord, forms just dorscal to the primitive gut during the embryonic development of all vertebrates. These cells become distended to a spherical form by a considerable con- tent of fluid and by their turgidity may give some support to the early embryo. The nuclei are small and centrally located. The position of the nuclei and the spherical shape of these cells help to distinguish them from fat cells, which they resemble in routine preparations. This tissue is supplanted by cartilaginous tissue forming from mesenchyme in the surrounding region. REFERENCES. Arey, I.. B. 1932. Certain basic principles of wound healing, Anat Rec 51, 299. AscHOFF, L. 1924. Reticulo-endothelial System: Lecture.s on Pathology, New York, Paul B. Hoeber, Inc. Bensley, S. H. 1934. On the presence, properties and distribution of the intercellular ground substance of loose connective tissue, Anat. Rec, 60, 93. Carrel, A., and Ebeling, A. 1926. Fibroblast and macrophage. Jour Exp Med., 44, 261, 285. Dawson, H. B. 1934. Further studies on epiphyseal union in the skeleton of the rat, Anat. Rec, 60, 83. DoDDS, G. S. 1932. Osteoclasts and cartilage removal in endochondral ossi- cation of certain mammals, Am. Jour. Anat., 50, 97. Fr.\n§ois-Franck, L., Back, A., and Faure-Fremiet, E. 1932. Etude microcincmatographique du mesenchyme des salmonides, Compt. rend. Assn. anat., 27, 284. Ham, a. W. 1931. The variabihty of the planes of cell division in the cartilage columns of the growing epiphyseal plate, Anat. Rec, 51, 125. Key, J. A. 1928. The cytology of the synovial fluid of normal joints, Anat. Rec, vol. 40. Lewis, W. H. 1922. Is mesenchyme a syncytium? Anat. Rec, vol. 23, 177. Rice, H. G., and Jackson, C. M. 1934. The histological distribution of fats in the liver, kidney, trachea, lung and skin of the rat at various postnatal stages, Anat. Rec, 59, 135. Ruth, E. B. 1934. The Os Priapi: A study in bone development, Anat. Rec, 60, 231. Singer, E. 1933. Structure and relations of the leucophores of Rana pipiens as revealed by the ultra-violet and fluorescent light, Anat. Rec, 58, 93. Summer, F. B., and Wells, N. A. 1933 The effects of optic stimuli upon the formation and destruction of melanin pigment in fishes. Jour. Exp Zool 64, 377. See Appendix for general text references. CHAPTER IV. THE BLOOD. Blood might well be considered under connective tissue as a type in which the intercellular material, the plasma, is a fluid carrying a variety of free cells. Both the fluid and the cellular elements circulate through the body in endothelial-lined vessels and act as media for metabolic exchanges. The blood also plays a vital part in integrating and regulating the activities of the other tissues through the endocrine secretions which it carries. THE PLASMA. Although the plasma ordinarily presents no structural features in histological preparations, its physiological importance should not be overlooked. Biochemical and biophysical studies show the plasma to be composed largely of water with numerous substances in solution. Various salts, sugar, proteins, and fat are held at relatively constant concentration levels in the blood. During cir- culation they are made available to the various cells of the body by dift'usion through the capillary walls into the tissue juice at the same time that metabolic wastes are entering the blood through the capillaries from the tissue juice. The balance is kept constant by a continuous replenishing of the various nutritive substances coupled with an equivalent removal of wastes. Since some knowl- edge of the role of the plasma in relation to processes concerned with the internal environment of the organism is essential to an understanding of the functioning of the tissues and organs, the student is advised to consult the references given at the end of the chapter. THE BLOOD CELLS. The cells of the blood are divided into two main groups: the the erythrocytes, or red blood cells, and the leukocytes, or white blood cells. In addition to these there may be small cytoplasmic bodies, the platelets in the case of mammals, and spindle cells or thrombocvtes in lower forms. THE BLOOD CELLS 71 Erythrocytes. —These cells take their name from the red color they give the blood en masse, but single cells have a pale yellow-green color. In mammals the erythrocytes are enucleated, that is they lose their nuclei before entering the circulating blood, but in other vertebrates they are true cells, usually flattened ovals in shape, with a central nucleus causing a cellular bulge. There is great variation in their size in different species, as evidenced by the following list of measurements: in Amphiuma (Fig. 40), which has the largest, the cells are about 80 microns in diameter; in the frog they are 22.3 by 15.7 microns; in the lizard they are 15.8 by 9.9 microns; in the rabbit the diameter is about 6.9 microns; in the cat, (3.3 microns; in the musk deer, 2.5 microns; and in man, 7.5 Fig. 40. — Photograph of blood cells of Amphiuma, showing erythrocytes and heterophils. microns. In many species, especially those with nucleated erythro- cytes, there may be a considerable variation in size in the same individual. When a drop of fresh mammalian blood is spread out in a thin film on a glass slide, the erythrocytes tend to collect in groups called rouleaux, strings of bi-concave discs. The formation of such strings indicates a certain cohesiveness in the limiting membranes. Two components of these cells ha\-e been distinguished, a stroma or framework which is an optically homogeneous colloidal substance, and hemoglobin which is a conjugated protein composed of globin and a colored compoimd of iron called hemochromogen. The 72 THE BLOOD hemoglobin is discharged from cells subjected to various agents in the process known as hemolysis. It can be induced experimentally by increasing the concentration of salts in the fluid surrounding the cells, by repeated freezing and thawing of blood, by addition of chloroform or ether, and by the action of electric currents. Ilemo- lyzed blood cells are said to be laked, and when this occurs in the circulating blood, as it does in the case of certain pathologic condi- tions, the hemoglobin is eliminated from the plasma in the kidneys and passes out with the urine. The surface of the red cell is somewhat more dense than the interior protoplasm. It is semipermeable and in osmotic equilib- rium with the solutions normally present outside and is said to be isotonic with them. Mammalian plasma has about the same osmotic pressure as an 0.85 per cent sodium chloride solution, which is isotonic with the protoplasm within the red corpuscle. Frog plasma is similar to a 0.7 per cent sodium chloride solution. If distilled water is added to a drop of fresh blood on a slide, microscopic ob- servations show the erythrocytes increasing in size as they absorb water. If the swelling is allowed to continue the cells become spherical, and hemolysis occurs. This change is a result of endos- mosis, since the solution has a concentration less than that of the protoplasm within the red cells. Such a solution is said to be hypo- tonic. A reversal of this process occurs if the external medium is more concentrated, or hyj^ertonic. \Yater is then extracted from the red cells which become wrinkled or crenated, and finally hemo- lysis takes place. Oxygen diffuses through the membranes of the respiratory surfaces into the tissue juice and then through the endothelial walls of the capillaries. As the erythrocytes circulate slowly through the capillaries in\'esting the air sacs of the lungs, or through the ca])illaries in the gills and skin of aquatic animals, the hemo- globin unites in a weak chemical combination with oxygen to form oxyhemoglobin. In this form oxygen is carried to the various tis- .sues of the body and is given up as needed. The surface area of all the red cells may be computed on the basis of indi\idual cell size and the estimated number of red cells in the body. In the case of man, estimates from 2500 to 4500 square meters \m\e been worked out, areas many times that of the body. Depending largely upon the severity of service, there is a limit to the functioning of the red cells. When the colloidal composition ages beyond a certain ])oint a granular condition results and the red cells are broken up. These THE BLOOD CELLS 73 cells are removed from the circulation in the s]>lcen and liver and new cells arising in the blood-forming centers take their places. The reasons given for the segregation of hemoglobin in special cells or corpuscles, a condition characteristic of vertebrates and rarely found in animals of the lower phyla, is that capillary walls are so constructed that if the hemoglobin were free in the i)lasma it would leave the capillaries and be eliminated from the circulation, as occurs in cases where red cells are destroyed, as in malaria. ?\u-ther- more, the retention of hemoglobin within the ca])illaries, should it be free in the plasma, w'ould necessitate the capillary walls being so constructed as to prevent its passage. If this were so, various Erythrocyte Lymphocytes Monocyte Neutrophil Eosinophil- Basophil Fig. 41. — Diagram showing types of leukocytes. The size is showTi in comparison with that of an erythrocyte. other substances whose ])assage through such walls is essential would also be retained. In its present arrangement the hemoglobin held in the red cells is siu'roimded by a fluid stroma whose constantly balanced ])roperties ])ermit it more efficient functioning than would be possible if it were free in the ])lasma of the blood, where the sodium chloride concentration would interfere with its function. Leukocytes.— These elements of the blood are called white l)lood cells and all are nucleated. Special stains make possil^le a differ- entiation of five distinct types, which can be separated into two classes. One class lacks granules in the cytoplasm and its cells are know^i as agranulocytes; the other class includes those cells with a distinctly granular cytoplasm, the granulocytes. (Fig. 41.) 74 THE BLOOD Agranulocytes.— Two types of cells are differentiated in this class, lymphocytes and monocytes, although there are numerous apparently transitional forms and variations. Lymphocytes.— Lymphocytes are small cells about as large as the red cells in mammals, but smaller than the erythrocytes in many lower vertebrates. The cell body is round in form, with a relati\'ely large spherical nucleus and only a thin rim of basophilic cytoplasm. The nucleus stains darkly, showing a chromatin network and usu- ally a nucleolus. The cytoplasm takes a light blue tint with the usual blood stain (Wright's) made from methylene blue and eosin. Variations in the size of lymphocytes are common and three sizes, large, medium, and small, are often designated. The small lymphocytes are usually meant when lymphocytes are mentioned without any qualification as to size. The medium and large lympho- cytes are limited to the lymph glands and bone-marrow, occurring but rarely in the circulating blood under normal conditions. The lymphocytes cannot be looked upon as fully differentiated cells, for they are believed to have the ability to develop not only into lymphocytes of various sizes but into monocytes and indirectly into fibroblasts and histiocytes (macrophages). Lymphocytes leave the capillaries by ameboid motion to invade the tissues, so that they are mainly extravascular elements. Their invasions often carry them into the lumen of the alimentary tract and great numbers are lost in this manner. They have a slight phagocytic action and are found invading all tissues under conditions usually associated with inflammation. Nothing very definite can be said of their role as lymphocytes, although as wandering elements capable of differen- tiation into other cell types their role is important. Their source is mainly the lymph glands and the spleen in mammals, but in lower forms they may have their origin in the same centers as the other blood cells. Monocytes.— Monocytes are larger than lymphocytes, with a spherical or indented, more lightly staining nucleus located eccen- trically in the basophilic cytoplasm. These cells have a marked motility intravascularly and are actively phagocytic for foreign materials, including cell debris and bacteria. They are believed by some investigators to transform into macrophages when becoming actively phagocytic, and into fibroblasts when they pass from the capillaries into surrounding connective tissues. Many of the features of enlarged monocytes, macrophages, and fibroblasts make clear differentiations of types impossible. Various gradations of cells THE BLOOD CELLS (0 from typical lymphocytes to monocytes are t'oiind, indicating their source as the lymphocyte. Under conditions of chronic infiannnation, cells derived from lymphocytes are present. These cells, called plasma cells, have an eccentric nucleus in which the chromatin material radiates from the center like the spokes of a wheel. Gr.\^ulocytes.— This group of cells shows a more restricted development. The various tjT)es of cells constituting it are differ- entiated as end-products and do not ])ossess the ability to multiply or give rise to other cell types, as in the case of the agranulocytes. The\' are formed in the bone-marrow or in other hemapoietic centers and are all about the same size. On the basis of the reaction of the cytoplasmic granules to certain stains three types of granulo- cytes are differentiated, namely, neutrophils, eosinophils, and basophils. Neutrophils ( Ilctewphils) .— Amon^ the mammals these cells are a little larger than the red cells, while in the lower forms they may be snuiller. The cytoplasm is filled with numerous uniformly small granules. In man they are neutrophilic, staining a lavender color, but in lower forms they may take either acid or basic, or both acid and basic dyes. The nucleus is ])olymorphic, with several small deeply staining lobes connected by thin strands of nuclear material. Neutrophils are the most motile of the granulocytes. The\' are markedly phagocytic for many bacteria, and when thus actively engaged are called microphages. Great numbers are found in areas of infection and numerous dead neutrophils appear in pus, though they do not normally occur extra\ascularl\-. They are formed mainly in the bone-marrow or in other hemapoietic centers. Eosinojjhils.— These cells have less polymorphic or even spherical nuclei that are lightly staining. The cytoplasm contains numerous uniformly large acidophilic granules. P'osinophils are less motile than neutrophils, have but a slight phagocytic action for bacteria, and are often found extravascularly. They are found in increased numbers in cases of i)arasitism by worms and under other circum- stances that \m\e led to the conclusion that they have a role in detoxification and in allergic conditions. In some forms, such as cats, rats and mice, eosinophils appear to be absent. Basophils.— In these cells the nucleus is slightly lobed or spherical, it stains faintly, and is located centrally. The cytoplasm contains large variable basophilic granules, usually less numerous than those of either neutrophils or eosinophils. This type of cell is extremely 76 THE BLOOD rare in man, but occurs in greater numbers in lower vertebrates. It has less motility than either of the other types of granulocytes and little is known concerning its function. The invasion of foreign materials may produce an increase in their number. They closely resemble the mast cells found in loose fibroelastic connective tissues. Platelets.— In addition to the red and white cells of the blood there are found in mammals certain cytoplasmic elements called platelets. These are much smaller than the red cells and in smears take the blue basic dye and appear in clumps or masses. They are thought to be associated with blood clotting, though not found in clots. Their origin is traced to large giant cells in the bone-marrow, known as megakaryocytes, which are thought to extend pseudopodia into the marrow sinuses where they are cut off as platelets. Thrombocytes or Spindle Cells.— In the blood of vertebrates below mammals platelets are not usually found, but a cellular element, the spindle cell or thrombocyte, is thought to have an analogous role in blood clotting. These cells are about the size of the red cells or lymphocytes and have a heavily staining spherical or oval nucleus in which a nucleolus is usually not present. The cytoplasm is clear and varies in its reaction from a more or less neutral to a lightly basophilic condition. The cells appear oval or spindle-shaped in outline and several cells may fuse and give the appearance of a cyto- plasmic mass with several nuclei. The origin of these cells is traced to differentiation of the endothelial cells and of cells indistinguishable from lymphocytes. Blood Cell Formation. Hemapoiesis is the name given to the process of blood cell formation, and a study of it in different groups of vertebrates shows some variation in its location. Embryologically blood cells as well as the blood vessels originate in mesenchyme, occurring first as isolated masses or cords, called l)lood islands, in the wall of the yolk sac. The peripheral cells of these masses be- come flattened and form the endothelium of the vessels, while the central cells become surrounded by plasma and form the first blood cells. Other similar centers arise from the mesenchyme of the embryo proper and with subsequent development a vascular net- work is formed that gradually gives rise to a system of blood vessels and developing blood cells. During the early stages blood cells are formed by division of the primitive blood cells in the vessels and to a lesser extent from the surrounding endothelial cells. After this early embryonic stage, blood cells no longer proliferate in the Aessels, but THE BLOOD CELLS 77 certain centers are dcNeloped from uicscnchyme in (liH'erent regions of the body. A study of the vertebrates, l:)eginning at the lowest end of the scale with the fish and progressing through to the mam- mals, presents a series in which evolution of l)l()()d-forming centers may be traced and compared with similar stages in the embryonic development of mauniuils. The earliest hema])oietic center to ap])ear is found in the case of the hagfish, where a diffuse arrangement of proliferating and differ- entiating blood cells occurs in the connective tissue of the gastro- intestinal tract. In other forms, blood-forming tissue becomes more localised and concentrated in certain regions of the tract, as in the spiral valve of the lami)rey. A still further condensation gives a spleen that is bound into the wall of the stomach or intestine. Such centers are supported by a network of connectixe tissue in which the blood cells are proliferating in close contact with sinusoidal cajiillar- ies leading into the venous system. In ganoids the spleen is an extra-enteral organ attached to the mesentery; the submucosa of the intestinal tract may still retain the capacity to develo}) granu- locytes, but red cell formation centers in the spleen. In the higher fishes and in the amphibia, the spleen becomes the main center for blood cell formation. Mesenchyme cells in other localities, such as the cai)sule of the gonads and in the liver, also play a role in blood cell formation. With the development of hollow bones in reptiles, birds, and mammals, the center for production of the red cells and granulocytes shifts to the bone-marrow, the spleen playing its major role only in early devel()i)ment. With this shift to the bone-marrow the production of agranulocytes, ])rimarily lym])h()- cytes, is taken care of by lym])hoid tissue in the form of lymph nodes and the spleen. Except in embryonic life, where blood cells form in the developing vessels, blood cell formation usually takes place extravascularly. In the hemapoietic centers there is a network of reticular cells and fibers supporting the proliferating primitive blood and lymph cells derived from mesenchyme. Following a period of differentiation into the ^•arious types of cells the fully differentiated red or white cells enter the venous sinuses which form a network through such centers. In mammals, where lymphocyte production does not com- monly occur together with that of the other myeloid elements, which are primarily produced in the marrow, the lymphocytes enter lymph vessels supplying the lymphoid tissue, or, as in the spleen, enter 78 THE BLOOD sinuses collecting into veins. In the case of bone-marrow a number of types are found, grading from the stem cells, or hemoc\toblasts, to the fully differentiated granulocytes and erythrocytes. Bone-marrow.— As already noted in the study of bone, there are two types of marrow, red and yellow. Yellow marrow is composed mainly of fat cells and is located in the medullary canal of the shaft of long bones, but red marrow with few fat cells and many blood-forming cells is located in the spaces between spicules of spongy bone. Such places occur in the epiphyses of long bones, in cranial bones, ribs, and the sternum. These locations are well protected, richly vascular, have a low blood-pressure and a slow circulation apparently essential for blood cell formation. There are two main constituents of red marrow, the stroma and free cells. The stroma consists of a network of argyrophil fibers and reticular cells and a small amount of fibroelastic connective tissue supporting small arteries, veins, lymphatics, and nerves. The capillaries are sinusoidal in character, and fully developed erythrocytes and granu- locytes formed in the intersinusoidal tissues make their way through them into the general circulation. In the stroma are cells in ^'arious stages of differentiation, ranging from stem cells to fully differentiated erythrocytes and granulocytes. (Fig. 42.) Hemoci/tobJasi.'^.— These are generalized cells similar to large lymphocytes and are the stem cells from which erythrocytes and granulocytes develop. They may have an ameboid appearance. Their cytoplasm is non-granular and basophilic; the nucleus is large and oval with a coarse chromatin network. Erythrocyte Formation. — CertSim hemocytoblasts differentiate into round erythrocytoblasts with a spherical nucleus, and following a series of mitoses accompanied by increasing differentiation they become erythrocytes. In the erythrocytoblast stage the cytoplasm reacts to both acid and basic dyes. As the hemoglobin increases the baso]:)hilic reaction diminishes, and in the normoblast stage the cells have considerable hemoglobin in the cytoplasm and the nucleus is relati^'ely smaller. These changes become more prom- inent with further mitoses, until mitotic activity comes to an end. In mammals the nucleus is extruded from the cell body, which then passes into a sinusoid and so into the vascular system. Series of stages from hemocytoblasts to newly formed erythrocytes may be found in red marrow. Granulocyte Formatio)i. — Cerhun hemocytoblasts undergo mitoses during which a differentiation of another kind takes place and gives rise to one or another of the three types of graiuik)('ytes. An early THE BLOOD CELLS 79 stage is recognized as tlic ])remyelo('yte, of which three forms can be distinguished on tlie hasis of cytoplasmic granulation. The nuclei are spherical or reniform at first, but each type undergoes further mitoses with further differential granulation of the cyto- plasm and lobulation of the nucleus until neutrophil myelocytes, 4 "a; 8 \:^ Fig. 42. — Diagrammatic representation of the components of red bone-marrow. 1, fat cell; 2, hemocytoblast; 3, erythro blast; 4, normoblast; ,5, erythrocyte; 6, myelo- cyte; 7, neutrophil; 8, eosinophil; 9, basophil; 10, megakaryocyte; A, reticular tissue; B, cell of sinusoid wall; C, sinusoid. eosinophil myelocytes, and basophil myelocytes are formed. P^ach of these types undergoes its own special development leading to the formation of completely differentiated neutrophils, eosinophils, and basophils which enter the circulating blood by way of the sinusoids. Megakaryocytes. These giant cells are usually spherical in form and are found extravascularly in the red marrow of mammals. They also arise from hemocytoblasts by a series of transitional forms. Xormally they remain in the marrow, where they are 80 THE BLOOD believed to form pseudopodial-like processes, the ends of which are pinched off and pass into the blood stream as platelets. Develop- ing and degenerating megakaryocytes are to be foimd in red marrow. The Destruction of Blood Cells. — The different tyjjes of blood cells apparently have diff'ering periods of life. Some are relatively short lived, as the erythrocytes of mammals which have an estimated life of about thirty days, but others may live over much longer i)eriods. It has been observed that many red cells are phagocytized l)y histio- cytes, the macrophages, in the red pulp of the spleen; in certain pathological conditions so many are destroyed that the red pulp becomes brown in color. Old granulocytes are also apparently phagocytized by Kupffer cells, the macrophages in the liver. The lymphocytes are continually degenerating in lymph tissue and many are lost as a result of their migration through the epithelial lining of the alimentary tract and various other ducts. REFERENCES. Dawson, A. B. 1931. Supravital studies on the erythrocyte of the catfish (Ameiurus nebulosus, Lesureur ), with special reference to the Nittis stigma, Anat. Rec, 49, 121. 1932. The reaction of the erythrocytes of vertebrates, especially fishes, to vital dyes, Biol. Bull., 63, 48. 1933. A reinterpretation of the findings of Komocki (1932) on the blood on the Urodele (Batrachoseps attenuatus), Anat. Rec, 58, 31. 1935. The hemopoietic response in the catfish, Ameiuris nebulosus. to chronic lead poisoning, Biol. Bull , 68, 335. JoRDON, H. E. 1932. The histology of the blood and the blood-forming tissues of the Urodele (Proteus anguineus), Am. Jour. Anat., 51, 215. 1933. The evolution of blood-forming tissues. Quart. Rev. Biol., 8, 58. 18, 1. 1934. Extramedullary erythrocytopoiesis in man, Arch. Path., 1934. The transformation of adipose tissue into hemocytopoietic tissue, Anat. Rec, 59, 461. JoRD.\N, H. E., .\ND Flipper, ,1. C. 1913. Hematopoiesis in chelonia, Folia ha?matol., 15, 1. JoRD.'VN, H. E., AND SpEiDEL, C. C. 1930. Blood formation in Cyclostomes, Am. Jour. Anat., 216, 355. ■ 1931. Blood formation in the .\frican hmgtish, under normal conditions and under conditions of prolonged esti\ation and recovery. Jour. Mori)h., 51, 319. Kindred, J. E. 1932. A study of the tinctorial reaction of hemoglobiniferous cells, Russell body cell, plasma cells and lymphocytes of the albino rat by a new method of selective staining, Anat. Rec, 53, 43. Mayer.son, H. S. 1930. The blood cytology of dogs, Anat. Rec, 47, 239. Redfield, a. C. 1933. The evolution of the respiratory function of the blood, (iuart. Rev. Biol., 8, 58. Sabin, F. 1928. Bone-marrow, Physiol. Rev., 8, 191. See Appendix for general text references. CHAPTER V. THE mus(;le tissues. The most oiitstan' flivision of the mature cells which apparently retain their ability to divide by mitosis. In newly formed tissue the fibrils appear to continue from cell to cell, l>ut this cannot be seen in preparations of older tissues. In general the myofibrilhe of this type of muscle are difficult to distinguish. The sui)erficial myofibrillae appear coarser than those in the interior of the cell. The non-fibrillar sarcoplasm is best seen as a lighter area at either end of the nucleus where the myofibrils diverge in ])assing. By soaking pieces of tissue composed of smooth muscle in weak acid or SMOOTH MUSCLE 83 alkaline solutions it is possible to dissolve the substance cementing adjacent cells and shake them apart, so that isolated smooth muscle cells may be observed. Smooth muscle cells are variously organized. In the connective tissue of the villi or folds of the intestine a few isolated smooth muscle cells occur; associated with hairs are small bundles which form the arrector pili muscles; in more complex organizations, the cells are arranged in bundles or sheets. When organized into groups or muscle coats the ta])ering ])ortions of the cells overlap and a network of elastic and reticular fibers extends between adjacent cells from the surrounding fibroelastic connective tissue. When such sheets or bands of smooth muscle are cut in longitudinal section (Fig. 44), the characteristic spindle shape of the cells is _ »L -Inner circular coat Fig. 44. — Diagram of cross-section and longitudinal section of smooth muscle. evident, but in cross-sections roughly circular sections of different sizes appear adjacent to each other and nuclei appear only in those sections through the central region of the cells. This is what one would expect from o\'erlapping of the tapering ends. In the wall of the alimentary tract from the lower esophagus to the anus or cloacal o])ening there are usually two distinct sheets of smooth muscle ; an inner coat of muscle cells encircle the tube, and an outer coat has cells arranged lengthwise. (Fig. 45.) Constric- tions of the inner coat decrease the lumen, and constrictions of the longitudinal coat cause a shortening of the tube at the points affected. During life, waves of contraction pass along these coats simultaneously and cause the peristaltic movements essential in the functioning of the digestive system. ■^A . Vk 84 THE MUSCLE TISSUES Sometimes when pieces of the intestinal wall are fixed, indications of the contraction wave have been preserved. Small band-like swellings running across the muscle sheet appear at regular intervals, these contraction swellings involve different portions of adjacent cells which do not function as independent units. When the intes- tine or other organ involved is greatly distended, the muscle sheets appear much thinner than in the relaxed state. With relaxation after such expansions the cells slide back into their former position and form a thicker coat. Capillaries extend through the connec- tive tissue network surrounding the muscle cells and follow their Fig. 45. — Photograph of cross-section and longitudinal section of smooth muscle in wall of frog's stomach. disposition as do the sympathetic nerves controlling their involun- tary action. In the repair of smooth muscle there are evidences of some degree of mitotic activity on the part of fully formed cells, but in cases of extensive injury lesions are closed by scars of con- nective tissue. CARDIAC MUSCLE. Beneath the foregut of the embryo, an endothelial tube surrounded by mesenchyme is the forerunner of the heart and the base of its connecting vessels, to which this ty])e of muscle is limited. The processes of the early mesenchyme cells appear to continue with the adjacent cells to form a syncytium, and from these the cardiac muscle is derived by contiiuied multi])licati()n, gradual elongation, and differentiation of intracellular fibrils. The developing cells, or myoblasts, elongate and granules are said to a])pear first in the CARDIAC MUSCLE ])eripheral cytoplasm. Tliese orraniiles increase in number and appear to become arrangetl in linear fashion and fuse to form coarse fibers, the cardiac myofibrils. The cells grow in length but preserve their fiber-like form and lateral attachments with adjoining myoblasts. (Fig. 4(>.) The myofibrillar increase in number presumably b\- longi- tudinal di\ision and become more numerous in the peripheral region, leaving a central portion with a core of undifferentiated cytoplasm and the nucleus. With dcNelopment the fibrils show striations resulting from alternate differences in composition; these appear as dark and light bands which occur at the same level in all the fibrils of a given fiber, so that the whole fiber has a striated ap- pearance. In the lower vertebrates the myofibrils are less numerous and form a peripheral layer, but in higher forms they are scattered throughout, except in the immediate region of the nucleus. The fibers of higher verte- brates are limited by a thin membrane called the sarco- lemma, which is usually con- sidered to be a condensation of sarcoplasm. Among fishes and amphibians, cardiac muscle appears to lack the interstitial tissue found in higher forms, and the bundles of muscle fibers are separated from the blood only by the covering endothelium, a condition resembling the embryonic state of higher forms. A longitudinal section of cardiac muscle does not have the appear- ance of separate cells as does smooth muscle. The general picture is that of a network of long fibers in which the nuclei are at regular intervals in the center of the fibers. The clefts between fibers are small in the higher vertebrates but are easily seen in lower forms, such as the fish and frog. A cross-section shows sections of the fibers irregular in outline in places where branching occurs, but in other portions of the fibers the size is more uniform and the outline quite regular. This is in contrast with the marked variability in cross-sections of smooth muscle, where the outlines are regular but the size varies with the region of the cell cut. After treatment in Fig. 46. — Photograph of an isolated portion of cardiac muscle of the frog, showing a fiber with a central nucleus, striated myofibrils, aud branches. 86 THE MUSCLE TISSUES dissociating fluid, cardiac muscle can be shaken into separate units resembling single cells. They are roughly rectangular in shape, with parallel sides and very uneven ends. One or both ends may have short stubby branches that connected it with other adjacent fibers. Each of these units is cross-striated and shows longitudinal fibrillae. It is not certain that the apparent ends are actually boundaries of cells; they may be artefacts brought about by the treatment. The isolated units of cardiac muscle correspond to cells having a nucleus and a surrounding portion of cytoplasm. Howe\er, the most reasonable conclusion drawn from the lack of definite cell boundaries, and the fact that the myofibrils extend continuously through several such units, is that the cardiac muscle is a syncytium. The myofibrils appear to be similar to those of skeletal muscle, Fig. 47. — Diagram of cardiac muscle cells, showdng intercalated discs. where they are more easily studied. At times even in fresh heart tissue, and particularly after certain techniques, peculiar bands, the intercalated discs, may be obser^'ed extending partially or inter- ruptedly across a fiber. (Fig. 47.) These discs were formerly thought to represent end-boundaries of the cells, but this seems unlikely since some of them pass across a fiber at the level of the nucleus and others delimit portions of a fiber without a nucleus. They usually present a staircase appearance and the separate portions do not o^'erlap. Some investigators interpret them as places where the fibers were in a state of contraction. They are more commonly found in older cardiac tissue and from this the inference has been drawn that possibly they represent lines where normal functioning is breaking down. The exact nature of these structures remains to be demonstrated. The blood supply for cardiac muscle is supplied directly from the clefts between the fibers in the case of the lower vertebrates, CARDIAC MUSCLE 87 as may he observed in the fish or amphibian heart. In mammals, ho\ve\er, there is a rich ca])inary network carried between the fibers by the interpenetrating connective tissue. The syncytial organization of the heart is probably directly concerned with the rhythmical contractions so characteristic of it, but it is also essential that a free circulation takes place regularly, for interferences in the l)lo()(l flow aft'ects the normal rhythmical activity. Cardiac muscle contractions are shorter in duration than the resting phase and under normal conditions fatigue rarel}' occurs. Regeneration does not seem to be possible in cardiac muscle and enlargements of the heart occurring in some adult animals result from increase in the size of the muscle fibers, not their number, or possibly from increase in the connective tissue if present. Neurogenic and Myogenic Theories of the Heart-beat. ^Although this ap])ears to be primarily a functional problem, histological dis- coveries have had much to do with our knowdedge of the subject. The heart of an elasmobranch is two-chambered ; there is one auricle and one ventricle. The sinus venosus draining blood from the body carries this blood into the auricle; from the latter, the blood passes into the ventricle whose contraction drives the blood into circulation through the body. In action, the sinus contracts first, then the auricle, then the ventricle, then the bulbus arteriosus. In this order, one after the other, repetition occurs rhythmically. It appears that whatever the nature of the stimulus is, it begins in the sinus wall. The amphibian heart has two auricles and one ven- tricle; reptiles have two auricles and the beginning of two ventricles; birds and mammals have two auricles and two ventricles. In each case the old elasmobranch heart organization is represented roughly by tissue at the junction of the vena cava with the right auricle, and careful observation shows that the rhythmic contraction of these higher hearts begins at this tissue and is followed by con- traction of the auricles and ventricles. The heart is provided with branches of sympathetic nerves which have a sensory function, and with branches of the tenth cranial and the vagus over which the impulses regulate the speed of the rhythmic beat. There is no evidence, however, that either set of nerves is concerned with the origin and continuance of the rhythm. Rhythmical contractions of so-called hearts of invertebrates are eflfected by nerve impulses, and the occurrence of nerves in vertebrate hearts suggested that the heart-beat was due directly to nerve stimuli. However, this explanation does not apply to vertebrate hearts. 88 THE MUSCLE TISSUES The embryo heart beats rhythmically before nerves ha\'e developed in it, and when the heart of a cold-blooded vertebrate is removed from its body, rhythmic beating may be continued for many days if proper conditions are maintained. Also, small pieces of heart of the chick or rabbit live, grow, and contract in tissue culture after the ner\'es have degenerated. Evidence is still needed to prove the neurogenic theory of the heart-beat. The myogenic theory, on the other hand, has much in its favor. According to this idea, the stimulus arises in cardiac tissue and is transmitted by this tissue to various contracting portions. The question then arises as to whether impulses pass over ordinary muscle fibers or whether there are special fibers for this function. Such fibers have been found in mammals, where a small mass of especially modified fibers occur at the junction of the superior vena cava and the right auricle. These fibers are poor in fibrils but rich in sarcoplasm; they are small, poorly striated, and form a network. This mass is called the sino-auricular node and is the place where the automatic rhythmic contractions begin. The impulses continue over the auricle via a network of the same type of fibers, the so-called Purkinje fibers, which become continuous with typical cardiac fibers. The auricles are separated from the ventricles by rings of connective tissue aroimd the openings between them and Purkinje fibers converge there to form the auricvilar-ventricular node, a second mass of modified cardiac fibers. This node is located near the ventricles and Purkinje fibers extend on into the* ventricles. Impulses beginning at the junction of the vena cava with the auricles cause the latter to contract, and then the impulses continue into the ventricles, causing a progressive contraction in them also. The rest period between each cycle of contraction is longer than the contraction period. The heart is refractory during this rest period and it does not respond to stimuli. Its sensitivity to stimuli increases toward the end of the rest period. If the im})ulses to the ventricles are interfered with, the ventricles do not respond, or may set up an independent rhythm of their own, in which case the heart as a whole fails to function properly. SKELETAL MUSCLE. As the name implies, this type is associated with skeletal ])arts. The unit of structure is the fiber, as it was in cardiac muscle, but here the fibers are unbranching, elongated, multinucleated cylinders of varying length. Such fibers likewise are formed from myoblasts SKELETAL MUSCLE 89 which are derived from mesodermal cells. Though not branching and forming networks, as in the case of cardiac muscle, they are like it in being syncytial. Distinct cellular limitations are not evident and man\- nuclei are distributed along the length of each fiber. These fibers do not break into semblances of cells u])on treatment with dis- sociating fluids. The development of the elongate multinucleated fibers is held by some to occur by repeated divisions of the nuclei of myoblasts without accompanying division of the cytoplasm, which increases in quantity and elongates. Others believe they arise through fusion of the ends of adjacent myoblasts. Fibrils appear first about the periphery and increase in number and dis- External perimysium {epimysium) Internal perimysium r> Fiber Fig. 48. — Diagram of a cross-seotion of a skeletal muscle. tribution during de\'elopment. The nuclei of the myoblasts at the first appearance of fibrils are central, but later in development they appear more peripherally located. The fibers are organized into skeletal muscles which are enclosed in a relatively thick fibroelastic connective-tissue sheath. (Fig. 48.) This is the external jjerimysium (epimysiimi) which continues inter- nally as the internal perimysium to divide the muscle into bundles of fibers, or fasciculi. (Fig. 49.) The individual fibers are surrounded by a thin sheath of connective tissue, called the endomysium, which contains fibrous and cellular elements of loose fibroelastic and reticular connective tissues. (Fig. 50.) Fibrocytes and histiocytes and undifferentiated mesenchymal cells in the connective-tissue sheaths play a part in the repair of lesions of skeletal muscle which does not exhibit marked powers of regeneration. The capillaries 90 THE MUSCLE TISSUES and nerve fibers as well as the larger blood vessels and nerve trunks are supported in the connective-tissue networks surrounding the individual fibers and the fiber bundles. Internal perimysium Sarcolemma ■Endomysium Fig. 49. — Diagram of a bundle of skeletal muscle fibers. Individual fibers may be observed in fresh skeletal muscle by teasing with needles, but it is difficult to find uninjured ends of such fibers. In fixed and stained preparations, fibers are seen to Fig. 50. — Photograph of a cross-section of skeletal nmsclc n\ the turtle, showing fibers surrounded by endomysium. Capillaries may be seen between some fibers. end in various ways. Some are rounded and blunt, others tai)er oft" within their comiectivc-tissue sheath, which becomes continuous with other sheaths to which the fiber attaches within the nniscle or SKELETAL MUSCLE 91 with tendhioiis tissue with which it is thus attached to })oiies, carti- lage, or other structures. The fibers are clearly limited within their endom>sial sheath by a continuous, thin, trans])arent membrane, known as the sarcolemma. In injured regions of teased fibers the fibrillar contents are often broken and separated, making it possible to observe this membrane more easily. Its origin is disputed; some believe it to be formed by the connecti\'e sheath, and others attribute its presence to the activity of the protoplasm of the muscle fiber itself. The latter seems more likely, for it has been shown to have none of the reactions of collagen or reticulum of connective tissue. Within the sarcolennna the fibers are composed of a fluid proto- plasmic substance, the sarcoplasm, and numerous highly developed myofibrils which run ])arallel to each other lengthwise of the fiber. The fibrils appear to originate, as in the case of those in cardiac muscle, from linear fusion of fine granules forming in the embryonic myoblasts. Contraction first appears when the myofibrils have formed. They increase in number with development of the early fiber and are arranged into groups separated by intervening sarco- plasm. Such groups of fibrils are called sarcostyles and are apparent in cross-sections as Cohnheim's areas. The separation of such groups from each other depends upon the amount of intervening sarcoplasm; in some muscles they are not easily discovered. Fixa- tion may also play some part in producing these decidedly localized groups of fibrils. Although each fibril is a continuous thread of protoplasm, it appears to be composed of plates, or discs, of two alternating kinds of material. These give rise to the dark and light bands forming the cross striations characteristic of this and cardiac muscle. The discs show better when the muscle tissue is soaked in dilute aqueous solutions of acids and alkalies. A number of bands have been identified, but only four are easily demonstrated and ordinarily only two of these are outstanding. The relatively broad, dark, ref^acti^'e band stains with hematoxylin and is called the "Q" or anisotropic band. (Fig. 50a.) Alternating with these are the less re- fractive, pale discs that ordinarily remain unstained ; these are the " J" or isotropic bands. Each of these bands is apparently divided by another narrower band of opposite character. The " Q" band is thus seen to have an indistinct light band, the "J/" band, running through its center; and the "J" band has an indistinct thin dark band, the "Z" band, or intermediate disc, running through its 92 THE MUSCLE TISSUES center. These latter bands represent differences in the densities of the "Q" and "J" bands. The region between two "Z" bands is considered to be a functional and structural unit, the sarcomere, of the fibril. Various theories of muscle contraction have arisen, based on the different peculiarities associated with the parts of these sarcomeres, but little is really known of the mechanism of contrac- tion. The dark "Q" discs are doubly refractive, poor in extensi- bility, poor in water content, do not shrink, and stain darkly in hematoxylin. The lighter "J" discs are singly refractive, pale, rich in water content, extensible, shrink in reagents, and do not stain well, if at all. M Baml~i. rJ Band ^-—\ \.--, - ^ -'----~,^,. .—.:.,...--:..,, ^ - ,./| ■^v-7r-Sarcolem ma >^-^^^^ ■•■■ T •■ T BOB >Q5-« 1 JOB I JIH rBH I HrK; 1 ~a^E£,c< 1 f^QS^r^~^f yofibril Sarcolemma- \ / \\Sarcomerc Q Bandy X.^ jj^„^ Fig. 50a. — Diagram of a skeletal muscle fiber in longitudinal section. The nuclei of skeletal muscle fibers are oval in shape, but their size and location varies, depending upon the animal. In lower vertebrates the nuclei occur scattered throughout the fiber among the fibrils, but in some cases show a tendency to be located more alKuidantly near the j)eriphery. In mammals the nuclei are most commonly located in the sarcoplasra, directly below the sarcolemma, but in some muscles of the red type the>- may be scattered more generally through the fiber. The nuclei vary from an oval to a fusiform shape, but are disposed lengthwise in the fiber in either case. The sarcoplasm filling in between the fibrils may vary greatly in amount and the density and size of the fibers also show variations with species. There is usually an accunnilation of sarcoplasm about the nuclei, especially at either end. Variations in the size of muscles in mature animals is dependent mainly u])()n increase in sarcoplasm. The nerve sui)])ly from the cerebrosi)inal ner\es i)laces this type of muscle under voluntary control in contrast to the two other ty])es of nnisclc. Capillaries form a meshwork with the longer capillary portion running lengthwise of the fibers, and short and SKELETAL MUSCLE 93 thicker cross-pieces, or anipulla?, extend across the fibers and may dilate with blood when the muscle is contracted. If a muscle is seriously injured its destroyed ])ortion is re])lace(l by scar tissue, but if the sarcolemma of a fiber is not injured the injured myofibrils are digested and the fiber remaining acts like a myoblast and forms new nuclei and fibrils, possibly also new units by mitosis. The manner of attachment of skeletal muscles to tendons is thought by some to involve a continuation of the myofibrils with the collagenous fibrils of the tendon, by others to involve continua- tions of the connective tissue (endomysium) with the tendon. The latter appears to be the more likely association. REFERENCES. Brinley, F. ,J. 1932. A physiological study of the innervation of the heart of fish embryos, PhysioL ZooL, 5, 527. Cardwell, .J. C, AND Abram.son, D. I. 1931. The atrioventricular conduc- tion system of the beef heart, Am. Jour. Anat., 49, 167. Carr, R. W. 1931. Muscle tendon attachment in the striated muscle of the fetal pig. Am. Jour. Anat., 99, 1. Jordan, H. E. 1934. Structural changes during contraction in striped mu.scle of the frog, Am. Jour. Anat., 55, 117. Katznelson, Z. S. 1934. Histogenesis of muscle in Ami)hibia: 1. Develop- ment of striated muscle from mesenchyme in urodeles, Anat. Rec, 61, 109. Lewis, M. R., and Lewis, W. H. 1917. The contraction of smooth muscle cells in tissue cultures. Am. Jour. Physiol., 44, 67. Lloyd, W. 1930. The form and function of the auriculo-ventricular bundle in the rabbit. Am. Join-. Anat., 45, 379. Mather, V., and Hines, M. 1934. Studies in the innervations of skeletal muscle: The limb muscle in the newt, Triturus torosus, Am. Jour. Anat., 54, 177. Pollister, a. 1932. Mitosis in non-.striated muscle cells," Anat. Rec, 53, 11. Zschiesche, E. S., and Stilwell, E. F. 1934. Intercalated discs of the heart of the guinea-pig, Anat. Rec, 60, 477, See Appendix for general text references. CHAPTER VI. THE NERVE TISSUE. All cells are to a certain extent irritable and conductive; that is, they receive stimuli from external sources and transform them into impulses which are conducted to a portion or the whole of the cell to stimulate some reaction- by that portion or by the entire cell. In unicellular animals and the simplest metazoans, no special organization appears to be developed to carry on these fundamental functions of protoplasm. x\mong the metazoans generally, how- ever, an association of special cells has evolved to form the nerve tissue, which functions primarily as a receiver of stimuli and con- ductor of impulses. The vital unit of this tissue is the nerve cell, or neuron, which takes on varied forms but invariably has one or more cytoplasmic processes making contact with closely adjacent or more remote cells or tissues of the body. Typically, each cell has a large nucleus sin-rounded by a cytoplasmic mass from which slender processes grow out for varying distances to form nerve fibers. Each neuron is a separate unit, but the processes of one cell come into contact with others at synaptic junctions, or synapses, so that impulses pass from one nerve cell to another, and by chains of such cells impulses may be conducted over considerable distances to finally effect responses in other cells or tissues. Little is known concerning the exact nature of stimuli, or how they are transformed into impulses, or how the latter are transmitted along nerve cells, but the essential part played by these cells in coordinating the other tissues of the organism has been proven repeatedly by careful experimentation. The nerve cells and their processes are organized into organ centers such as the lirain, spinal cord, and ganglia, but the jh'o- cesses alone form the nerves which are organized into an intercon- necting system associating the ^'arious tissues and organs with the nerve center and making possible integrated action. HISTOGENESIS OF NERVE TISSUE. The foundation of all the nerve tissue appears in the de\'eloping embryo as a thickened region of ectoderm, the neural plate, along the mid-dorsal line. Following rapid and unequal growth of the THE NEURON 95 Axon Collateral cells of this ])late, a neural gi'oove is formed and deepens until the thickened folds fuse dorsally to form a neural tube which lies l)elovv and sei)arate from the ectoderm. The cells of this neural tube ^i\'e rise to the major i)art of the ner^•e tissue. The anterior portion of the tube forms the brain anrl the posterior portion the s])inal cord. Between the nein*al tube and the ectoderm a longitudinal band of cells a])])ears on each side and forms the neural crest. From these cells are derived the s])inal and cranial ganglia and indirectly the sym])athetic ganglia. The cells of the neural tube undergo a series of divisions and finally differentiate into the nerve cells or neurons and the neuroglia cells which form the supporting tissue of the nervous system. THE NEURON. A study of nerve tissue involves an understanding of the neuron, or nerve cell. A description of it includes a consideration of the cyton, which consists of the nucleus and surrounding portion of cytoplasm, and the cytoplas- mic processes which extend out from the cyton. (Fig. 51.) The Cyton.— The size of this portion of the neuron \'aries from a few to several himdred microns in diameter. The limiting sur- face is a denser cytoplasm but not a distinct cell membrane, as found in other types of cells. There is considerable variation in shape, some cell bodies are spherical, others oval, pyriform, fusiform, or even stellate. Such variations are largely due to the nmnber and location of the processes which extend out from the cvtons. Telodendrion Fig. 51. — Diagram of a neuron. 96 THE NERVE TISSUE NucleKS. ^The large spherical nucleus is bounded by a distinct nuclear membrane. Within it is a large nucleolus which stains intensely with basic dyes. The chromatic material within the nucleus is generally scant, as compared with that of other cells. In large cytons it appears as a network concentrated about the nucleolus. Fine protoplasmic granules staining with both basic and acid dyes occur abundanth'. In some neurons several nucleoli may be observed. Mature neurons differ from other cells in that they do not undergo mitosis. Nissl Bodies.— These irregular masses of basophilic material occurring in the cytoplasm have been called tigroid bodies, chromo- phil substance, and other names. (Fig. 52.) S])ecial techniques Fig. 52.— Spinal ganglion cells of the cat, showing numerous Nissl bodies. Each cell is surrounded by a number of smaller satellite cells. are often used to make them stand out clearly, but almost e\ery stained preparation shows them scattered through the cytoplasm. It is belie^'ed that these bodies contain iron and store oxygen, and that their presence is essential to the functioning of the nerve cell. Their appearance as definite bodies nvA\ be due to fixation, since different fixatives result in differences in their form. In the living cells the substance of which they are com])ose(l may be in a diffuse state. ]\Iodifications in their appearance occur with changes in the conditions of the nerve cells. In pathological conditions they often disappear entirely. THE REFLEX ARC 97 Neurofibrils. — \n \\\m^ cells it is difficult to demonstrate anything but a more or less homofjeneous cyto])lasm, hut in fixed and stained preparations, especially siher impregnated tissues, very fine long thread-like structures api)ear to extend throughout the cell body and its })rocesses. At first they were thought to be the primary means by which impulses were transported, but there seems to be little proof as to their function, though they appear as characteristic featiuTs in nerve cells. Golgi Apparatus .—^y special techniques, using osmic acid and silver, a blackened network appears in the cytoplasm usually con- centrated about the nucleus. This a])])aratus of (lolgi disappears in cells subjected to injin-y and cannot be demonstrated in li\ing unstained cells. Chondrlosomes (Mitochuudria).— Tiny rods and granules appear scattered through the cytoplasm and may be demonstrated in living cells with Janus green B. Certain methods also preserve them in fixed and stained jireparations. Little is known of their functional significance. Cytoplasmic Processes. — It is customary- to recognize two types of processes extending from the cyton, namely, axon and dendrites. There is no Nissl substance in the axon. It is a slender fiber-like extension of uniform diameter throughout and has a smooth clean surface. Usually the axon arises from a particular place in the cyton marked by a conical extension called the axon hill. Slender collateral branches may arise from the axon along its course and are at right angles to its surface. In many neurons the axon ends dis- tally in a brush of finer branching processes known as the terminal arborization or telodendria. The dendrites are thick, irregularly branching, cytoplasmic processes extending out from the cyton and contain the Nissl substance and other cytoplasmic elements gener- ally present in the cyton. THE REFLEX ARC. The simplest physiological organization of neurons is called a reflex arc. It involves at least two neurons, as in the following example. A stimulus on the skin is translated into a nerve impulse in the peripheral terminus of a dendritic process of neuron A. (Fig. 58.) The nerve impulse travels centrally to the cyton of this neuron, which is located in a spinal ganglion. From this cyton the nerve impulse passes over its axon into the gray matter of the coid to terminate in the ventral horn. Here it connects with the den- 7 98 THE NERVE TISSUE drites of neuron B, and passes into the cyton of this second nerve celL From cyton B the impulse travels into the axon which extends outward in the spinal nerve to muscle tissue and stimulates the muscle to contract. Most reflex reactions involve more than two neurons. This is especially true of higher mammals, where volition exercises control over simpler reflex mechanisms. Dorsal septum of spinal cord Ganglion cell ganglion Sensory root Spinal nerve Motor root Ventral , fissure of Cyton of motor nerve cell ending in muscle spinal cord Fig. 53. — Diagram illustrating a reflex arc. ^Epiderm is Derm is Muscle THE SYNAPSE. Neurons are associated with each other through the synapse, at which point the terminals of the axon of one neuron come into contact by contiguity with the dendrites or cell body of another neuron. Such association may be effected by a basket work of neurofibrillar processes from the terminal end of one neuron fitting against the dendrites or cyton of the neuron receiving the impulse. The synaptic type of association is accepted as the usual one, but there is evidence showing that nerve fibers may be continuous at these points, especially among the lower vertebrates. TYPES OF NEURONS. Neurons located in different regions of the nervous system have definite functional demands made upon them, and associated with their functioning is a certain arrangement of their processes so that several types of cells may be classified as follows: unipolar cells, neurons with a single process which arises from one side; bi])olar cells, in which a single axon and a single dendrite process project from opposite ends of the neurons; multipolar cells, in which numer- ous processes project from different regions of the cytons; and ganglion cells, in which two ditfcrciit processes, axon and dendrite arise from one side, a i)scu(l()-uiiip()lar condition. TYPES OF NEUHOXS D!) Unipolar Cells. l)urin-mal cells lose their cilia, become cuboidal, and act as a secretory epithelium. Astrocytes.— The astrocytes are stellate cells with processes attached to blood ves- sels, and are the largest type of neuroglia element. The nucleus is large and oval, with scant scattered chromatin and no nucleolus. Two types are usually recognized, fibrous astrocytes and protoplasmic astrocytes. Fibrous astrocytes, also called spider cells, are characterized by long, usually unbranched processes containing fibrous elements. They occur abundantly among the myelinated fibers of the white matter of spinal cord and brain. (Fig. (U.) The protoplasmic astrocytes, which occur in gray matter, have a stellate cell body with granular cytoplasm and many short, stubby, branching processes. The abundance of the processes, as shown in Golgi ])reparations, has led to calling them mossy cells. Microgliocytes.— These elements appear as small cells in their resting state and have fine-branched processes, but are capable of becoming ameboid and phagocytic. The nuclei of these cells are the smallest and stain more deeply than those of other cells found in the spinal cord and brain. Fig. 61. — Types of neu- roglia cells. A, protoplas- mic astrocyte; B, fibrous astrocyte. GANGLIA 107 The function of the astrocytes is associated with sup])ort and heahng in the case of injury and possibly assist with the formation of myehn. The microghocytes may he considered as analogous with phagocytic cells in other regions of the body, and form ]jart of the reticulo-endothelial system of the nervous system. GANGLIA. A ganglion may be defined as a small aggregation of neurons (cj'tons and processes) outside the central nervous system. (Fig. 02.) Each dorsal root of a spinal nerve possesses a spinal ganglion. The dorsal root of such a nerve is sheathed in external perineurium. I. ^ < ■ • ,. . ^r. ■■% ,, . _,ijy Fig. 62. — Photograph of spinal ganglion of the cat. Longitudinal section showing groups of ganglion cells separated by fiber tracts. The loose fibroelastic connective- tissue sheath is partly torn away from the ganglion. This connective tissue covers the ganglion and extensions from it continue internally, partly separating masses of cytons and fibers. The cytons are large spherical cells of different diameters. Within each is a large nucleus which usually contains a nucleolus. Around each cyton may be seen a row of nuclei, indicati\'e of a sheath of indifferent cells— the so-called satellite cells, which are homologous with neurolemma cells. P^lsewhere are clumps of fibers, usually with myelin and neurolemma. The single process from the cyton is rarely visible in a section. A short distance away from the cyton it divides into a peripheral and central process. The peripheral 108 THE NERVE TISSUE branch is regarded as a specialized dendrite. The central process is regarded as an axon which extends from the ganglion into the dorsal horn of gray matter on that side of the spinal cord. Outside the cord and brain are several sympathetic ganglia of such size and uniformity of location that they have received ana- tomical names. But in addition to these which can be located by dissection, there are a great many small ganglia that can be seen only in microscopic preparations. The cells of sympathetic ganglia have long axons and dendrites which in some cases extend out beyond the capsule cells to form connections with adjacent cells. In other cases the dendrites are entirely wuthin the capsule. Con- nective tissue surrounds the ganglion and penetrates among the fibers and cells. Myelin is not usually found surrounding the sympathetic fibers, but a neurolemma sheath does occur, DEGENERATION OF NERVES. Degeneration changes appear soon after a peripheral nerve is severed. Before this occurs, however, there is an almost immediate chemical change which extends in both directions from the lesion. This effect is known as traumatic degeneration and extends cen- trally to reach the cytons of the neurons involved. The Nissl sub- stance disintegrates and disappears. Although the cytons are affected by the lesion they possess the power of recovery. This recovery phase soon follows and extends out along the nerve fibers as far as the lesion. But the portion of the nerve distal to the lesion does not recover and disintegration of the myelin and axon pro- gresses outward from the lesion. A great increase in neurolemma nuclei has been observed to accompany these changes. In about three days after the lesion is made, the nerve no longer conducts and the structure innervated by this particular nerve is no longer served. If the nerve supplies certain muscles, these will be par- alyzed, since motor impulses are no longer being sent to them. Paralysis will be permanent unless the nerve regenerates. REGENERATION OF NERVES. If, soon after the lesion is made, the cut end of the peripheral portion of the nerve is brought into contact with the cut end of the central portion and kept in this position, then the neurolennna cells which absorbed the broken-down axon and myelin material will form protoplasmic bands. These protoplasmic bands serve as THE BRAIN AND SPINAL CORD 109 tracks along which the sprouting ends of the cut central portions of the axon will grow, and the (le\eloping new axons will find their ])ro])er terminations. It is \ery important to have no scar tissue formed at the lesion where the cut ends of the injured nerve are brought together. Regeneration is more rapid in young animals than in old and also more rapid in warm-blooded types. Degeneration proceeds centrally in some cases, and involves cytons and dendrites. Xo regeneration of such cells takes i)lace. Nor is there complete regeneration in the central nervous system. The phenomenon of \Yallerian degeneration has been of great value in aiding the deter- mination of the central origin and ])eripheral termination of groups of nerve fibers. An experimental lesion is made involving a small area in the spinal cord. Proper time for degeneration of the nerve fibers concerned is permitted to elapse. The animal is then killed. The cord is removed and serial sections made following the proper technicjue devised for the purpose. Disintegrating fibers and cytons have a distinguishing ai)i)earance and enable the investigator to follow the route of the degeneration and thus determine the origin of the fibers occupying the region of the cord involved in the experimental lesion. THE BRAIN AND SPINAL CORD. Both the brain and sjMual cord are suspended within the carti- laginous or l)ony ca])sule by several connective-tissue membranes. The outermost, the dura mater, is a thick fibroelastic connective tissue attached to the bony or cartilaginous capsule. A narrow cleft, the subdural space, contains fluid and separates the dura from an innermost membrane, the arachnoid, a thin connective-tissue membrane. Connecting strands from the arachnoid to the dura mater divide the subdural space into compartments, others join it to an innermost membrane, the pia mater, immediately surrounding the brain or cord. The spaces of the arachnoid are filled with fluid, the cerebrospinal fluid, a term applied also to the fluid within the ventricles of the brain and the canal of the spinal cortl. The brain like the spinal cord has a region of gray matter occupied by cytons, nerve processes, and neuroglia; and a white matter occupied by the processes and neuroglia only. Among the lower forms the cytons are fewer. The cerebrum and cerebellum of mammalian brains may be recognized by the type and arrangement of cells; in the cerebral cortex the pyramidal cells are characteristic, and in the cerebellar region the Purkinje cells are diagnostic features. 1 10 THE NERVE TISSUE Structure of the Spinal Cord.— The gray matter in the cerel^riim and cerebelhun is external and eo\'ers the white matter. In the spinal cord the o;ray matter is internal and more or less surrounded by white matter. The cord is almost divided into two halves by a deep median dorsal septum and a shallower, wider, median ventral fissure. However, the two halves are connected by a white and gray commissure. The gray matter resembles the letter "X" or letter "H." The narrower dorsal horns of gray matter extend almost to the periphery. (Fig. ()o.) The ventral horns are much wider ^Dorsal median septum ^^o^>?T^cV, ^or fal column of white matter ^^^— Origin of dorsal root Dorsal horn of /i^i' graij matter f/'^^^^ mm White matter^fm ^ 'M -Lateral column Spinal canal pixSi'iV'j Doff^ Gray matter \i™ — Ventral horn of ^^fc^o's) gray matter fcr^il i ^o^E'Kl of white matter -Origin of ventral root Ventral median fissure^ Ventral column of white matter Fig. 6.S. — Diagram showing regions of the spinal cord. and do not extend to the ventro-lateral surface. The dorsal median septum extends down to the gray commissure, and the latter con- tains the canal of the spinal cord which is lined by ependymal cells. Ventral to the gray commissure is a white commissure. The white matter between the dorsal septimi and the gray horn adjacent to it is known as the dorsal funiculus or column of white matter. \'entral and lateral to the dorsal horns on either side is the ventro-lateral column or funiculus. The latter white matter may be subdi\'ided into a ^•entral coliunn betw^een the ventral fissure and the gray matter of the ventral horn adjacent to it. The white matter dorsal and lateral to this is known as the lateral column of each side. The white matter consists of myelinated and unmyelinated axons, and neuroglia but no neurolemma. The surface of the cord is immediately covered with ])ia mater and this extends into the white matter as short septa in places. Studies of \ari()iis kinds THE BRAIN AND SPINAL CORD 111 have shown that the axons of the white matter oeeur in strands of well-defined location. The axons of the dorsal columns carry sen- sory impulses up the cord; those of the ventral columns carry motor impulses down the cord; and the lateral cohmins are suhdi\ided into tracts, some of which are sensory and some motor. The fjray matter of the cord consists of neurogHa; axons entering from the columns; entire short neurons; cytons, dendrites, and the initial portions of axons which extend further beyond the gray matter into the ventral roots of spinal nerves and on out into such nerves. The neurons of the gray matter are multipolar in type and have already been described. Axons of some gray matter neurons extend out into the white matter and are incorporated as part of a white column. REFERENCES. Bailey, P., and Heller, G. 1924. The interstitial ti.ssue of the nervous system: a review, Jonr. Nerv. and Ment. Dis., 59, ;^37. Bartelmez, G. W. 1920. The morphology of the synapse in vertebrate.s, Arch. Neurol, and Psychiat., 4, 122. Bartelmez, G. W., and Hoerr, N. L. 1933. The vestibular club endings in Ameirus: Further evidence on the morphology of the synapse, Jour. Compt. Neurol, 57, 401. Clark. E. R., Clark, E. L., and Williams, Roy G. 1934. Microscopic observations in the living rabbit of the new growth of nerves and the establishment of nerve-controlled contraction of newly formed arterioles, Am. .Jour. Anat., 55, 47. DE Renyi, G. S. 1931. The structure of cells in tissue as revealed by micro- dissection: V. The physical properties of nerve cells of the frog, Join-. Comp. Neurol, 53, 497. Einarson, L. 1933. Notes on the morphology of the chromophil material of nerve cells and its relation to nuclear substance. Am. Jour. Anat., 53, 141. Gerard, R. W. 1931. Nerve conduction in relation to nerve structure, Quart. Rev. Biol, 6, 59. Hill, A. V., Fenn, W. O., Gerard, R. W., and Ga.sser, H. S. 1934. Physica and chemical changes in nerve. Supplement of Science, vol. 79, No. 2. Loo Yu Tag. 1931. The forebrain of the opposum, Didelphis virginiana: II. Histology, Jour. Comp. Neurol, 52, 1. Mather, V., and Hines, M. 1934. Studies in the innervation of skeletal muscle: V. The limb muscle of the newt, Triturus torosus. Am. Jour. Anat., 54, 177. Ngowyang, G. 1930. On the growth of the motor cells, fiom birth to matur- ity, at four levels in the spinal cord of the albino mouse. Jour. Comp. Neurol, 50, 231. Parker, G. H. 1935. Neurohumors; Novel agents in the action of the nervous system. Science, 81, 279. Parker, G. H., and Paine, V. L. 1934. Progressive nerve degeneration and its rate in the lateral line nerve of the catfish. Am. Jour. Anat., 54, 1. Penfield, W. 1924. Olegodendroglia and its relation to classical neuioglia, Brain, 47, 430. 112 THE NERVE TISSUE Ranson, S. W. 1934. Anatomy of Nerves, 4th ecL, Philadelphia, W. B. Saunders Company. Sheldon, R. E. 1912. The olfactory tracts and centers in Teleosts, Jour. Comp. Neurol., 22, 177. Speidel, C. C. 1935. Studies of living nerves, Biol. Bull., 68, 140. TiEGs, O. W. 1931. A study of the neurofibril structure of the nerve cell. Jour. Comp. Neurol., 52, 189. Van Campenhotjt, E. 1930. Historical survey of the development of the SJ^npathetic nervous system, Quart. Rev. Biol., 5, 217. WiNDLE, W. F. 1933. Neurofibrillar development in the central nervous system of cat embryos between 8 and 12 mm. long, Jour. Comp. Neurol., 58, 643. WiNDLE, F. W., AND Clark, S. L. 1928. Observations on the histology of the synapse. Jour. Comp. Neurol., 46, 153. See Appendix for general text references. CHAPTER Vll. THE VASCULAll SYSTEM. In various regions of early embryos, groups of mesenchyme cells begin the development of the vascular system. The central cells of such an area become rounded and separated by a fluid inter- cellular plasma. The peripheral cells of these regions unite to form an endothelial tube enclosing the free primitive blood cells and the plasma. The thin walls about these spaces are interconnected with others, so that gradually a network of endothelial-walled tubes forms the first capillary system. Some of the early ca])illaries develop into arteries, others into veins, and a tubular part is later differentiated into the heart. In the development of an artery and a vein, not only is an enlargement of the tube brought about, but there is also an addition of fibroelastic connective tissue and smooth muscle organized in sheaths about the endothelial lining. THE CAPILLARIES. These narrow, delicate, endothelial-walled tubes form a vast net- work in the connective tissue throughout the body. The diameter varies from a minimum of slightly less than the diameter of an Fig. 64. — Diagram of part of a capillary network, showing endothelial cells erythrocyte to a diameter several times this. (Figs. 64 and 65.) In cross-sections of very small capillaries only one or two endothelial cells form the wall, l)ut in larger tubes a number of cells are present. The boimdaries of the endothelial cells show as irregular black lines after silver nitrate treatment. The cells are elongated in the direc- 114 THE VASCULAR SYSTEM tion of the flow in the himen and each cell has a nucleus in the center of a clear cytoplasm. In fixed preparations, the capillaries contract and in cross-sections the nuclei appear to protrude into the lumen, a condition not true of living capillaries. Some investigators conclude that capillaries are intrinsically contractile, and others believe that the contractility is due to certain flat irregular cells, similar to reticular cells, which lie outside the capillaries but in close contact with the wall. It is well known that plasma and leukocytes from the ]:)lood stream pass out into tissue spaces through the thin capillary cell and also that wastes in fluid form pass from tissue spaces into the stream within the capillary. Some investigators claim that the exchange is facilitated by minute openings where adjacent cells meet, but in general the endothelial wall appears quite unbroken. The Fig. 65. — Sectional view of a capillary in the gill of a dogfish and erythrocytes within it. chemistry and physics of passage of fluids and white cells from the capillary and back into it are incompletely known. Usually the blood-vascular system of a vertebrate is regarded as a closed system in that the erythrocytes do not normally leave it. In every other respect it is an open system. There is a constant passage of food compounds from the intestinal tract into capillaries and lymphatics in the villi where they lie near the epithelium. Oxygen diffuses through the wall of the air sacs of the lung and is taken up by erythrocytes in capillaries adjacent to the air sac wall. Wastes of metabolism also pass from tissue juice into capillaries. The arrangement of the capillary network is determined to a considerable degree by the disposition of the cells or tissue organiza- tion supplied by the capillaries. Capillaries supplying skeletal muscles are long tubules between adjacent nuiscle fibers and are connected laterally. In the kidney there is a rich caiiillary network between adjacent uriniferous tubules. In each villus of the small intestine there is a basketwork of (•ai)illaries in the stroma just within the epithelial wall. Secretory vesicles of the thyroid gland THE ARTERIES 115 are enclosed in delicate connective tissne in which lies a network of capillaries. The richness of the ca])illary network is related to the functional activity of the organ supplied. Where great functional activity of the organ is constant, the meshwork is close and the capillaries are large. A good illustration of this is seen in the capillary network about the alveoli of the lung and the tubules of the kidney. The capillary system of the liver and spleen is atypical and will be described when considering these organs. Healing of tissue lesions may involve development of new capillar- ies and of small arterioles and venules. In case such structures form, they first appear as buds from existing capillaries. An examination of the circulation in the web between the toes of the frog's foot reveals a rapidly flowing stream of blood cells in the small arterial branches and a much slower streaming in the network of capillaries. Erythrocytes are carried along like lea\'es in a stream, some having to bend in making a turn from one channel into another. The slow flow through capillaries permits diffusion of food and oxygen out into the tissue spaces and a return of organic wastes to the blood stream from the tissues. As the capillaries unite to form small veins, the speed of the current increases again, but is not as rapid as in the corresponding arteries. THE ARTERIES. The transition from capillaries to arteries is marked by the gradual appearance of smooth muscle and fibroelastic connective tissue. (Fig. (iG.) In general, three coats are usually indicated in a study of the wall of an arter}.- an inner, tunica intima; a middle, tunica media; and an external, tunica externa. The intima is com- posed of the lining endothelium with a slight amount of fibroelastic connecti\'e tissue. The media is characterized b}' smooth muscle associated with varying amounts of fibroelastic connective tissue. The adventitia is chiefly fibroelastic connective tissue. Arteries are usually divided into three groups on the basis of size and composition of the media. The large or elastic arteries include the aorta, pul- monary arteries, carotids, and a few others with a similar structure. The medium-sized, or muscular, arteries include other arteries named by anatomists; these vessels agree in structure, although they vary in size. To the small arteries belong a great number that have no special names. Two more groups might be added, namely, arterioles and precapillary arterioles. Although the composition of 116 THE VASCULAR SYSTEM all these groups varies, there is a gradation of the type of construction all along the line, as can be understood best b}' studying the smallest arteries first. Small Arteries.— The smallest branches adjacent to capillaries have a small amount of connective tissue supporting isolated smooth muscle cells outside the endothelium. These are pre-capillary arter- ioles. They are branches of larger arterioles which have a definite smooth muscle sheath surrounded by a sheath of fil^roelastic tissue. The small arteries have thick walls in comparison with the size of the lumen. The intima has a thin subendothelial laver of fibro- 1 Capillary S [ Arterioles 4 Fig. 66. — Diagram of small arterioles and a capillary. elastic connecti^'e tissue separated from the media by an internal elastic lamina, a membrane of elastic fibers. The media is composed mainly of smooth muscle cells interspersed with fibro- elastic connective tissue and is separated from the adventitia by an external elastic lamina which resembles the internal membrane. (Fig. 07.) The adventitia is composed of fibroelastic connective tissue with a very few smooth muscle cells. It is usually not as thick as the media but much thicker than the intima. With the addition of the muscle tissue the vessels take an acti^'e i)art in distributing blood by rhythmic contractions or peristaltic action. These small arteries grade into medium-sized arteries. Medium-sized or Muscular Arteries. These are larger than the preceding vessels and the predominant tissue of the media is smooth THE ARTERIES 117 muscle. Ill the larfjer branches the proportion of elastic fibers in the media increases. The intima is often ])o()rly preserved, the endothelial cells bein^ indicated by their nuclei which protrude into the lumen. The subendothelial connective tissue is generally inconspicuous but is better seen in the larger \essels of this class. The internal elastic membrane appears as a clear, wavy line and the thick media shows numerous layers of smooth muscle separated by connective tissue. In larger vessels, elastic fibers appear more prominently in the media. The adventitia is as thick as the media or may be thicker, with elastic fibers becoming more numerous toward the media. Fig. 67. — A photograph of a small artery of the cat, showing internal and external elastic membranes out- lining the muscular and elastic media. Fig. 68. — Aorta of the dog, fixed in Bouin's fluid and stained with hematoxylin and eosin, showing the heavy elastic media and fibrous adventitia. , Large or Elastic Arteries.— These ^TSsels, such as the aorta and the pulmonary arteries, have a relati^•ely thick wall which appears thin in view of the large size of the lumen. When compared with a medium-sized artery the muscle has decreased in amount and there is a greater supply of elastic fibers. The intima is thicker due to the presence of more subendothelial connective tissue. The internal elastic lamina is not evident and in the media are many layers of fenestrated elastic membranes which are seen best when orcein or resorcin fuchsin is used as a stain. (Fig. ()8.) Alter- nating with the elastic tissue are smooth muscle and collagenous fibers. The media appears to be very much thicker than the adventitia, which is composed of fibroelastic tissue with few, if any, smooth muscle cells. At the origin of the aorta and pul- 118 THE VASCULAR SYSTEM monary arteries from the heart, the walls may be composed mainly of cardiac muscle. The fibroelastic tissue forming the outer coat of the adventitia in all vessels merges with the connective tissue supporting the vessel. The main function of arteries is to conduct blood away from the heart. The contraction of ventricles forces blood into the great arteries originating from them, and these vessels already filled with blood are distended by the added supply. When the force of ven- tricular contraction is spent, the fluid tends to return to the ven- tricles but is prevented by the closing of the valves at their entrance. Then the distended elastic walls contract as the stretched elastic tissue recoils. The blood is thus sent on into the medium-sized arteries where the smooth muscle takes a part and active peristaltic waves propel the blood onward. THE VEINS. Passing from a capillary toward the heart, there are postcapillary veins, venules, small veins, medium-sized veins, and large veins, showing varying structural additions and modifications. Outside the endothelial lining smooth muscle and fibroelastic connective Adventitia Media .J , VEIN Intima Internal elastic membrane . ^r^T^T^^r External elastic membrane ARTERY Fig. 69. — Diagram of small artery and vein. tissue form the added tissue in the wall of veins, but there is such a great variation in structure that each vein nuist be studied for its particular type of organization. On the whole, however, it can be said that the wall of any vein is thinner than that of its accompany- ing artery, and consequently veins often appear collapsed in micro- scopic preparations. (Fig. t)9.) Elastic lamiiue are not usually present and in many cases it is difficult to distinguish all three THE LYMPH VESSELS lU) coats. The structure seems to be influenced by gravitational diffi- culties, so there appears to be more muscle in veins of the extremities than in those nearer the heart. Veins differ from arteries in that all except very small veins possess valves which are pocket-like extensions of the intima with a core of subendothelial connective tissue. (Fig. 70.) Such valves •% Valie p '^^^^^-^^ms^^f-s-^s^^^^m^ f Intima f- V_"?^>"-i ..^ Ph%3 ^m itSsL'^^tlif^' ^i^. ,-•.'' 1 m H^P^ ■*' ■^ ■ j%i'-*K>6asv ?3| ^H Bw^' ^■^^*^ m P piiniijiui ^ -• Fig. 73. — Mesenteric lymph node of the gray squirrel, showing dense cortical region and lighter medulla. The connective tissue of the capsule extends into the medulla at the hilum and forms trabeculae in the cortex. regions. Lymph nodes are prominent in the mesentery near the junction of the small with the large intestine and also in the con- nective tissue in the region of the axilla and the groin. (Fig. 73.) Each node is invested with a capsule of fibroelastic connective tissue in which smooth muscle cells are usually scattered. The capsular tissue is more evident at the hilum, or concave depression, where the efferent lymph vessel, an artery, vein, and nerves are connected with the node. In examinations of stained preparations of mammalian nodes, two regions, an outer cortex and an inner medulla, usually stand out clearly. (Fig. 74.) Cortex. — Licomplete partitions, or trabeculse, of fibroelastic con- nective tissue extend from the capsule into the interior, thus dividing the cortex roughly into compartments. In these compartments are THE LVMFII NUDES 125 lymph nodules, varying in sliajx', tli()iiti|;li usually coiiiposed of oval or ])yriforni masses of lym])h()i(l cells. Kxteudini; from the coarser connective-tissue framework is a finer network of reticular cells and fibers in whose meshes the lymi)h()id cells are held. The reticulum extends between the nodules and trabecnhe and capsule, forming cortical sinuses. x\fferent lym])h vessels entering; the capsules break into capillaries connecting with these sinuses through which the infiowing lymph passes into the interior. The lymphocytes pro- duced in the nodules move through the reticulum into the cortical lymph sinuses through which lym])h is slowly ])assing toward the medulla. The size of the germinal centers varies with the degree of mitotic activity in them, the active nodules having more marked ■Afferent lymph vessel apsiile Traheeula Reticular tissue network Efferent lipnph vessel Fig. 74. — Diagram of a connective tissue and reticular tissue skeleton of a lymph node. germinal centers. Cells of the sinus walls become macrophages and remove foreign elements from the hinph as it filters tlu'ough (Fig. 75.) Medulla.— The internal ends of the connective-tissue trabeculge form a network of coarse fibers between which extends the reticulum of argyrophil fibers and reticular cells. Clustered along the coarser connective-tissue network are rod-like masses of lym])hoid cells known as medullary cords. Between them are inter- connecting spaces, the medullary sinuses, with which the cortical sinuses are continuous. The walls of the sinuses in the cortex and medulla are formed by reticular and endothelial cells which, in case of invasion by foreign substances, such as bacteria, become active phagocytic cells, or macrophages. There is a slow distribution or circulation of lymph from the lymph vessels at the cortex through 126 THE LYMPHATIC SYSTEM the sinuses into the efferent lymph vessels at the hilum. During this passage through the sinuses a number of lymphocytes are added so that lymph leaving the node is richer in cells than when it entered. Three varieties of lymphocytes (small, medium, and large) have been distinguished in the node, but by far the greater number are the small variety. When mitotic activity is at its height the germinal center is filled with lymphocytes of the small variety similar to those in the peripheral portion of the nodule. Then proliferation ceases for a time and during this resting period the nodule has a uniform appearance throughout. Even isolated nodules have periods of rest and acti^•ity. Afferent lymph vessel TrabecuJa Capsule Trabecula Lymph nodule Medullary cords Retficular- nehvork Efferent lymph vessel Fig. 75. — Diagram showing structural plan of a lymph node. The cortex is the outer zone containing lymph nodules. The medulla is the central region with cords of lymphocytes. The artery entering at the hilum branches throughout the fibro- elastic connective tissue skeleton, breaking up into capillaries which connect with venules and the latter eventually with the vein or veins which leave from the hilum. Small arterioles in trabecule give rise to a capillary net which surrounds the nodules. So far as erythrocytes are concerned, they remain within the vascular system in the node, i. e., there are normally no free red blood cells in the sinuses. Trabeculse appear to be better developed in perii^heral lymph nodes, such as those in the axilla or groin. Nodes within the body cavity have relatively greater medullary regions. Trabecid;e of small nodes are difficult to locate, and in some nodes the cortex is massed chiefly at one end while the medulla is concentrated at the other. THE SPLEEN 127 Lower vertebrates do not show such marked lyini)h()id or^jjaiiiza- tion. In the frog there are scattered and ^•ery small oval or spJierical bodies composed of accumulations of lym])hocytes in a network of connective tissue surrounded by a fibroelastic cai)sule. Fine trabecuke may dixidc tlie structure, but medulla and cortex are not differentiated. Presumably the lym])hocytes ])roduced here enter lymph capillaries su])])orted in the connective tissue of the organ. THE HEMOLYMPH GLAND. Although the lym])h node appears limited to mammals, somewhat modified structures, called hemolym])h nodes or glands, are found in mannnals and birds. In these lymphoid organizations, which structurally resemble the lymph nodes, blood instead of lymph is distributed in the cortex from arterial capillaries and filters through sinuses to be collected by efferent veins. Lymph vessels are limited to the scanty trabecular tissue. THE SPLEEN. The spleen is the largest single lymph gland. It is similar to hemal nodes, which are often called accessory spleens, in being associated with the blood-vascular system instead of lymphatics. Its shape and location varies in different vertebrates; among the fishes it may be an irregular mass closely associated with the stomach and intestinal wall; in others, as in the frog, it occurs as a spherical mass in the mesentery supporting the intestine; among the mam- mals it is a flattened structure near the stomach. . The spleen is surrounded with a capsule of fibroelastic connective tissue and smooth muscle covered with mesothelium, but there is no division into a distinct cortex and medulla. Trabecula? of con- nective tissue and smooth muscle extend from the capsule to form a coarse internal framework. A reticulum like that in the nodes extends between the trabeculse and forms the inner supjiort for the lymphoid tissue. The larger arteries and veins supplying the spleen are carried by the trabeculse. Examination of slices of fresh mammalian or avian spleen re\eals small, whitish masses, the white pulp, distributed throughout a red tissue. Each white pulp mass is a splenic nodule (splenic corpuscle or Mal])ighian corpuscle) which is composed of a mass of lympho- cytes enmeshed in a reticular network, and is com])arable to a solitary lymph nodule or to a cortical nodule of a lymph node. 128 THE LYMPHATIC SYSTEM (Fig. 76.) Within each is one or more small arteries, usually eccen- tric; a feature which serves to identify spleens so organized. The red pulp is composed of a reticular network, diffuse lymphoid tissue, and sinusoids filled with blood. Fig. 76. — Spleen of the woodehuck, showing the large splenic corpuscles scattered through the diffuse red pulp. The nodules have light germinal centers and arterial branches appear in their peripheral portion as small, open, white areas. The microsco]jic structiu-e of a mannnalian spleen is clarified by an understanding of the distribution of blood vessels in it. The main splenic artery and splenic vein enter at the hilum and each Splenic 1^\^ sinusoid Fig. 77. — Diagram showing scheme of circidation in the spleen. vessel dixides into branches which arc su})p(»rtc(l by the branching tra})eculie. As the trabccuhe divide, each supports a l)raiich of an artery and an accom])anying vein. Small arterial branciies lea\'e the trabecular tissue and enter the lymphoid tissue. (Fig. ~~.) THE SPLEEN 129 The adventitia of such arteries has loose fil»roelastic connective tissue in which Iymi)hocytes become aggref^ated into rod-like or oval masses, the splenic nodules, or Malpighian corpuscles. The branches of these small arteries emerge into the red ])ulp, where they are known as ])ul]) arteries. Each pulp artery di\ides into a group of short vessels, the penicilli, which have a pulp, a sheathed, and a terminal region. The tunica media of the first division is rich in smooth nuiscle and is embedded in red i)uli); the sheathed portion is devoir! of muscle and has a narrow lumen surrounded by connective tissue infiltrated with the lymphocytes and connected with the reticulum of the red pulp; the terminal portion is an endothelial tube which unites with the peculiar sinu- soids in the red pul]). Endothelial cells Nucleus Membrane Reticular fibers Fig. 78. — Diagram of part of a splenic sinusoid which consists of long endo- thelial cells, a non-cellular membrane, and encircling a network of reticular fibers. Openings are indicated from the lumen of the sinusoid through the membrane. (Redrawn from Braus, in Bailey's Histology, Williams & Wilkins C'ompany.j The sinusoids (Fig. 7S) have a wider lumen than ordinary capil- laries, and their elongated endothelial cells are separated from each other at intervals and are associated with the reticular cells which form a network through the red pulp. At intervals, fusiform cells and reticular fibers encircle the endothelial cells. The many slit- like spaces in the walls of the sinusoids communicate with the loosely organized lymphoid tissue of the red pulp. The sinusoids join complete endothelial-walled venous capillaries, which continue into venules and veins carrying the blood back into the trabecular and out of the spleen. (Fig. 79.) The sheathed portions of the penicilli where the lumen is small slow down the blood issuing from the small arteries of the splenic sinusoids. The sheathed penicilli may act as valves, pre\enting backward flow of blood. In the red pulp of the spleen and of hemal nodes, definite openings in the blood-vascular system 9 130 THE LYMPHATIC SYSTEM occur. Cells in the blood stream can pass through the slits in the sinusoid wall into the red pulp proper and cells in the red pulp can pass into the blood stream by way of the sinusoid. In the reticulum and diffuse lymphoid tissue of the red pulp there are the same red and white cells found in the circulating blood. The lymphocytes produced in the nodules enter the reticulum of the red pulp and thus may pass directly into the blood stream. Also in the red pulp are numerous giant cells, macrophages presumably derived from reticular cells in which phagocytic activity is pronounced. Foreign elements and disintegrating blood cells are ingested by these cells. Fig. 79.— Photograph of spleen of Necturus, showing the vascular distribution; the vessel on the left is shown with several branches opening out into sinusoids. Mallory stain. In certain pathological conditions so many red cells are disinte- grating and so many are being phagocytized that the red pulp acquires a brown color. The endothelial cells also appear capable of phagocytic activity and ameboid movement. In embryonic mammals and in lower vertebrates the spleen is hemopoietic, and lymphocyte production is not so clearly localized in nodules. The structure remains fundamentally the same, except that nodules are no longer clearly differentiated, lymphocytes and erythrocytes both being produced in a conunon reticulum, as already observed in the chapter on blood. Although erythrocytes THE THYMUS 131 are imKluced in the s])leeii of the eiiil)ryo inammal, this ])()\ver is later transferred to tlie red marrow of bones. lIowe\er, in certain path()lo,u:ical conditions, erythroblasts and the three ty])es of myel- ocytes can he found in the spleen, showin"; that in such cases production of erythrocytes and fj;ranul()cytes can again occur here. A recent review of lymphatics by 1 )rinker and Fields corroborates an early account of Mollier concerning the ojjen nature of this ])orti()ii of the vascular system — open to the extent that blood with its cells can freely though sluggishly pass into diffuse lymphoid tissue of the red pulp. The s])lenic sinusoids join com])lete \-enous ca])illaries, which continue into small venules ])rojecting through the red pulj) to enter the trabecular. In the s])leen, blood can leave the vessels and mingle with surrounding cells without clotting. Blo()d-])ressure is very low here, and ])eriodic })ulsations of the entire spleen have been observed; a ])eriodic contraction forcing bloofi from the ])ulp into the sinuses and veins is followed by a dilatation. The smooth muscle in the capsule and trabecuhe effects this con- traction. The connective tissue of the capsule limits the dilatation or the accunuilation of too much blood as the contraction of the smooth muscle imparts a gentle pressure forcing the blood l)ack into the veins. The absence of cellular injury when blood enters the red i)uli) from the sinusoids apparently accounts for the failure of clotting of such extraxascular blood. THE THYMUS. The thymus occurs throughout the vertebrate groiips as a variable structure beginning as invaginations of the ei)ithelium of the gill clefts that in\-ade the underlying mesenchyme. The thyroid and parathyroid anlages are similarly derived. In some fishes the epithelial tissue appears predominant, but in most forms the asso- ciated mesenchymal elements develop a lymphoid tissue about the epithelial elements. This lymphoid tissue becomes more ])rominent among reptiles, birds and mammals. The epithelial tissue is usually cut off from the gill pouches during embryonic development. In the lower forms the thymus may remain as a varying number of small buds associated with the gill pouches from which they were derived. In the mammals a conniion lol)ulated mass is usually formed and lies along the lower part of the esojjhagus and extends into the thorax over the pericardium. In general, a thin-walled capsule of fibroelastic connective tissue 132 THE LYMPHATIC SYSTEM surrounds the entire gland and extensions of it divide the gland into small lobules. (Fig. SO.) Under the microscope each lobule appears to be a mass of lymphoid tissue with a darkly staining peripheral cortex of more dense lymphoid cells and a central diffuse medulla. The medullary portion of adjacent lobules connect with each other. The lymphoid cells of the cortex are very similar to small lymphocytes. At the border between cortex and medulla there is an abrupt change to the diffuse distribution of cells in the medulla. Each lobule has a network of reticular cells and fibers continuous with the coarse framework of loose fibroelastic connec- tive tissue. The lymphoid cells are considered by some not to be Fig. 80.— Photograph of a section through a portion of a child's thymus gland. Several lobules are shown with cortex and medulla. Note connection of the medullary portions of some adjacent lobules. typical lymphocytes, and as derivatives from embryonic endoderm are differentiated as thymocytes. However, their appearance, their ameboid motion, and other reactions are so similar that the usual view is that they are lymphocytes derived from mesenchyme ele- ments that migrated into this region. The reticular cells forming a network through the tissue are considered as derivatives of mesoderm into which the e])ithelial thymic invagination extended, and as derivatives of endodermal e])ithelium. From whatever source, these reticular cells of the thymus appear very similar to those foimd in other locations. Fusiform cells, called myoid cells, have striated ffbrils and occur commonly in the thymus of a number of forms, but not in mammals THE THYMUS 133 after birth. Aluch remains to be done to determine the exact natnre of the elements (•omi)()sin<^ this ()r' the loose connective tissue separating it from the epidermis. Chromatophores occur in this loose connective tissue closely surrounding the scales. The scales are formed bv the activity of osteoblasts derived from the coruun. INTEGUMENT OF AMPHIBIA. The skin of amphibians is usually soft and nu)ist and carries no scales. The epidermis is characterized by a stratified epithelium through which lunnerous nnilticellular alveolar mucous glands open. (Fig. 83.) The individual surface cells are not desquanuited, but there is a periodic shedding of large patches or sheets of cells of the corneum. The corium is thin and may be separated by lymph spaces INTEGUMENT OF AMPHIBIA 137 from the niiderlyin^ tissues. The rich siij)])ly of lilood vessels which extend into the coriuni faciUtate respiratory exchanges through the skin, which in these forms may he the main respiratory organ. Tlie gills of such forms as Xecturus are tuft-like ])rojections of skin richly supplied by the capillaries of the corium. In such forms as toads, which are primarily land-living forms, the epidermis is hard- ened and thickened to prevent loss of moisture. The skin of the frog is surfaced with stratified squamous epithe- lim, five to six rows of cells dee]). The basal cells are low columnar Fig. 83. — Photograph of integument of the frog with an epidermis composed of stratified squamous epithelium and associated simple alveolar mucous glands. Im- mediately below the epidermis there is a narrow region of loose vascular connective tissue in which contracted chromatophores occur; below the gtands the corium is composed of a dense fibrous tissue. or cuboidal, the intermediate cells flatten out toward the surface, and the superficial scaly cells adhere to one another so as to form a cuticular covering which sloughs off in sheet-like patches. Just underneath the epithelium there is a loose vascular connective tissue in which chromatophore cells are scattered, while beneath this is a zone of more densely organized connective tissue. The deepest region of the corium is densely organized, with ])arallel collagenous bundles. Alveolar glands extend into the superficial corium beneath the chromatophore network, each gland opening among the basal cells of the epithelial covering. The excretory duct leading from the mouth of the gland forms a channel passing through the epithe- lium to the surface. 138 THE INTEGUMENT Two types of alveolar jijlands are differentiated. A smaller mucous secreting gland occurs generally in the connective tissue below the epithelium. Another and larger type, called the poison gland, extends more deeply into the corium and is surrounded by smooth muscle cells. It is composed of cells filled with acidophilic granules; the major portion of these cells disintegrate, liberating the secretion into the lumen and leaving the nuclei scattered along connective-tissue sheath. The function of these glands is protective, their secretions are poisonous and presumably discourage many animals from eating frogs. To the mucous glands are attributed the function of keeping the skin moist. The skin of Xecturus has a stratified cuboidal epithelium forming the epidermis and a network of chromatophores in the connective tissue just below. Among the epithelial cells are scattered spherical cells filled with acidophilic secretion. As in the frog, there are two types of glands. One is a smaller mucous gland, while the other is a larger serous type, in which the contents of the cells disin- tegrate and fill the lumen with secretion, leaving the nuclei scattered aroimd the periphery. INTEGUMENT OF REPTILES. With reptiles the land habitat is assmned, and a dry skin makes its appearance. The epidermis of the reptile is relatively thin and the superficial region in contact with the air is composed of dead, cornified squamous cells. Folds of the cornified epidermis alone form the scales of reptiles. Some reptiles shed their skin periodically, the horny superficial epidermis (the corneum layer) separates from the softer subjacent cell layers so that this dead upper covering of the epidermis is shed in one piece. Glands are restricted to regions about the mouth and anus. The corium in reptiles is relatively thick and is composed of dense connective tissue, with numerous chromatophores located imme- diately below the epidermal covering. When bony ])lates are formed they are derived from the corium in a manner similar to that described in intramembranous bone formation. In most turtles, both scales and bony plates are formed. In turtles and alligators, the tissue of each plate is not continuous with the l)()ny tissue of adjacent ])lates, so that growth in size of the animal is accompanied by continuous peripheral additions to the indi\i(hial plates. When claws are present, they are formed by accunuilations of the corneal layer of the epidermis. INTEdUMENT OF MAMMALS 139 INTEGUMENT OF BIRDS. In birds the skin is thin hut is covered by scales and feathers formed by the epidermis. The epidermis has a superficial layer of dead cornified squamous cells resting on a few layers of poly- hedral cells. The scales covering the legs and the claws of the toes are formed by accumulation of cells in the corneum. Glands are limited to the base of the tail, where the uropygial glands secrete an oily substance used mainly in dressing the feathers. The dermis is composed of dense fibrous connective tissue adjoining the epi- dermis, but in the deeper region there is a looser fibroelastic con- nective tissue carrying blood vessels and containing numerous fat cells. Pigment cells are generally absent from the corium, the coloring of birds being due to pigment secreted in the epidermal cells of the feathers. The bill, or beak, of lairds is formed by the accumulation of horny epidermal cells. Feathers.— The beginning of feather development is very similar to that of scale development: a dermal pajiilla thrusts out with a covering of epidermis and then the base sinks into the dermis. The dermal pajjilla carries blood vessels and nerves to this epidermal covering which alone is responsible for the feather formation. INTEGUMENT OF MAMMALS. Among the mammals, both layers of the skin are usuall\' much thicker than in the forms previously described. IVIulticellular glands and epidermal formations, the hairs, are abundant. The structin-e of the epiflermis varies considerably in different parts of the mammalian body. In exposed regions where there is consider- able friction, the epidermis is extremely thick because of an accu- mulation of cornified cells in the corneum. Over the body generally the skin proper is broken by numerous projecting hairs, as well as by the ducts of sweat and sebaceous glands deri^'ed from the activity of the stratum germinativum. Pigment may be found in the ejji- dermal cells of most mammals. The dermis composed of interlacing bundles of fil)roelastic con- nective tissue divisible into two blending layers, an upper narrow one, called the subepithelial or ])a])illary layer, and a dee])er, denser, wide reticular layer. Immediately below the basal epithelial cells of the epidermis lies a fine network of reticular fibers and reticu- lar cells. These fibers intermingle with the elastic and collagenous fibers to form a network generally paralleling the skin surface. 140 THE INTEGUMENT The dermis projects into the epidermis in the form of ridges and papillae. In the connective tissue near the epidermis are cells con- taining pigment granules and resembling the dermal chromatophores which are so very prominent in the dermis of amphibians. Some of the dermal papilhe contain ca])illary networks furnishing nutri- ment to this region and to the overlying e])idermis. In papillae adjacent to those possessing capillary networks are a number of types of sensory end-organs. Sir. corneum Str. gernunativum \;^^i'^^(^ (. " Sweat gland Subcutisl ■•^/ Str. corn rum Str. lucuhilum Str. granulo.sum Str. germinativnm > Subcutis A B Fig. 84. — Diagrams of skin striiftiire. .1, from back of hand; B, from palm of hand. Skin of Palm or Sole. — The skin covering the palm of the hand or sole of the foot offers an example of the extreme development of the skin of mammals. (Fig. 84.) The epidermis of these regions ])resents four distinct strata. Proceeding upward from the dermis these are: the stratum germinativnm, the stratmn granulosum, the stratum lucidum, and the stratum corneum, or superficial layer. It is to be understood that cells pass through all these layers before being cast off from the surface of the skin, the ditfercnt layers merely repre- senting various stages in the transformation of the epidermal cell, from the time it leaves the stratum gern.'nativum, until its death and desquamation. Stratum Gerininativum. — The lower border of the strat m germi- nativum is not a smooth surface because of the projc^ctioii of dermal INTEGUMENT OF MAMMALS 141 papillfie. Since the outer surface of the epidermis is smooth, its di- ameter must be thicker at some places than at others. The stratum germinati\um, or Mal])ighian layer, consists of a base of short colum- nar cells ()\erlying which are several strata of polyhedral cells, the outermost being somewhat flatter than the inner. Frequent cell di- visions occur in the cells of the Malpighian layer, and at each division one of the daughter cells is pushed out toward the surface. Since this process is continuous, older cells are constantly being pushed further and fiu-ther away from the basal layer. Consequently, each cell passes through the three outer zones and is eventually cast off from the surface. So-called protoplasmic bridges connect adjacent cells of the stratum germinati\'um and have led some to call them "prickle" cells. Skin color depends on dift'erent colored Fig. 85. — .Section through the foot-pad of a squirrel, showing four regions of epidermis. pigment granules in the lower cells of the stratum germinativum. These pigments range through black to yellow and red and may be present only in the lower layer of cells, or may extend to all the cells of the stratum germinativum. Stratum Granulosum. — Thh is a zone of several layers of cells outside the stratum germinativum. The cells have a flattened poly- hedral shape. Irregular-sized granules which are composed of a substance called keratohyaline represent some definite chemical change in the protoplasm of these cells. The nuclei of the outer cells of this granular zone disintegrate. Stratum Lucidum. — This layer appears as a bright, clear band just above the stratum granulosum and consists of closely packed 142 THE INTEGUMENT translucent cells. The granules so conspicuous in the granulosuni are, in this layer, dissolved into a semifluid colloidal substance called eleidin. (Fig. S5.) Stratuvi Corueum.— This is the superficial layer of the epidermis composed of dead scale-like cells in which the eleidin has become keratin and the nuclear regions appear as clear spaces. These cells are constantly being shed or desquamated. In other regions of the skin, however, these four zones do not normally appear and only the stratum germinativum and the stratum corneum are distinct. Sweat Glands.— Distributed widely through the skin of some mammals are the simple coiled tubular sweat glands. (Fig. 84.) Their coiled portion lies well down in the reticular layer of the der- mis, or even in the subcutaneous region. The excretory duct formed by cuboidal cells runs a more or less straight course. In the epidermis the duct is merely a spiral passageway between the cells. The internal twisted secreting portion is composed of pyra- midal cells, each with a basally placed nucleus and granular or vacuolated cytoplasm. The free end of the cells adjacent to the lumen may swell with secretion and become detached from the basal nucleated part to fill the lumen. The secretion is watery and may be slightly colored from pigment present in the cells. In cats, rats, and mice the sweat glands are restricted to the toe pads; in rabbits, to the region around the lips; in deer, to a region near the base of the tail; in the hippopotamus, to the inside of the ears; and in the cow, to the surface of the snout. These glands play a part in the regulation of body temperature and the excretion of wastes. Hairs. — Hair development is similar to that of feathers and scales. A hair originates from groups of cells in the deep layers of the epidermis which grow down into the subjacent dermal connecti\e tissue and produce an epithelial rod at an oblique angle with the surface. From these epithelial cells surrounded b>- the connective tissue of the dermis, shafts of hairs are formed and grow out beyond the surface of the skin. The epithelial cells and surrounding con- nective tissue of the skin form the hair root. (Fig. SG.) The root is surrounded by the hair follicle, a blind tube-like sac, internally comi)()sed of epithelial sheaths, and surrounded by con- nective-tissue sheaths. Indenting the base of the follicle there is a knob-like extension of vascular connective tissue called the hair papilla. Cells multiplying in the basal portion of the epithelium INTEGUMENT OF MAMMALS 143 above the pa])illa form the shaft, which, with its dilVerentiating and cornifying cells, is thrust upward into the canal leading through the epidermis. Between the hair itself and the follicle wall is a space, and into this, not far from the surface, opens the excretory duct of a sebaceous Den Sebaceous (jlatul -— Cortex of hair Vessel Dermic coat Outer root f:^" Stratum corneum ^~ Stratum lucidutii ^ Stratum granulosa in ' Stratum mucosum " Stratum germinatimim oj nair - 7-,^:Kr — -^^T" - r --- 'y^ uc coat ^-^^^^^^jr^^^^^^ Inner root sheath - V-- —s\^^'^-'<^~rr ^ r c^ "r'.^y; Arrector pili -~>~''^-'^'-'"~ ' muscle Dermic coat Z::^ • Medulla of hair Bulb of hair - Papilla of hair — -'^S^S"'^-^..^ — Fig. 86. — Diagram of skin structure in region of a hair. (Gray's Anatomy.) gland. The epidermis of the follicle, from the surface as far down as the sebaceous gland, has the usual e])idermal layers of the skin; but further down, first the corneum, and then the granular layer, disappears imtil only the Malpighian layer, or stratum germi- nativum, persists in the deeper portion of the follicle. This per- sisting layer thins down to form the external root sheath around 144 THE INTEGUMENT the shaft of the hair; further toward the base of the folHcle, a special epithelial layer, the inner root sheath, surrounds the root, and separates it from the external root sheath. At the enlarged base of the follicle the sheaths blend with the root into a wall of indifferent epithelial cells above and around the papilla, and from these cells the hair continues to develop. It is e^'ident that cross- sections made through a hair follicle at different \eye\s will show distinctly different compositions. Hairs are set obliquely in the skin, and on the acute angle side there is a small strand of smooth muscle, the arrector pili muscle, which extends through the dermis from the side of the follicle diagonally upward toward the epidermis. When this muscle con- tracts the hair is pulled more erect to form a temporary ridge in the skin adjacent to the hair. When such contractions occur generally over the body they produce the condition of the skin known as "goose flesh." In man there is a constant falling-out of hair and growth of new hairs, quite independent of the seasons; in most mammals the shedding of hair takes place annually, in late spring or early sununer. Sebaceous Glands. — Between the arrector pili muscle and the hair follicle there is usually a sebaceous gland which is simple branched, alveolar in type. The gland is composed of large, clear cells derived from the epithelia of the outer root sheath, and is surrounded by a capsule of connective tissue derived from the follicle sheath. The excretory duct is relatively wide and has a layer of stratified squa- mous epithelium. (Fig. 86.) The alveoli are filled with stratified cells which are relatively small and mitotically active basally. Toward the center of the alveolus, one can follow the accumulation of fat globules in the cytoplasm with concomitant increase in the size of the cell. Ulti- mately the entire cell disintegrates and the secretion, together with the disintegrating products of the cell itself, is liberated at the side of the hair near the skin surface as an oily compound, the sebiun. After such disintegration of the cells and the discharge of the sebum, regeneration of new epithelium of the gland takes place from the basal cells. Possibly the muscular contraction of the arrector ])iH muscle aids in the discharge of the secretion at the neck of the hair follicle. Meibomian glands are modified sebaceous glands which occur in mammals along the edge of the eyelids. Tlicy prochice a INTEGUMENT OF MAMMALS 145 secretion liberated at the base of the eyelash. Somewhat similar ceruminons glands lining the external auditory meatus s(>orete a waxy su})stance. ]\Iost mammals have mucus-])rodncing and lubri- cating epidermal glands associated with the skin of the external reproductive structures. In the skin near the anus of some forms there are modified sebaceous glands producing odoriferous secre- tions. Mammary Glands. These glands, which are for the most part located in the dermis, arise from invaginations of the embryonic skin. They form in both male and female, but normally develop to functional maturity only in the female and undergo regressive Fig. 87. — Mammary gland of human in intermediate stage of activity, showing two lobules with an interlobular duct breaking into two mtralobular ducts that extend into the secreting end-pieces. A dense connective tissue surrounds the lobules and groups them into lobes. changes in the male. Each mammary gland (Fig. 87) is compound aheolar in form. The excretory ducts open in the nipple, which is a conical extension of connective tissue covered with epidermis. The alveolar portions of the mammary glands are large and com- posed of pyramidal cells, but there is considerable ^•ariation in size depending on the secretory state of the gland. When acti^'e, fat globules accumulate in the cytoplasm toward the free end of the cells and the enlarged terminal portions are discharged into the lumen. The remaining nucleated basal portion of each cell soon repeats the process. Secretions accumulating in the lumen pass along small ducts which open into the larger lactiferous ducts. The number 10 146 THE INTEGUMENT of these ducts in each nipple Aaries from one to many. They open to the surface at the end of the nipple, where the stratified squamous epithelium of the covering skin continues inward to line their outer- most portion. Before reaching the nipple, the lactiferous ducts dilate into reservoirs for the storage of the secreted milk. In the inactive state the alveoli are collapsed and the gland is composed mainly of connective tissue. Nails.— The nails are comparatively simple derivatives of the skin, which cover the dorsal surface of the tips of the fingers and toes of primates. (Fig. 88.) They are horny plates, slightly convex and Fig. 8S. — Cross-section of the finger tip and nail of human, showing nail bed, grooves, nail, and skin of the finger. roughly rectangular. The skin underneath the nail bed rounds later- ally and proximally into folds which form the nail wall. The over- lapping of these walls on the nails make deep proximal and shallow lateral nail grooves in which the nail lies. Under the proximal wall the nail is soft and is composed of stratified squamous epitheliinn corresponding to the stratum germinativum, proliferation from which gives rise to cells that harden as they push outward. The nail plate is formed of fused, flat, scale-like, cornified, ei)ithelium cells, and corresponds to the stratum corneum of the skin. In the proximal region the underlying epithelium is thicker and forms the matrix from which new nail formation takes place. REFERENCES. Alvey, C. H. 1932. The epidermal "glands" of Ceratodus and Protopterus, Anat. Rec, 54, 91. Cooper, Z. K. 1930. A histological study of the integument of the Armadillo, Tatusia novemcincta, Am. Jour. Anat., 45, 1. David, L. T. 1932. Histology of the skin of the Mexican hairless swine (Sus scrofa), Am. Jour. Anat., 50, 283. REFERENCES 147 Dawson, A. B. 1920. The integument of Nectunis inaeulosus, Jour. Morpli., 31, -187. Dawson, H. L. 1930. A study of hair growtli in the guinea-pig (Cavia cobaya) Am. Jour. Anat., 45 461. DuNiHUE, F. W. 1934. Histolysis and regeneration of anuran tail skin, liiol. Bull., 67, 381. Erickson, T. C. 1931. The postnatal development of the caudal integument in the rat, Am. Jour. Anat., 47, 173. Herrick, E. H. 1933. The structure of epidermal melanophores in frog tadpoles, Biol. Bull., 64, 304. See Appendix for general text references. CHAPTER X. THE RESPIRATORY SYSTEIVI. In describing the blood and integument we have ah-eady con- sidered one of the means whereby respiratory needs are supplied. Depending upon the environment of the animal in question, there are certain other arrangements of tissues to accommodate gaseous exchanges required by metabolism, THE RESPIRATORY SYSTEM OF FISHES. In the case of fish, respiration is effected mainly by means of gills, which are specialized structures arising from the pharyngeal region to accommodate respiration in water. Water enters through the mouth, passes into chambers surrounding the gills, and bathes them before passing outward through external gill slits. A typical gill is composed of a median septum supporting two lamellae of connective tissue and muscle and an exposed epithelial surface. The septa are supported by cartilaginous or bony arches. The lamellar surface is usually much folded and near it is an extensive capillary supply bringing the blood close to the water for the exchange of gases through the epithelial membranes. The air sac or swim bladder appearing in the majority of the higher fishes is formed as a thin-walled pouch from the esophageal region and is lined with thin e])ithelial mem})ranes. It has been considered by some as being associated with respiration and also as a hydrostatic organ. THE RESPIRATORY SYSTEM OF AMPHIBIANS. In amphibian larvjie and in adults of all mature water- dwelling forms, thin membranous external gills are develo])ed as epidermal folds. (Fig. 89.) They are lost in the adults of those species adapted to terrestrial life. These membranes are well supplied with blood vessels (Fig. 90) and ser^'e for respiration during aquatic life. The skin is also vascularly e(iuii)i)ed and takes ])art in respiration in many forms. THE RESPIRATORY SYSTEM OF AMPHIBIANS 149 III terrestrial aini)liil)iaiis, air is taken in thronj^;!! the nostrils with the aid of nniseles of the mouth and throat, and passes into the two internal sae-like diverticula of the pharynx, the lun bronchi, air passes through smaller branches to THE RESPIRATORY SYSTEM OF MAMMALS 151 bronchioles and CAeiitually reaches the microscopic end-pieces, the respiratory alveoli, where o;aseous exchange takes place. The nose is an organ composed of bone, cartilage, mnscle, skin, and epithelial membranes, so constructed as to provide open ways for the passage of air. The olfactory area of the passage has epi- thelium composed of three types of cells. There are small basal cells and long, thin supporting cells; scattered between these cells are long fusiform olfactory cells. The nasal cavity continues into many irregular spaces, or sinuses, lined with a thin epithelial membrane. The nasal passage continues posteriorly into the pharynx, where the epithelium consists of pseudostratified ciliated epithelium. ]\Iany of the long superficial cells are goblet cells, and the surface of the membrane is generally well moistened with mucus. Under the epithelium is fibroelastic connective tissue carrying lymphatics, blood vessels, nerves, and small mixed mucoserous glands. The Larynx.— The larynx is posterior to the pharynx in quad- ruped mammals and is sometimes called the voice box, for it contains the vocal cords. It has a skeleton of a number of cartilag- inous pieces, some of them single units, others occurring in pairs. In general, these cartilages are of the hyaline variety. They are covered with perichondrium which merges with the fibroelastic con- nective tissue forming the external covering of the larynx. Many small muscles of the voluntary striated type are present. The cartilages are covered with loose fibroelastic connective tissue which may be called a submucosa or tunica propria. It contains much lymphoid tissue and possibly scattered single lymph nodules. The epithelium of the mucosa is of the pseudostratified ciliated variet,y, except over the vocal cords and under the epiglottis where it is stratified squamous. The vocal cords are a pair of shelf-like tissue masses extending from the lateral walls of the larynx into the laryngeal cavity. P^ach vocal cord has an external skeletal muscle mass. Internal to this, toward the lumen, is elastic tissue and the stratified squamous epithelium covering the vocal cords. The Trachea.— The trachea is a dilated tube extending from the larynx to the bronchi. It is just dorsal to the skin of the neck in the mid-ventral line and dorsal to it is the esophagus. Laterally the muscles and blood vessels of the neck are associated with fibroelastic connective tissue which also forms the outer covering of the trachea. The th\-roid gland lies against its ventral surface 152 THE RESPIRATORY SYSTEM just back of the larynx. As it enters the thorax, the trachea divides into two branches, the bronchi, one of which enters each king. Embedded in the tracheal connective tissue are the rings of cartilage which keep this air passage dilated. In old animals they may undergo partial ossification. The cartilages are not complete rings, but usually have an open space between the ends adjacent to the esophagus. This space between the ends of the cartilage is filled by a band of smooth muscle and connective tissue. The cartilages are enclosed in perichondrium and the superficial portions of the perichondrium merge with -Epithelium the adventitial connective tissues and with similar fibroelastic con- ^i&'is^""''^''"""'"'"""'"" nective tissue l.ying between "^^^ Gland adjacent cartilages. The internal connective tissue may be called a -Submucosa submucosa and is rich in elastic fibers. In it are many small -Ptnchondrmm compound mucous and mixed ^^::S.j^ s^-j^ ^c^eD©^® mucoserous glands. ine sub- ' Advent it ia «>@® ® '^to^J^^S"^'"''^^'^^^ mucosa also contains manv scat- „@ ^ '^ %% t'S^^'^ tered lymphocytes which may ^Ssgj^%f^..s;£^ — Perichondrium be organized into lymph nodules. The mucosa is composed of ciliated pseudostratified epithe- FiG 94 —Mammalian trachea cross- hum, in whicll are nUmerOUS ^''*'*""^ goblet cells which add mucus to that from the submucosal glands, thus keeping the internal surface of the trachea moistened. (Fig. 94.) The Lungs. — Since the respiratory system of manunals arose embryologically as an invagination from the embryonic foregut, the trachea represents the principal excretory duct which divides pos- teriorly into two bronchi which branch into bronchioles. The lungs are invested with a thin double membrane, the pleurie, similar in structure to the peritoneum, with a potential space between the two membranes. The pleural membrane adjacent to the lung contains much smooth muscle in its connective tissue and di])s down into the spaces between adjacent lung lobes to connect with the con- nective tissue between the lobules. Bronchi and Branches. ^Each bronchus has about the same structure as the trachea but the size of the tubes decrease as THE RESPIRATORY SYSTEM OF MAMMALS 153 the branching increases. (Fig. 95.) Histological changes also appear. The cartilage rings change to flat cartilage plates which become smaller and smaller in the branchings until finally no cartilage su])])()rts the smallest bronchioles. Patches of smooth muscle arranged concentrically around the tube a])pear just within the cartilage ])lates in the tunica pro])ria. As the branches decrease in size the muscle increases proportionately until in the very small or terminal bronchioles there is no cartilage but a distinct Trachea Bronchioh Fig. 95. — Bronchial tree of mammal. circular sheet of smooth muscle is present outside the tunica propria. The epithelium changes gradually from a pseudostratified variety until in the smallest bronchioles there is but one layer of columnar ciliated epithelium. The goblet cells have disappeared also. The glands in the tunica propria, which in the larger bronchi were so numerous, have decreased so that there are none in the smallest branches. The mucosa and tunica propria in smaller branches show longitudinal folds. Terminal hronchioks (Fig. 90) are separated from the respiratory tissue by their adventitia, a thin layer of fibroelastic connective tissue. The tunica propria of connective tissue is rich in elastic 154 THE RESPIRATORY SYSTEM fibers arranged lengthwise of the tube and supports the mucosa of ciliated columnar epithelium lining the lumen. The lung is composed of many lobules which are somewhat conical or pyramidal in shape and are composed of a number of terminal bronchioles and their branches. Adjacent lobules are separated from each other by interlobular connective tissue. Each terminal bronchiole divides into two or more respiratory bronchioles. (Fig. 97.) Near its origin, the mucosa of this subdivision has ciliated columnar epithelium; distally the cilia are lacking and Terminal bronchiole Mm Connectne -— ^ - tissue Arteri/- -vy i) / Aluoh Alveoli Fig. 96 Fig. 97 Fig. 96. — Diagram of section of a terminal bronchiole, the lumen lined with ciliated columnar epithelium. Fig. 97. — Terminal bronchiole of mammal and its branches. the cells are cuboidal. Outside the mucosa the wall is very thin, consisting of fibrous tissue containing numerous elastic fibers and smooth muscle cells. It has a few small saccular diverticula, or pulmonary air sacs, whose wall apparently consists of flat, squamous epithelium invested with a rich capillary network where gaseous exchanges can take ])lace. At its distal end the respiratory bronchiole di\'i(les into two or more short, thin-walled, irregidar alveolar ducts. The simple squamous epithelium of the thin wall is supported by a very small amount of fibrous tissue with a few scattered elastic fillers and smooth uniscle cells. The wall is broken along its length b\- man^• THE RESPIRATORY SYSTEM OF MAMMALS 155 saccular diverticula, either alveolar sacs or siii(i;le i)uhnouary alveoli. (Fi^. 9S.) The fil)rous tissue, elastic fil)ers, and smooth muscle cells are chiefly in the wall of the aheolar duct, around the openings to the alveolar sacs and pulmonary alveoli. The alveolar sacs are not single bladder-like enlargements, but each consists of a cluster of respiratory or pulmonary alveoli which are homologous with the secretory end-pieces of a gland like the parotid. Alveolar sacs and respiratory alveoli are mere sacs whose walls appear to consist of squamous epithelium and re- ticular tissue. The alveoli often contain large cells called dust cells, since they contain black particles. Their source is debatable. Each pulmonary unit, there- fore, consists of a respiratory bronchiole and its alveolar sacs and pulmonary aheoli. The air sacs and alveoli are in- vested with a fine, close- meshed network of large capil- laries, so thick-set that the spaces between adjacent capil- laries is less than the width of the capillaries. Between adjacent alveoli there is also a rich meshwork of reticular fibers, some elastic fibers, and scattered smooth muscle cells. Vascular Supply. ^The lungs are supplied by two sets of blood vessels, namely, («) the bronchial arteries and veins, and (b) pul- monary arteries and ^•eins. The l^ronchial \'essels arise from the systemic system and their branches extend in the connecti\e tissue down into the interlobular connective tissues. The pulmonary artery arises from the right ventricle and has a branch to each limg, w^here its branches follow those of the bronchi. Alongside a ter- minal bronchiole is a very small branch of a pulmonary artery. This branches as the respiratory bronchioles are formed, so that near the alveolar ducts and sacs the pulmonary arterioles break into capillaries which invest the air cells. At the lateral borders of the alveoli of a pulmonary unit, the small venules originate which Fig. 9s. — Photograph of the king of a rabbit, showing a small bronchiole in upper right. In Ihe center a respiratory bronchiole is shown breaking into two alveolar ducts and their alveoli. 156 THE RESPIRATORY SYSTEM unite to form small veins of the pulmonary venous system. In a section of a lung, the branches of the pulmonary vein are somewhat distant from a bronchiole. The pulmonary venous system carries to the left auricle blood partially depleted of carbon dioxide and enriched with oxygen to be distributed to all organs and tissues. The lungs are innervated by branches of the sympathetic and vagus (tenth cranial) nerve. The abundant supply of smooth muscle and elastic tissue present play an important part in the recurring movements of inspiration and expiration. In addition to these are the muscular diaphragm and the intercostal muscles which play a role in these movements. Before birth the lung resembles a compound tubulo-alveolar gland, the terminal pieces being lined with cuboidal epithelium. At birth, the contact of the air with the body usually- acts as a stimulus to start up the series of inspirations and expirations that continue until death. With the first few inspirations, the inspired air fills and expands the lungs and the cuboidal cells of the terminal pieces are flattened. REFERENCES. Bremer, J. L. 1932. Accessory bronchi in embryos; their occurrence and probable fate, Anat. Rec, 54, 361. JossELYN, L. E. 1935. The nature of the pulmonary alveolar lining, Anat. Rec.,' 62, 147. Macklin, C. C. 1929. The musculature of the bronchi and lungs, Physiol. Rec.,' 9, 1. Olkon, D. M., and Joannides, M. 1930. Capillaroscopic appearance of the pulmonary alveoli in the hving dog, Anat. Rec, 45, 121. RoTHLEY, H. 1930. Ueber den feineren Bau der Luftrohre und der Lunge der Reptilien, Ztschr. f. wissensch. Biol., 20, 1. Stewart, F. W. 1923. An histogenic study of the respiratory epithelium, Anat. Rec, 25, 181. WiSLOCKi, G. B. 1935. The lungs of the Manatee (Trichechus latu-ostris) compared with those of other aciuatic mammals, Biol. Bull., 68, 385. See Appendix for general text references. CHAPTER XL THE DIGESTIVE SYSTEM. Arising from tlic simple eml)ry()nic invaginations which form the fore and hind gut, there develops in vertebrates a relatively elaborate system for service in the digestion, absorption, and defecation of food. This system is composed not only of the long canal that extends from mouth to anus, or cloaca, but includes also the tongue, teeth, a few large and a great many small glands derived from embryonic outjjocketings of the endodermal (epithelial) lining. Food is taken in at the mouth, where it is usuall,y subjected to the mechanical ])rocess of mastication by the tongue and the teeth. The secretions of the glands of this region pass into the oral cavity to aid in lubricating the food for passage. Food thus crushed and lubricated passes into the esophagus, which conducts it to the stomach, where digestive juices are poured upon it from the glands of the stomach walls. Very little, if any, absorption occurs in the stomach where the food is stored temporarily and is actively worked upon by digestive ferments (enzymes). It is passed from the lower end of the stomach by peristaltic waves into the small intestine, whose surface is greatly increased by folds. Secretions from small glands in the intestinal wall, together with the secretions from the pancreas and the liver, which are introduced here by ducts from these two organs, are all mixed with the food mass in the lumen. Under the combined chemical acti^'ities of the secretions added to it, the food is broken into products which are capable of being absorbed through the epithelial wall of the intestine. Passing through the small intestine the unabsorbed portion of the food enters the large intestine where absorption of water takes place. The glands lining the walls of the large intestine are probably mainly acti\'e in lubricating unusable materials and waste by- products of digestion for passage and elimination. THE MUCOUS MEMBRANE. The intestinal tube extends from the lips to the posterior aperture. There is a line of demarcation at each of these places where the skin ends and the membrane lining the cavity of the intestinal tract 158 THE DIGESTIVE SYSTEM begins. This lining is a mucous membrane, or mucosa. A mucosa consists of at least two different tissues: an epithelial membrane and the connective tissue, or tunica propria, upon which it rests. A third membrane of smooth muscle may also be present and form a muscularis mucosae. A typical mucous membrane, therefore, consists of an epithelium, a tunica propria, and a muscularis mucosa. Mucous membranes rest upon a submucosa, which is a loose fibroelastic connective-tissue layer. The latter is extensible, thus allowing for the passage or pres- ence of quantities of food in the tract and passively contracts as the food disappears. The apparent thickness of the submucosa depends usually on the distention of the canal. Where no muscularis mucosa is present the connective tissue just underneath the epithelium is more dense and corresponds to a tunica propria; the deeper zone which corresponds with the submucosa is more loosely organized. THE LIPS. A longitudinal vertical section through the lip, perpendicular to the surface, shows that the outer surface consists of skin tissue with an epidermis of stratified epithelium and a dermis of a dense fibroelastic connective tissue. Hairs, sebaceous glands, and sweat glands or other typical skin features are present, depending on the animal. These structures change at the inner surface of the lip proper, where the epithelium becomes gradually thicker and loses the integumental features. Small sulicpithelial connective tissue papillae appear. \Yhere the inner surface is continually moistened by the fluids present in the mouth, the epithelium is still thicker and the connective tissue papillae are more pronounced. The epithelium of the oral surface rests upon a denser connective tissue (tunica propria) which emerges into a more loosely organized, deeper con- nective tissue (submucosa), supporting the larger blood vessels, lymph vessels, and nerves. Small compound glands of the mucous- serous type are usuall\- present, those grouped in the submucosa near the margin of the skin area being called labial glands, and others further l)ack are known as buccal glands. The region between the dermis of the skin surface and the sub- mucosa of the oral mucosa is composed mainly of voluntary muscles. THE ORAL CAVITY 159 THE ORAL CAVITY. This region is lined with stratified epithehmn resting \\\Kn\ a subepithelial C()nnecti^'e tissue which is thicker in some ])laces than others. In the hard palate, when ])resent, the mucous mem- brane is in close relation with bone, but o\-er the jaws striated muscle lies below the mucous membrane. ]\Iucous, serous, and mixed mucoserous glands are often present. Reptilian, avian, and mammalian oral epithelium is usually stratified squamous in char- acter. The mucous membrane which lines the oral cavity of the frog is covered with stratified ciliated columnar epithelium in which goblet cells are numerous. There are also frequent pocket-like involutions lined with large goblet cells. In the tunica propria there is diffuse lymphoid tissue and occasionally a lymphoid condensation. Teeth. Very conspicuous as oral modifications are the teeth of mammals. They are hard structures derived in part from epithe- lium and in part from connective tissue covering the jaws. They develop from large papillse of connective tissue covered by epithe- lium. Both tissues undergo a peculiar hardening process through chemical transformations and deposits. The de\'elopment of the teeth resembles that of the placoid scales of Elasmobranchs. The gum consists of muscle, connective tissue, and stratified squamous epithelium. Each tooth is di\"isible into a crown which projects above the gum, and a root, or roots in cases of the larger teeth with divided basal insertions, which tapers to fit into an alveolus or pocket in the jaw bone. The tooth is composed mainly of dentin, \vhich surrounds a pulp cavity and is thickest in the crown region where it is covered by an epithelial deri^'ative, the enamel. Below the gum level, a thin layer of cementum covers the dentin of the root. The dentin and cementum resemble bone in structure, but the enamel is formed mainly of deposits of inorganic (only 3 to 5 per cent organic) salts in the epithelial cells and is a harder material than bone. A canal passes through the root carrying blood vessels and nerves to the pulp cavity. Tongue.— Although the tongue is generally represented in all vertebrates, it varies widely in the extent to which it develops. Among the fish it is poorly represented, and is called a primary tongue. Such a tongue is composed of a projection of dense con- nective tissue from the floor of the mouth and is covered by a mucous membrane similar to that of the oral cavitv. 160 THE DIGESTIVE SYSTEM The iirodeles show evidences of the formation of a secondary extension of the primary tongue, and this consists of muscle, con- nective tissue, and epithehum, together with glands that secrete a sticky mucous substance used in capturing food. When at rest the tongue of the frog lies along the mid-line of the mouth's floor, being attached in front and free behind. It can be flipped out, and when extended assumes a spoon-like form for catching its moving prey. The upper and lower surfaces are covered by stratified columnar epithelium. The epithelium of the upper surface of the resting tongue is comparati\'ely thin, with many Fig. 99. -Photograph of the tongue of the frog. The upper portion shows the glandular lower surface of the tongue. goblet cells and may be folded to form numerous papillae. The lower surface has a great many simple or branched tubular glands with dilated ends separated from each other by a minute amount of connective tissue. (Fig. 99.) The superficial epithelium is of the stratified columnar type, but the glands are lined with simi)le colum- nar cells, mainly mucous in nature. Considerable diffuse lymphoid tissue may be found below the epithelial surface, and occasionally a lymphoid condensation resembling a nodule. In the connective tissue below the mucosal covering are bundles of striated muscle fibers. The main blood vessels and nerves follow a central course through the tongue. Chelonia and Crocodilia have a fleshy secondary tongue capable THE ORAL CAVITY 161 of limited movement. In snakes tlie ton^ne is a slender l)ifurcate muscular orfjan that is usually retractable within a sheath in the base of the mouth. It is covered with stratified squamous ei)ithe- lium, below which a thin coat of coimective tissue with chromato- phores adjoins the underlying- muscles. The majority of the muscle fibers run longitudinally. The main blood vessels and the large nerve trunks run through the center of the tongue on either side of a median band of peri)en are much fewer in number than the filiform and are scattered irregularly over the papillary region among the filiform papillae. They are formed by extensions of the connective tissue that broaden out as they rise above the general \eye\ of the tongue. The e])ithelial covering is thin, so that these paiiilhe ai)pear THE ORAL CAVITY 163 red, (kie to the hlood of the uii(I(m-|\ iiii;- coiincctixt' tissue sl)()\viiiitlicr of the other salivary glands. THE ORAL CAVITY 165 Submaxillary Glands.— 'V\\\^ pair of inlands is located in the floor of the mouth and the secretion is carried into the oral cavity by an excretory duct which opens on the side of the tongue well forward. The division into lobes and lobules and the arrangement of ducts Excretory duct Secretory duct Intermediate duct End piece- Serous cells PAROTID Excretory duct Intermediate dud End piece Mucous cell with serous demilunes SUBMAXILLARY Fig. 103. — Diagrams showing structure of the parotid and submaxillary glands. Fig. 104. — Intermediate duct breaking into secretory end-pieces of the submaxillary gland of the cat, showing mucous cells surrounded by crescents of serous cells. 166 THE DIGESTIVE SYSTEM is similar to that found in the parotid. (Fig. 103.) There are two types of cells in the alveoli or secreting end-pieces. A serous type of cell similar to that found in the parotid may be found forming many of the alveoli. Another type of cell is present also, the mucous cell, with lighter, clearer cytoplasm, which takes a basic stain and has basally located nuclei. (Fig. 104.) Outside of some of these mucous cells are crescents of serous cells. The secretory ducts are more extensive in the submaxillary than in the parotid gland. In man, serous cells predominate, but in many other animals, such as the woodchuck and the cat, the mucous cells predominate. Suhlingval Glands.— The sublingual glands are located at the base of the tongue and, like the parotid and submaxillary, are divided into lobes and lobules. The intercalated ducts are lacking as may also be a large proportion of the secretory ducts. Most of the glandular end-pieces (in the cat) are composed of cells of the mucous type, w^ith more numerous crescents of serous cells than in the submaxillary. The parotid, submaxillary, and sublingual glands are uniformly present only in mammals. There are few, if any, glands associated with the mucous membrane of the mouth cavity of fishes, but mucous glands appear in the oral cavity of Amphibia. In reptiles, mucous glands are located in the mouth ca^dty near the edge of the jaws, in the tongue, in the wall of the mouth cavity, and also under the tongue. The poison glands of snakes are specialized labial glands. ]\Iucous glands are well represented in this region in herbixorous mammals that feed largely on dry grass. Pharynx.— The pharynx is posterior to the oral cavity. The pathways of food from the oral cavity to the esophagus, and of air from the nasal passages to the larynx, cross in the pharynx so that this chamber is part of both the digestive and respiratory systems. Embryologically, it is associated with gills, with the trachea and lungs, with the thyroid gland, parathyroid glands, and the thymus, although presenting a great deal of variation in different ^■erte- brates. It is somewhat funnel-shaped, exjjanded anteriorly and tapered down posteriorly. THE ALIMENTARY CANAL. Througiiout the alimentary or digestive canal, but not including the month or anus, there is a similar structural plan for the wall, which is com])osed throughout of four coats of tissues. THE ALIMENTARY CANAL 167 The innermost coat, the mucous membrane or the tunica nnicosa, is com])()se(l of the hnint:; e])itlielium resting on a l)asement mem- brane l)eio\v which is fibroeiastic connective tissue called the lamina proi)ria or tunica ])roi)ria. A thin, double layer of smooth muscle, the muscularis mucosae, is generally i)resent and nuirks the external boundary of the mucosa. Adjoining the mucosa there is a region of looser fibroeiastic connective tissue, the tunica submucosa, rich in blood vessels and nerves. The symi)athetic ner\-e plexus carried in this region is called Meissner's or the submucosal ])lexus. Surrounding the sub- mucosa externally there is a coat of muscle tissue, the tunica mus- cularis, consisting generally of an inner circular layer and an outer longitudinal layer separated by a narrow region of connective tissue which carries a sympathetic nerve plexus called Auerbach's jjlexus or the myenteric plexus. The outermost coat of the wall, the tunica adventitia, is composed of fibroeiastic connective tissue. This structural plan shows greatest variation in the mucosa of the different regions, es])ecially as regards the glandular development. The Esophagus. — 77?f Ampldbian Esophagus.— This part of the digestive tract has the typical four coats. The submucosa extends into a number of long folds carrying the mucosa into the lumen. The epithelium of the mucosa is usually of the stratified ciliated columnar type with niunerous mucous cells, although the esophagus of Amblystoma has a mucosal epithelium which resembles the pseudostratified variety, and in Cryptobranchus it is more nearly stratified cuboidal. The muscularis coat is usually represented by a prominent circular sheath, but the longitudinal muscle sheath is in many cases represented by separate bundles. The ad^'entitia connects externally with the skeletal muscles of the neck region. In the submucosa of certain portions of the frog esophagus large compound serous glands occur. The Reptilian Esophagus.— A study of the esophagus of the lizard (Fig. 105), snake, turtle, and alligator demonstrates that the submucosal folds extending into the lumen are i)resent here also. The mucosal epithelium is in general of the stratified columnar variety. The columnar zone cells ma\^ be ciliated and there may be many goblet cells present. The muscularis coat is represented by a distinct circular coat aufl a longitudinal coat formed by separate strands of smooth muscle cells. The adventitia is of loose connective tissue which sujjports })lood vessels, nerves, and lymphatics. 168 THE DIGESTIVE SYSTEM The Mammalian Esophagus. — Connecting the pharynx with the stomach is a tube with the characteristic four coats of the digestive canal ah-eady outhned. (Fig. lOG.) In the region of the neck the adventitia consists of loose fibroelastic tissue connecting the esoph- agus with neighboring structures in the neck. As it passes through the thorax this outer coat becomes a serosa (visceral peritoneum), which is composed of an outer thin mesothelial membrane and an underlying thin layer of connective tissue. The muscularis mav have striated \'oluntary muscle fibers in the upper region of Fig. 105. — Diagram of a cross-section of the esophagus of a lizard. Note the ruga- like extensions of the submucosa into the lumen. These are covered with stratified columnar epithelium with many goblet cells. The submucosa supports blood vessels. The muscularis coat is represented by a well-defined circular sheath. The longi- tudinal coat is represented by relatively few muscle cells. the esophagus, a mixture with smooth muscle in the middle region, and finally a lower portion composed entirely of smooth invohmtary muscle. Its composition is variable, however, for in some animals striated muscle may be found reaching to the stomach. A very loose fibroelastic connective tissue forms the submucosa which unites the muscularis with the mucosa. The submucosa contains large blood vessels and numerous mucous glaufls whose ducts o])en through the mucosa. A muscularis muco.sa marks the beginning of the mucosa, except in the upper })()rtions of the eso])hagus where it may be missing. Within the nuiscularis nuicosa is the fibroelastic tissue of the tunica propria, which projects as papilhe-like extensions THE ALIMENTARY CANAL 169 into the stratified squamous epithelium restinji; upon it. In addition to the deep mucous glands found in the suhmucosa there may also be simi)Ie branched tubular glands lying in the tunica proi)ria and resembling those to be found in the cardiac portion of the stomach. When the esophagus is contracted, the mucosa is disposed in longi- tudinal folds and the lumen is closed. AdventiHa Fig. 106. — Diagram of a cross-section of a mammalian esophagus. The Stomach. —Beginning with the stomach and extending through the large intestine, the adventitia is covered by mesothe- lium, and the coat thus formed is called a serous membrane or serosa. The Fish Stomach. ^The submucosa has broad ruga-like expan- sions extending toward the center of the lumen. The mucosa con- sists of closely packed, slender, simple columnar epithelium lining the lumen and tubular glands of variable depth. (Fig. 107.) The peripheral submucosa is not so dense as that adjacent to the epithe- lium. The muscularis coat consists of a thick circular muscle sheath and a thin outer longitudinal coat. The ad^'entitia is very thin. Evaginations occur in the posterior region of the stomach 170 THE DIGESTIVE SYSTEM Fig. 107. — Diagram of a cross-section of the stomach of a dogfish {Sqimlvs acanth- ias). This shows ruga-like folds of submucosa extending in toward the lumen. The mucosa contains closely arranged simple tubular glands. A muscularis mucosa separates these glands from the submucosa. The muscularis coat is represented by a circular smooth muscle sheath and a thin longitudinal sheath. The adventitia is dense and is associated with the mesentery. Fig. 108.— a cross-section of the stomach of a frog. The mucosa consists of simple branched tubular glands whose basal secretory cells are serous in character and acidophilic and the cells at the mouth of the gland appear to be mucous and basophilic. There is a well-defined muscularis mucosa. The submucosa is thickest in the region of the ruga-like extensions. The muscularis coat is represented by a thick circular and a thin longitudinal sheath of smooth muscle. The adventitia is a thin serosa. THE ALIMENTARY CANAL 171 of teleosts as pyloric ceca, small fiiiger-like pouches structurally similar to the stomach. The Stumach of ^[inphibid. The mucosa consists of simple branched tubular glands lined with simple columnar epithelium and separated from each other by tunica ])r()])ria. The submucosa has extensions forming folds into the lumen. There is in some s])ecies, such as the frog (Fig. 108), a well-defined muscularis mucosa. The muscularis coat has ])rominent circular muscle and a much thinner longitudinal coat. The Stomach of Reptiles. — Submucosal folds are present here also. The mucosa is composed of simple tubular glands lined with colum- Fig. 109. — Photograph through a ruga of the fundus of the dog's stomach. Note the broad extension of the submucosa surmounted by the glandular mucosa. The muscularis has a thick circular and thinner longitudinal coat. A muscularis mucosa follows the outline of the glandular mucosa. nar epithelium. The superficial ei)itheliiim appears to be stratified columnar in some forms and goblet cells may be nimierous. The circular coat of the muscularis is prominent, but the longitudinal coat is much narrower or represented by scattered bimdles. The adventia is usually thin. The Mammalian Stomach.— The mucosa at the lower end of the esophagus is marked by an abrujjt transition from a relati\ely smooth surface and stratified squamous epithelium to the folded and glandular mucosa of the stomach lined by simple columnar epithelium. Below the mucosa the transition is more gradual, the deep mucous glands of the esophagus often extending over into the stomach. Folds in the stomach wall involving submucosa and mucosa form ridges, or rugge, observed best in the empty stomach. 172 THE DIGESTIVE SYSTEM i>. (Fig. 109.) Small pits, or gastric crypts, are easily visible with slight magnification of the internal mucosal surface. The mucosa is quite thick, due to the presence of simple tubular gastric glands which are roughly divisible into three types: the cardiac, fundic, and pyloric glands. Cardiac glands, found where the esophagus and the stomach join, are relatively few in number. In these glands the cells are chiefly of the mucous type. Cardiac glands are small, simple branched tubular in form, with a short excretory duct lined by col- umnar cells. The twisted secretory tubules are formed from cuboidal or columnar cells. There is great variation in the number of cardiac glands, and in some cases they are absent. Fundic glands are most numerous and produce the essential elements of gastric juice, the cardiac and pyloric glands may function mainly as mucous glands. The fundic glands are branched tubular in form, with a relatively short excretory portion extending into the gastric pit, and glandular portions that are generally very much longer. (Fig. 110.) The cells of the gastric pit walls are columnar in type. A constricted portion of the gland near the gastric pit is called the neck, and from it each secretory tubule leads to a dilated end. Two types of cells, the chief or central cells and the parietal cells make up the secreting tubules. The chief cells are roughly pyramidal, their cytoplasm shows secretion grainiles in the end toward the lumen, and nuclei are located in the basal half. The secretory acti\ity of these cells is associated with the production of zymogen granules, which give rise to the pepsin of the gastric juice. Scattered along the secreting tubule, between the chief cells and the basement mem- brane, and more numerous toward the neck, are the parietal cells Fig. 110. — Diagram of tissues in the body or fundus of stomach of a mammal. A, mucosa with simple, branched tubular glands. The black dots represent parietal cells. Note mus- cularis mucosa at base. B, submucosa with artery and vein. C, muscularis. This being a longitudinal sec- tion, the inner circular mus- cle is cut across and the outer longitudinal muscle is cut lengthwise. D, adven- titia, which is a serosa with an external limiting mem- brane of mesothelium. THE ALIMENTARY CANAL 173 which are larger than the <'hicf cells. These cells are oval or polyji;onal, their finely irraniilar cytoplasm has an affinity for acid (lyes, and the large spherical nucleus is centrallx located. Parietal cells are associated with the production of the hydrochloric acid present in the gastric juice, and are often called oxyntic or acid cells for this reason. The pyloric glands intermingle with the fundic type in that portion of the stomach near the small intestine. The transition is gradual; parietal cells become less and less numerous and finally no longer appear in the more typical pyloric glands. The gastric pits become longer, and the secreting tubules become relatively shorter imtil they are about as long as the excretory portions but are more twisted than in the fundic region. The cells of the pyloric glands are distinctly mucous in appearance. The tunica ])r()i)ria of the stomach extends in between and around the secreting tubules of the gastric glands, and the muscularis mucosa lies just below the deepest ends of the secreting tubules. Scattered diffusely throughout the tunica proi)ria are lymphocytes, but in some regions solitary lymph nodules occur. The submucosa is typical, being composed of loose fibroelastic connective tissue whose longitudinal ridge-like extensions form rugte. The muscu- laris may have three layers in some regions, an inner oblique, a middle circular, and an outer longitudinal layer. In the j)yloric region the two inner layers are thickened to form a sphincter muscle. The adventitia is composed of a coat of loose fibroelastic connective tissue enclosed by a single layer of mesothelium. Although the stomach functions mainly as a temporary ])lace for storing food, some digestion occurs as a result of glandular action. Proteins may be converted into proteoses and peptones by the action of pepsin, while another enzyme, rennin, if present in the gastric juice, plays a role in the digestion of milk. The Small Intestine. — Continuing from the stomach, the digestive tract becomes a smaller tube whose internal surface is much increased b}' numerous folds. The muscularis coats, both the circular and longitudinal, tend to encircle the tract in a gentle spiral. In general, with the exception of the birds and mammals, the glands that are a prominent feature in the stomach mucosa are lacking in the intestine where the main function is absorption of the material digested by the secretions passing into it from the stomach and from the pan- creas and liver. The epithelium of the mucosa may contain numer- ous mucous secreting cells which cover the membrane with their lubricating secretion. 174 THE DIGESTIVE SYSTEM The Small Intestine of Fishes. — In the small intestine of the dog- fish (yonng Sqiialns acanthus) several submucosal folds extend into the lumen. (Fig. 111.) The mucosa is composed of columnar epithelial cells which rest upon a tunica propria which, due to the absence of a distinct muscularis mucosa, is continuous with the submucosa. Folds of the mucosa form tubular pockets at the base of the folds; these resemble simple tubular glands but their cells show no special secretory activity differentiating them from the other cells of the mucosa. The muscularis coat has a broad circular region but the longitudinal portion is poorly represented. The connective tissue of the adventitia is covered by cuboidal mesothe- lial cells. Fig. 111.— Diagram of a cross-section of the duodenum of a dogfish. The sub- mucosa has ruga-like extensions into the lumen. The mucosa consists of simple tubular glands lined with columnar epithelium and a thin muscularis mucosa. The submucosa is thickest in the region of the ruga-like extensions. The muscularis is represented by a thick circular coat and a thin longitudinal coat which is present in separate strands. The adventitia is a serosa covered with cuboidal epithelium. Among the teleosts the structural plan is similar to that in the dogfish })ut the mucosal folds a])i)ear much more elaborate and the epithelial membrane a])pears to be commonly composed of stratified or pseudostratified columnar cells. The SniaU fntestinc of Aiuphihia. — Tlwre are wide, longitudinal submucosal ridges in the intestine of urodeles. In Xecturus, the epithelium is simple columnar in type, with maii\- goblet cells present in it. There is diffuse lymphoid tissue in tiie subnuicosa. The circular muscle coat of the muscularis is thicker than the longitudinal coat. Superficially, mesotheliuni (•o\crs the ad- ventitia. In the frog (F'ig. 112), numerous small folds com- posed of mucosa and a core of connective tissue e.xteiul into the THE ALIMENTARY CANAL 175 lumen. Larj>:er folds extend out from the submucosa carrying along these smaller folds. The submucosa does not form the wide ruga- like extensions, as in the urodeles, l)ut it is present between the adjacent epithelial surfaces of the \'illus-like folds. The circular coat of the muscularis is not \'ery thick, and the longitudinal coat is still thinner. The Reptilian Small Intestine. — In reptiles there are numerous longitudinal folds of the mucosa and submucosa. The muscularis mucosa may be present as a scattered region of circular cells, or the submucosa and tunica ])ro])ria may form a single narrow connective- tissue region. The circular layer of the muscularis is the thickest Fig. 112. — Photograph of a cross-section of the small intestine of the frog, showing villus-like longitudinal folds. Bloodvessels are showni in the supporting mesentery. coat; the longitudinal coat is \'ery thin. Mesothelium covers the thin adventitia. In the lower vertel)rates the intestine shows little or no marked histological differentiation at the \arious levels, but in the anterior portion the submucosal folds are more numerous, the lumen smaller, and the wall pr()])orti()nally thicker. The Small Intestine of Mammals.— The same four coats noted in the stomach form the wall of the small intestine, but here the muscularis is quite regular, with an outer longitudinal and an inner circular layer. (Fig. 113.) The mucosa possesses characteristic features that deserve closer attention. The inner surface of the canal generally shows circular and oblique folds involving the 176 THE DIGESTIVE SYSTEM mucosa and part of the submiicosa. These folds, parallehng each other and extending part way around the knuen at irregular inter- vals, are called valvule conniventes, or plictv circulares. The mucosa is modified by extension into the lumen of i)apilla-like pro- jections which may be leaf-like, finger-like, or broadly club-shaped. These structures are known as villi and are diagnostic features of Fig. 113. — Diagram of tissues in the wall of the small intestine of a mammal. A, mucosa showing villi with the glands of Lieberki'ihn extending basally between them. At the base of these glands is the museularis muc-osa. B, submucosa with an artery and vein. (', muscularis coat with an inner circular and an external longitudinal muscle coat. D, adventitia with its external limiting membrane of mesothelium. the small intestine of mammals. They .serve to increase the surface and function in the ])rocess of absorption. The villi in a relaxt^d condition of the tract nearly fill the lumen. Each villus has a core of the loose fibroelastic tissue from the tunica propria and scattered smooth muscle cells from the muscularis mucosa. Simple cohunnar epithelium with basally located nuclei cover the villi. The cells have a striated cuticular border, the striated appearance being claimed by some observers to be due to minute canaliculi through which absorption of digested material is effected. (Fig. 114.) Scattered among the simi)le columnar cells THE ALIMENTARY CANAL 177 are goblet cells. These appeal- in \aryinu- miinhcrs and hccoiiic more markedly abundant in the ileum. Within the epitheHum of each villus there is loose connective tissue, a basketwork of blood capillaries, a nerve net, and soin(> ditt'use lym])lioi(l tissue. A dilated blind lym])h cai)illary, a lacteal, occu])ies the central portion. The villi vary in sha])e, size, and number in various mammals. From the bases of the villi extend simple tubular glands which formed as invaginations of the embryo gut epithelium. These are called Liel)erkiihn glands and extend down to the nuiscularis mucosa. The epithelium lining them is continuous with that covering the -^l — Epithelium Capillary network Connective tissue After II ^~ I ein -^Lymphatic Fig. 114. — Diagram of a villus. villus, but the cells are shorter and have no striated border. Near the base of these glands occur coarsely granular, basally striated cells, the cells of Paneth, which secrete a serous fluid containing an enzyme. These cells are widely- distributed throughout the epithe- lium of the digestive canal of higher vertebrates. Another type, which reduces silver, is called an argentaffin cell and has a finely granular basal portion of cytoplasm. It has been demonstrated scattered among the cells lining the crypts and rarely among those covering the villi, but as yet its nature is not full>- understood. The tunica propria, or stroma, as it max- be called, ])acks in between the Lieberkiihn glands, as well as forming the core of the villi. Its light, loose reticular network and denser fibroelastic tissue supports 12 178 THE DIGESTIVE SYSTEM a diffuse lymphoid tissue, and solitary lymph nodules are relatively common. In the ileum, near the jejunum or lower, the lymph nodules form groups, called Peyer's patches, which may extend into the submucosa. Xo villi co\er some nodules which project into the lumen. The muscularis mucosa may be of varying dis- tinctness with an outer longitudinal and inner circular layer of smooth muscle. The submucosa of loose fibroelastic tissue carries the larger blood and lymph vessels, Meissner's nerve plexus and, except in the region of the duodenum, has no glands. Branched tubular glands, Brunner's glands, appear in the submucosa of the upper region of the duodenum and often extend o\'er into the adjoining region of the pyloric stomach. The secretory portion of these glands is composed of pyramidal or columnar epithelium. The secretion contains a proteolytic enzyme similar to pepsin in its action. Ducts from these glands lead up through the mucosa and open either between the villi or into the crypts of Lieberkiihn glands. The cells lining the excretory ducts are similar to those of the epitheliiun lining the duodenum. Outside the submucosa is the muscularis coat consisting of an inner sheath of circularly disposed smooth muscle and an outer longitudinal coat. Connective tissue containing Auerbach's plexus lies between them. The adventitia is composed of a thin layer of fibroelastic connec- tive tissue covered with mesothelium. The Large Intestine.— The Large Intestine uf Elasmubranchs. ^The large intestine of Selachians consists of the so-called spiral valve, in which the inner surface of the tube is a spiral meml)ranous shelf formed by an extension of submucosa covered with a mucosa. An adventitia with superficial cuboidal epithelium forms the external coat. Internal to this are scattered strands of circularly arranged smooth muscle cells, forming a circular coat of a muscularis. The submucosa of fibroelastic connective tissue forms the core of the spiral valve. The mucosa has a mnnber of sim])le tu])ular iuAolu- tions resembling glands but they are lined with a stratified or pseudo- stratified columnar epithelium. The epithelium covering the mucosa not so invaginated is of the same type as that found between the folds. The folds help to increase the absorption of material passing over the valve but have no secretory activity. Lymphoid and myeloid tissue may be consjucnous in the connective tissue below the epithelium. THE ALIMENTARY CANAL 170 In part of the intestine below the spiral Aahe in the Selachians, and over the entire lower portion in those forms not having a spiral valve, the strnctnre resembles that of the small intestine. The mucosa becomes smoother due to loss of mucosal and subnuicosal folds. The rectal gland of tlie Selachian is a short cylindrical structure attaching to the colon just posterior to the end of the spiral valve. A study of sections at various levels shows that it is a com])ound tubular gland. The tubular secretory end-pieces are composed of cuboidal cells apparently serous in nature. The function of the gland is not definitely known. The Large Intestine of Amphibia.^ The nuicosa is thrown into se\eral longitudinal folds by the submucosa. The epithelial lining is stratified or pseudostratified columnar with numerous goblet cells. Small glands may extend a short distance into the tunica projjria. The nuiscularis has an inner circular coat and an outer longitudinal coat, but the adventitia is reduced to a very thin membrane of connective tissue covered with simple squamous epithelium. In the frog, seromucous glands occur in the tunica propria, which ma}' be separated from the submucosa by a thin circular muscularis mucosa. The Large Intestine of Reptiles.— ^The mucosa has simple tubular glands that extend to a muscularis mucosa. The glands are lined with simple columnar cells among which are goblet cells. Diffuse lymphoid tissue is cjuite abundant in the submucosal tissue. The muscularis has a definite inner circular coat and a thin outer longi- tudinal muscular sheath. Extending lengthwise are branching submucosal folds which appear like long villi when the intestine is studied in sections. The Mammalian Large I ntestine. ^The same four coats make up the wall here as observed in the case of the small intestine, but certain characteristic modifications of the mucosa distinguish it. (Fig. 115.) There are no villi and generally no plicse circulares present. The mucosa has many tubular glands extending to the muscularis mucosa. These glands are homologous with the glands of Lieberkiihn of the small intestine, but are longer. The cells lining the surface of the lumen are tall columnar and may ha\e a thin striated cuticular border. Passing down into the glands, the cells become shorter and goblet cells become more numerous. Goblet cells are most numerous in the mid-region of the glands. The basal cells are less differentiated and are supposed at 180 THE DIGESTIVE SYSTEM times to give rise to cells replacing worn-out goblet cells. The goblet cells of glands secrete mucus to lubricate the intestinal lumen surface, and thus aid in the passage of the fecal material Mucos Subniucosa Musculuris Adventiiia Fig. 115. — Diagram of large intestine of mammals. which becomes more solid, due to absorption of water through the intestinal wall. The wall of the cecum usually has lymph nodules in the sub- mucosa. In some forms, as in the rabbit, these nodules may encircle Fig. 110.— Photograph of the ileocecal junction of the cat, showing the valve and the appendix, 3; 1, ileum; 2, cecum. the lumen and forui the greater ])art of the wall with a reduction in amount of coimective tissue and mu.scularis. The vermiform appendix is a blind pouch extending from the end of the cecum at its junction with the small intestine. (Fig. IIG.) THE PANCREAS 181 The wall has a inucosa with Lieberkiihn glands. The eharaeteristic feature is the ahiuuiaiice of lyni])h()i(l tissue which is j)resent, not only as nodules in the nuicosa and submucosa, hut also as diffuse tissue throughout the tiuiica propria. The circular coat of the muscularis is well represented, hut the outer longitudinal coat is thin. In some cases the glands may lie obliterated by lymphoid tissue and the lumen filled with lymphocytes that penetrate through the nnicosa. The Rectum and Anus of Mammals. — The rectum has long and large tubular glands and solitary lymph nodules are common in its mucosa and sulnnucosa. Longitudinal folds of the mucosa appear in the lower part of the rectum, and with them occurs a change in the character of the mucosa. The columnar epithe- lium and simple tubular glands of the rectum change abruptly to stratified squamous epitheliiun of the anus, and this epithelium is continuous with the skin in much the same fashion as already noted in the case of the lip at the other end of the digestive tract. The muscularis of the rectum has two complete layers, and near the anus striated muscle rejilaces the smooth muscle. The Cloaca. — In vertel)rates below mammals the rectum and uro- genital ducts open into a common chamber, the cloaca. In addi- tion to being a mere receptacle it shows modifications for transmis- sion of genital products and there is considerable variation in its structure. In fishes it is lined with stratified squamous ejiithelium supported by dense fibroelastic connective tissue and some muscle. In Amphibia it may be a sac lined with columnar cells, among which are munerous goblet cells, or it may have glandular linings associated with reproductive activities. Among the reptiles and birds, the \'entral wall of the cloaca may be developed into organs for copula- tion and portions of the wall may be glandular. The anus or cloacal opening is lined with stratified epithelium similar to that of the skin and is supported by loose fibroelastic connecti\'e tissue joining with the muscles underlying the skin of this region. THE PANCREAS. The pancreas has been called the salivary gland of the abdomen, and at first glance resembles a parotid (see Parotid) in the serous appearance of its secretory cells. It is a compound tubulo-alveolar gland lying behind the stomach and attaching to the duodenal wall. No distinct capsule covers it, and its lobules are separated by a 182 THE DIGESTIVE SYSTEM loose fibroelastic tissue. The secreting end-pieces, which may be tubular or short and roughly spherical, are formed of pyramidal cells. The cytoplasm of these cells is lighter staining toward the lumen and more deeply staining with striated appearance in the basal Fig. 117. — Diagrams showing structure of pancreas. ^4, a small excretory duct with its branches terminating in secretory end pieces. The intercalated ducts extend part way into the end-pieces. B, longitudinal section of an end-piece, showing centro-acinar cells (involuted intercalated duct) and the secretory cells which have basal striations and secretory granules toward the lumen. The secretory cells are serous in type. (', cross-section of a secretory end-piece distal to centro-acinar cells. region. (Fig. 117.) The lighter portion contains coarse zymogen granules, which are ])r('sumably forerunners of the enzymatic secre- tions. In the basal half of the cell there is a nucleus with a chro- matin network and one or more nucleoli. In sections, centro- acinar cells may occupy the lumen of many acini along the inner ends THE PANCREAS 183 ends of the secreting cells. I'hese cells represent a contiiuiation of the intercalated dncts which project a short way into the acinar lumen. Such cells have no secretory granules and possess a small deeply staining nucleus. Intercalated ducts hned with first cuhoidal then low cohnnnar pass to small excretory ducts lined with a tall columnar epithelium. The large excretory ducts are lined with a stratified columnar epithelium and pass into the main duct (duct of Wirsung), which opens into the intestinal lumen. Enzymes (trypsin, amylase, and lipase) present in the pancreatic secretion break down the proteins, starches, and fats into simpler compounds. ^^^^^r^ks^^^^^^' 1^ i'^Vj lESiL '^^B ^^ ^1 fci.,-1 ^:-rf^v^-^.:; 'V ' ^r-^^^asm ^m&k Fig. 11!S. — Section of pancreas of the water snake (Natrix>. It is similar to the pancreas of a mammal but relatively less connective tissue makes it more compactly glandular. The light gray areas are sections of islands of Langerhans. Though at first glance it resembles the parotid of manunals, the pancreas difl'ers in the nature of its secretory cells, in the looser arrangement of the interlobular connective tissue, in the absence of secretory ducts, and finally in the i)resence of certain groups of very distinct lightly staining cells forming the islands of Langerhans. The islands of Langerhans are composed of s])herical groups of from few to hundreds of lightly staining cells irregularly scattered among the acini and along the ducts. (Fig. US.) These cells are arranged in cords forming a network with munerous wide capillaries passing between the cords. Two types of cells are demonstrated on the basis of secretory granules which are not in either case like those of the acinar cells of the pancreas. One, the A cell, with fine 184 THE DIGESTIVE SYSTEM cytoplasmic i]:;raniiles and a large oval nucleus with little chromatin apparent is found most frequently in the center of the island. The second type, the B cell, is smaller and more numerous, with a smaller nucleus containing large chromatin granules. The secre- tion of these island cells, insulin, passes into the blood and governs the metabolism of carbohydrates. The appearance of these islands may \'ary considerably, even occurring as a separate gland outside the pancreas in some fishes. In lower vertebrates the pancreas is usually a more compact gland, but in general closely resembles the mammalian structure just described. Intercalated ducts are usually not present and centro-acinar cells are absent, THE LIVER. The liver arises as a diverticulum of the mid-gut and de\'elops into a compound tubular gland. Among fishes it may retain this simple glandular condition with blindly ending secretory tulniles. ^Yith the amphibia and reptiles the tul)ules fuse to form a network and the li\'er cells surround a central lumen into which secre- tions are ])oure(l from bile capillaries wdiich form as a network of grooves between the adjacent faces of the cells. The connective tissue is not abundant, and no lobulation occurs. In mammals there is an increase in amount of connective tissue and lobulations are indicated where connective tissue accompanies the larger })lood- vessels. Among the mammals the lumens of the embryonic end- pieces of the gland are lost, so that only cords of cells are apparent and an increasing amount of connective tissue results in a di\ision of the gland into lobules, and groups of these form lobes. Lower Vertebrates.— The liver of fishes is a typical com])ound tubular gland with the clear cells of the secreting end-pieces arranged about the lumen into which the secretion collects. Small bile ducts are formed of cuboidal cells. Between adjacent end-pieces and ducts and lobular masses is a small amount of connective tissue which supports capillaries and small vessels. Another type of cell with granular pigmented cytoplasm often occurs around the larger excretory ducts and l)lo()dvessels. The liver of amphibians is a modified compound tubular gland. (Fig. 119.) The peripheral end-pieces composed of the secreting cells form a series of differently directed short tubules that connect and form a network. The cells are large and pyramidal shaped. THE LIVER 185 with clear or vacuolated cyt()])lasin. The smaller hile ducts are of low cuboidal cells that have the same embryonic orifjin as the secretintj; cells. Between adjacent end-pieces is a small amount of connective tissue supportinj^; sinusoids similar to those of the mam- malian liver. Amonj? the secretinoj cells are o;rou])s of pij,nnent cells. The l)ile ducts are at first c()mi)osed of cuboidal cells, the larger branches are formed of cohnnnar cells, and the still larger branches have stratified columnar epithelium. In the Urodeles, the tissue of the inner zone of the capsule contains hemocytoblasts and neutro])hil and eosinophil myelocytes, and acts as a hemapoietic Fig. 119.— Section of liver of Amblystoma, showing rows of lange polyhedral hepatic cells separated by sinusoids. Cells filled with pigment are also shown. center. The mammalian li^•er has this property only during early embryonic life. The reptilian liver has the appearance of a compound tubular gland also. The secreting end-pieces are comi)osed of large ]x)ly- hedral cells. The bile ducts resemble in general those of a mam- malian salivary gland. Between adjacent tulniles is a prominent capillary network. In the connective tissue between lobules can be found the so-called portal canals, i. e., an artery, a Aein, and a bile duct. The Mammalian Liver.— This is the largest gland in the body and lies below the diaphragm in the upper region of the abdominal ca\'ity. The fibroelastic connective sheath, or capsule of Glisson, completely surrounds the liver. At the hilum, where the blood vessels enter 180 THE DIGESTIVE SYSTEM the connective tissue of the capsule, tissue of the same sort as the latter continues inward, accompanying the vessels, and di\ides the li\'er into se^'eral lobes, which in turn are subdivided into numer- ous lobules. (Fig. 120.) The liver lobule is the unit of hepatic structure in mammals, each lobule being roughly a five- or six-sided polyhedral prism in form. Each lobule is composed of anastomosing cords of liver cells that radiate outward from the center which is occupied by a central vein. The indi\'idual cords have two rows of cells whose boundaries are normally clearly defined. The indi- vidual cells are polyhedral in form, with a central nucleus, or possibly two or more nuclei, each wdth one or more nucleoli. Gran- ules of glycogen (animal starch) may be demonstrated in the Fig. 120. — Photograph of several lobules in the liver of the pig. cytoplasm. Fat droj^lets appear to be normally present and ^•ary inversely in amount with the glycogen. Pigment granules may also be present. Considering the variety of functions in which the hepatic cells participate, it is surprising that there is only one type of cell. Instead of the cells pouring their secretions into a lumen which the free ends of the cells adjoin, as is the case in other exo- crine glands, the hepatic cords have a network of small canaliculi, the bile capillaries, ruimiug between adjacent faces of cells forming the cords. To understand the C()mi)()sition of the liver it is imi)ortant to understand the distribution of the blood vessels to a lobule. Blood enters the liver from two sources, the hepatic artery and the i)ortal vein, and leaves through the hepatic veins which emi)ty into the vena cava. The ail'erent vessels, the he])atic artery and i)()rtal THE LIVER ISI vein, enter at the liiluni and divide into larji;e interl()l)ar branches following the septa separating the lobes. The interlobar vessels give off the interlobular branches which follow the septa between the lobules. The interlobular branches of the portal veins give oft' short branches ])assing to the surface of the lobules where they break up into an intralobular capillary network. (Fig. 121.) The hepatic artery follows the portal vein in its branching to the inter- lol)ular se])ta, where its finer branches break u]) into capillary net- FiG. 121. — Diagram of a cross-section of a lobule of the liver of a mammal with the hepatic cells radiating toward a common center. P, section of a branch of the portal vein in the interlobular connective tissue; H, branches of the hepatic artery near the branch of the portal vein; branches of this portal comp'onent extend to form the capillary system of the lobule. Some twigs of the hepatic artery components also join this capillary system. The diagram shows the capillaries forming a mesh- work among the cords of liver cells. The capillaries converge toward the intralobular vein (/L) in the center of the lobule. works, some of which supply the septa and then join the finer branches of the hepatic vein, while others enter the lobules to join with the intralobular capillary system. The intralobular capillary network is composed of wide lumened, hepatic sinusoids. These anastomose irregularly along the free surfaces of the cords of hepatic cells and everywhere separate the cords from each other, so that each liver cell has several of these sinusoids in contact with it, a disposition not found in the case of other gland cells. The sinusoids carry the blood toward the center of each lobule, where they empty it into the central vein which emerges at the base of the lobule. Each central vein joins a sub- 188 THE DIGESTIVE SYSTEM lobular ^'ein which e\'entiially unites with the hepatic ^•eins, the efferent blood vessels of the liver. In addition to the endothelial cell proper, which is small and flat with a darkly staining nucleus, there are present along the walls of the sinusoids other larger cells, the Kupffer cells. Kupffer cells have a number of cytoplasmic processes and a larger vesicular nucleus. By intravenous injection the Kupfl'er cells can be shown to be active in ingesting foreign particles from the blood passing through the sinusoids, i. e., they become macrophages. They may be considered as homologous with histiocyte cells and are part of the reticulo-endothelial system. Some believe thev are derived from the cells of the delicate reticular Fig. 122.— Photograph of injected bile capillaries in the liver of a rabbit. These are indicated by the network in black. Nuclei of liver cells are faintly stained and the cord-like arrangement of liver cells is evident. tissue enveloi)ing the sinusoids; others consider them derived from endothelial cells. Possibly both sources are correct. Histiocytes or reticulocytes and endothelial cells, forming the sin- usoids in the spleen and marrow, and Kupffer cells are considered homologous and constitute the reticulo-endothelial system, im- portant physiologically because of its phagocytic potentialities. Between the adjacent faces of the hei)atic cells the l)ile cai)illaries appear as grooves between the adjoining cell walls. By silver impregnation and injection the bile capillaries (Fig. 122) appear to be bounded by a delicate non-cellular membrane formed by the adjoining hepatic cell walls. These ca])illaries are functionally comparable to the lumens of other glands, and into them is collected THE LIVER 189 the bile secreted by tlie hepatic cells. Each hepatic cell uia\' have more than one bile capillary into which it cHscharjijes bile, but they occur only between adjoininjj surfaces of cells and not alonjj the free surfaces which adjoin the blood sinusoids. These capillaries form a branching network and join those of adjoining hepatic cords to carry the bile outward toward the periphery of the l()l)ule. At the periphery of the lobule they collect into small bile ducts in the interlobular septa. These interlobular bile ducts have walls formed of flat or cuboidal e])ithelium and coalesce to form larger ducts following the course of the portal vein and hepatic artery through the septa. With increasing size of the bile vessels, the epithelium of their wall changes to tall columnar which is surrounded by a coat of connective tissue and scattered longitudinal smooth muscle cells. The larger bile ducts from each lobe empty into a larger hepatic duct that in turn joins the common bile duct leading into the duodenum. The three vessels coursing close together through the connective- tissue septa of the liver, i. e., a branch of the he])atic artery, a branch of the portal vein, and a bile duct, form the so-called portal canal peculiar to the liver. The vein is the largest, the bile duct is second in size, and the artery is the smallest vessel of the three. As a gland the liver is peculiar in passing its secretions peripher- alh' to the collecting ducts and, although the hepatic cells are only of one type, they are presuma})ly equally ca])able of many different functions. Moreover, the blood com-ses not outward from a center, but in toward the center of each lobule where it is collected into efferent veins that follow at first a course different from the small afferent vessels and ducts of the gland. The amount of connective tissue varies in different animals, being particularly well represented in the pig liver, where the interlobular connecti\'e tissue clearly marks the boundaries of the lobules. The arrangement of the hepatic cells is unique as regards their relation to sinusoids, and the bile capillaries are mere troughs between the adjacent faces of two cells. The liver is associated with the production of l)ile which is carried to the duodenum, where it is mixed with food and ])ancreatic en- zymes. Part of the bile consists in secretion products from the liver cells, and in part it consists of excretions which the li\er cells have removed from the blood. Apparently liAcr cells are able to excrete foreign substances that have entered the blood. I3ile acids produced by the liver cells take j^art in the absorption of fats from 190 THE DIGESTIVE SYSTEM the intestine. The Hver also has important endocrine functions. It forms urea from ammonium carbonate and later on the urea is removed from the l)lood by the kidney and excreted. The most important endocrine function of liver cells invohes taking such a simple carbohydrate compound as orlucose from the blood and changing it to temporarily insoluble glycogen. Then as tissues, especially the muscles, need this fuel the li^'er recon\'erts glycogen back into simple sugar (glucose) in which form it is distributed by the blood-vascular system. The presence of insulin derived from islands of Langerhans in the pancreas is essential to the carrying out of this function. Tests show that li\'er cells, depending on the diet, in some cases show a preponderance of protein products, at other times of glycogen, and at still other times of fats. An extract, called heparin, obtained from liver tissue pre\ents the clotting of blood. Another substance obtained from liver tissue stimulates the production of erythrocytes. Li\'er tissue possesses great power of regeneration, but the remo\'al of the entire organ results in the death of the organism in a short time. THE GALL-BLADDER. The largest bile ducts from the lobes of the mammalian liver usually connect with a pouch, the gall-bladder, which lies along the posterior under surface of the liver. The cystic duct continues outward to join the common bile duct. The wall of the gall- bladder consists of three coats, a mucosa, muscularis, and an adventitia. The mucosa is much folded and has a co^'ering layer of tall columnar epithelial cells with basal nuclei. The tunica propria carries blood vessels, and toward the neck region of the pouch there occur small tubulo-alveolar glands. The muscidaris possesses interwoven bimdles of smooth muscles arranged as an inner longitudinal layer and an outer thicker circular coat. The outer part of the adventitia is not so dense as the inner and sup- ports blood and lymj^h vessels and nerves. In the fish, if present, it has a lining of simple or stratified columnar epithelium surrounded by fibrous connective tissue. In the frog, it is lined with columnar ei)ithelium surrounded by connective tissue which supports blood vessels. In the water snake (Natrix), it is lined with stratified columnar epithelium, and outside this is con- nective tissue with smooth muscle cells in it (lis})ose(l in strands around the wall. REFERENCES 191 REFERENCES. Arey, L. B. 1932. On the presence of so-called portal lobules in the seal's liver, Anat. Rec, 51, 315. Beams, H. W. 1930. Studies on the vacuome and the Golgi apparatus in the acinar cells of the pancreas of the rat, Anat. Rec, 45, 137. Beams, H. W., and King, R. L. 1932. Notes on the cytology of the parietal cells of the stomach of the rat, Anat. Rec, 53, 31. Berger, E. H. 1932. The distribution of parietal cells in the stomach, Am. Jour. Anat., 54, 87. Beust, T. B. 1934. Dental Histology, Philadelphia, W. B. Saunders Com- pany. Blake, J. H. 1930. Studies on the comparative histology of the digestive tube of certain Teleost fishes: I. A predaceous fish, the sea bass (Centro- pristes striatus). Jour. Morph., 50, 39. Jordan, H. E. 1931. The pigment content of the liver cells of inodeles, Anat. Rec, 48, 351. Kater, J. McA., AND Smith, D. M. 1932. The foimation of fat in the hepatic cells, Anat. Rec, 52, 55. O'Leary, J. S. 1930. An experimental study on the islet cells of the pancreas in vivo, Anat. Rec, 45, 27. RoGiCK, M.D. 1931. Studies on the comparative histology of the digestive tubes of certain Teleost fishes; II. A minnow (Campostoma anomalum), Jour. Morph., 52, 1. SiMARD, L. C, AND Van Campenhout, E. 1932. The embryonic develop- ment of argentafl!in cells in the chick intestine, Anat. Rec, 53, 141. SuRRARRER, T. 1931. Histology of the intestinal tract of two minnows, Notemigonus chrysoleuca (Mitchillj and Motropis atherinoides (Rafinseque, Ohio Jour. Sci., 31, 268. ToRREY, T. W. 1931. The relation of taste-buds to their nerve fibers, Proc Nat. Acad. Sci., 17, 591. Wharton, G. K. 1932. The blood supply of the pancreas, with special refer- ence to the islands of Langerhans, Anat. Rec, 53, 81. See Appendix for general text references^. CHAPTER XII. THE EXCKETORY SYSTE.M. The oxidation phenomena gronped under metabolism, and involving both synthesis and destruction of cell materials, are accompanied by the production of waste products. Such waste products include carbon dioxide, mineral salts, water, and numerous simple nitrogenous compounds. These are mainly remo\ed from the organism in aqueous solutions. We have already observed that in vertebrates carbon dioxide and some water are removed from the blood through the respiratory system, and that the integu- ment plays a part in the elimination of small amounts of waste. However, among most animals a distinct excretory system has been evolved to deal with nitrogenous wastes. In the chordates we find the presence of a segmental condition in the excretory system. In the primitive forms there is a system of segmental nephridial tubules with ciliated openings (nephro- stomes) into the coelom and outlets direct to the exterior. With the cephalochords such tubules have nephrostomes in the coelomic cavity and are also associated with coils of blood vessels, so that wastes are removed from both the coelomic fluid and the blood. With the vertebrates there is an increasing efficiency in the develop- ment of nephridial tubules for the remo^'al of wastes from the blood, and they become organized into one of three types of excretory organs called kidneys. These form in the embryo along the mid- dorsal region, one on each side, at different levels. The three types have a common source of origin in the mesoderm of the intermediate cell mass, the nephrotome, which lies lateral to the mesodermal segments and connects them with the somatic and splanchnic layers of mesoderm enclosing the c(elom. Tubules formed from this mesodermal region become grouped along the mid-dorsal region to form each type of kidney. In the cyclostomes, young fish, and young amphibians, the pronephros-, or head kidney, is functional; in adult fishes and amphib- ians, the mesonepliro.s, or middle kidney, is functional; in rei)tiles, birds, and nuinunals, the nictancpliro.s-, or ])()steri()r kidney, becomes THE MESOXEI'/Ilx'OS WV.y the functional organ. I )uring the enil)ryoloo;y of those forms having the metane])hros, the other two types are formed successively and may function tem])()rarily until the metane])hros takes ()V(>r the excretory function. THE PRONEPHROS. The pronephros consists of short, paired segmental tubules located well toward the head region, the tubules of each side connecting with a longitudinal duct on that side, and this opens posteriorly into the cloaca. Each pronephric tubule has a proximal, ciliated, funnel- like end, the nephrostome, opening into the ctelom. As they develop, the distal ])<)rtion of each tubule bends backward and unites with the distal end of the next tubule to form the common longi- tudinal collecting duct, the pronephric duct, for each sifle. As a result of caudad growth this pair of excretory ducts extend pos- teriorly until they open into the cloaca on its lateral wall. Near the nephrostome, but entirely separated horn the prone])hric tubule, a coiled portion of a small artery covered l)y peritoneum ])rojects into the cielom from the body wall. These simi)le vascular net- works, or glomeruli, filter wastes from the blood into the crelomic fluid. Such wastes, together with cnelomic fluid, are drawn into the pronephric tubules by the ciliary action of the nephrostomes and are then carried down the collecting tubule to the cloaca for expulsion. A urinary system of this type is the fimctional kidney of Amphi- oxus and myxinoid cyclostomes. In other cyclostomes it persists, but yields its i)lace as the functional kidney to the newly formed mesonephros a])pearing caudad to it. In the embryological de^'el- opment of other higher forms, the pronephric kidney is formed but degenerates, and its functions are taken over tem])orarily, or per- manently, by the mesonephros. The pronephric ducts, howe\'er, become the main urinary ducts of the mesonephros. THE MESONEPHROS. The mesonephros, or Wolffian l)ody, forms caudad to the site of the pronephros and is composed of more numerous tubules develop- ing metamerically in pairs. These tubules fuse mesially with the pronephric duct, which is now called the mesonephric or Wolffian duct. With development, the mesonephric tubules become gath- ered into a more or less compact mass, the kidney. In fishes, each 13 194 THE EXCRETORY SYSTEM kidney is usually an elongated body up uniler the peritoneum, close to the backbone. In the urodeles the kidneys become somewhat elongated, but in frogs and toads they are shorter and l^roader. In these latter forms, the kidney becomes free in the body above the viscera but is connected with the body wall by peritoneal sheets of tissue. The mesonephric tubule is more com])lex than the preceding pronephric tubule. In the embryonic tubules the proximal end has a distinct nephrostome, as in the case of the pronei)hric tu})ule, but difi'ers in that a glomerulus projects into each ne])hridial tubule in the region near the nephrostome. The arterial tuft is thus sur- rounded by a double capsule of reflected tu])ular tissue. Bowman's capsule, and the whole is called a renal corjiuscle. In such an arrangement the fluid from the blood is filtered through the inner capsule and ])asses directly into the lumen of the mesonephric tubule without first passing into the coelomic fluid, as in the case of the pronephros. With development the nephrostomes may close; in urodeles they persist through life but many of them degenerate in anuran kidneys. Continuing from the glomerulus there is a proximal secreting ])ortion of tul)ule that in turn connects with the mesonephric duct. The mesonephros is the functional kidney of most adult cyclos- tomes, elasmobranchs, teleosts, and Amphibia. It is functional for a short time in certain young lizards and for a short time after birth in the monotreme, Echidna, and in the marsupial, ])idel])hys. The kidney of the frog may be taken as an exam])le of the functional mesone])hros. Kidney of the Frog.^The kidney of the frog is a flattened, rela- tively broad, elongated body lying within the body ca\'ity dorsal to the viscera. It is composed of an aggregate of mesone])hric tubules whose distal ends unite with the main collecting duct, the mesonephric duct, which runs along the outer lateral edge and continues ])osteriorly to join the cloaca. Each tubule has several different regions. At the proximal end of each is the renal corpuscle (Eig. 123), formed by a glomerulus of arterial capillaries encapsulated by the double layer of thin, flat, epithelial cells forming Bowman's ca])sule. As the tubule contimies distally the cells liecome cuboidal, or low cohunnar, and are more distinctly outlined. The region of the tubule just distal to the capsule is formed by ciliated cuboidal cells and may receive the short l)ran(Ii composed of ciliated cuboidal cells that oi)ens to THE MESONEPHROS 195 the uephrostoiiie. After leaving the region of the renal eorpnsele, the tubule continues as a larger proximal convoluted portion. This convoluted ])ortion formed by low eohnnnar cells with a brush l)()r- der then joins a short constricted region of the tubule com]K)sed of ciliated cuboidai cells. Passing from the constricted portion, the tubule again becomes wider and convoluted, forming the distal convoluted ])ortion which is comi)osed of cuboidai cells often show- ing cytoplasmic striations, and fuses with a short, straight, con- necting ])iece that em])ties with others of the same kind into a larger collecting duct. A number of these collecting ducts join the meso- nephric duct which rims along the outer edge of the kidney. Fig. 123. — Photograph of a section of the bullfrog's kidney (Mesonephros), showing a glomerulus surrounded by Bowman's capsule which continues into the neck of the uriniferous tubule. Sections of nephridial tubules and adjoining capillaries surround the renal corpuscle. These dift'erent portions of the tul)ules are distributed in a more or less definite manner in the kidney. In the ventral region are located the distal convoluted portions; in the mid-region the renal corpuscles and constricted ])ortions; and in the dorsal region the proximal convoluted portions and the collecting branches. The adrenal gland appears as a wavy golden line located along the mid- ventral siu-face. In the male, the vasa efl'erentia from the testis unite with the main collecting duct along the lateral edge of the kidney. The Wolffian duct, therefore, ser\-es as a sperm duct during the breeding season. On the ventral surface adjacent to the renal corpuscles the nephrostomes are to be found when present. 196 THE EXCRETORY SYSTEM Amphibian kidneys have a pecuhar blood supply. Branches of the dorsal artery, called renal arteries, carry arterial blood to them, and venous branches from the kidneys unite to form the postcaval veins which return blood to the heart. In addition to these, renal- portal veins arising from veins in the legs also pass to the kidneys. The renal arteries are connected with the glomeruli of the renal corpuscles. The capillary network around the tubules is derived from the renal-portal veins. All the blood returns to the heart via the postcava. Despite many ph}-siological experiments with the frog's kidney, the manner of its functioning is not thoroughly understood. Obser- vations of the li^'ing kidney have shown an intermittent flow of blood through the glomeruli, with some glomeruli thus being retired from or recalled to active functioning as conditions demand. The neck region and the narrow ciliated portion at the lower end of the proximal convoluted tubvile have been observed to contract, and presumably regulate urinary flow. The flow of urine from the distal convoluted tubule to the collecting duct is rapid. Experiments indicate that the glomerulus filters urine from the blood, that the proximal convoluted tubules concentrate this filtrate by resorbing water to a greater extent than other substances, and that the ciliated portions before and behind this region act as valves and, together with the distal convoluted tubules, constitute the pressure regulating portion of the kidney. THE METANEPHROS. The metanephros develops from the caudad nephrogenic tissue near the cloaca at the base of the mesonephric duct, and is composed of a compact mass of tubules. Like the mesone])hros, which pre- cedes it in time and location, this kidney has a double origin. The Bowman's capsule and secreting portions of the tubules are formed from the caudad nephrogenic tissue, but the collecting ])ortions arise as outgrowths of the mesonephric duct, which was formed at the time of the pronephros appearance and retained after the degeneration of the tubules of that kidney. The glandular portion of the entire tubule, i. e., that part developing from nephrogenic tissue, is called a nephron. At about the time when nephrogenic tissue begins developing metanc])hric tubules, a bud evaginates from the bend of the mesonephric duct where it joins the cloaca. This bud pushes forward to the anterior lumbar region of the body cavity. THE METANEPHROS 197 where it expands and becomes associated with the nephrogenic tissue. The (hict ])ortion of this prolonged evagination becomes the lU'eter, or main excretory (hict of tlie metanephros. The ex- panded anterior portion is the primiti\'e pehis and gi\'es off anterior buds that form the calyces from which branching evaginations give rise to systems of branched collecting tubules. The latter fuse with the nephridial tubules that ha\e been developing during this same period. This collecting system forms a conical mass of tree-like branched collecting tubules that constitute the major portion of the medulla of the kidney and extend radially into the cortical portion occupied mainly by the secretory tubules with which they fuse. The nephridial tubules of the metanephros are more complex and more numerous than those of the mesonephros and do not at any time have a nephrostome associated with them. Bowman's capsule closely surrounds the glomerulus and continues distally to the neck of the tubule. Continuing from the neck, the tubide becomes con- voluted for some distance, then constricts and straightens to descend and loop liack to form a wider distal convoluted portion. The distal convoluted portion finally joins with a collecting tubule by a junctional tubide or in some cases by a short arched portion. This type is the permanent kidney of reptiles, birds, aufl mammals, but before its appearance the other two types successively' precede it in embryological de^'elopment. The caudad portion of the pro- nephric duct remains as the mesonephric duct, and this in turn by evagination from its caudad portion gives rise to the ureter of the metanephros, and the original main portion of it becomes converted into the male genital duct, the ^'as deferens, for these forms. As an example of this type the mammalian kidney may be used, since details of its structure are well known and give all the struc- tural characters. Mammahan Kidney.— Mammalian kidneys are bean-shaped organs located in the lumbar region against the dorsal body wall. (Fig. 124.) They appear to l)e free in the body cavity, but are held in place by the sturdy trunks of the renal arteries, renal veins, and by the ureters. The peritoneiun, which splits into connecti\'e-tissue sheets or fascia at the outside border of each kidne\', also aids in support. The posterior fascia unites with the front of the spinal column, and the anterior fascia covers the front of the kidney and its vessels, passing over the aorta to meet the corresponding fascia from the other kidney. There is usually considerable fatty tissue 198 THE EXCRETORY SYSTEM between the kidney and its fascia. The kirhiey of the cat is about 3 cm. long by 2.5 cm. wide and 2 cm. thick; the human kidney is about 11 cm. long, 6 cm. wide, and 2.5 cm. thick, with the long axis parallel to the backbone. The outer margin of each kidney is convex, but the mesial border presents an indentation, called the hilus or hilum. Here the renal artery, renal vein, and ureter con- nect with the kidney. The entire kidney is covered with a loose capsule of connective tissue which is similar to the peritoneum in composition. There is little connective tissue within the substance of the kidneys, although the capillaries are accompanied by a rich reticular network. Fig. 124. — Photograph of a longitudinal section of a rat's kidney, showing a single papilla extending into the calyx. The darker striated region surrounding the papilla (medulla) is the cortex. The adrenal is shown closely attached to the kidney on the left. If the entire kidney is divided lengthwise into two hahes by a plane parallel with the adjacent body wall, additional features can be seen macroscopically. i^t the hilus can be seen the expanded funnel-like continuation of the ureter, called the pehis, the internal end of which is further subdivided into small funnel-like divisions, called the major calyces, each of which divides into minor calyces. The main collecting tubules open into the minor calyces. (Fig. 125.) The body of the gland is divided into two zones, an external zone just within the cai)sule, called the cortex, and an inner zone, the medulla, toward the pelvis. Toward the hilus of the human kidney the medulla is divided into a number of triangular masses, with the base of each directed toward the cortex and the apex of each fitting into a minor calyx of the pelvis. These triangular appearing masses are known as renal pyramids and are in reality conical masses of THE METANEPHROS 199 tubules. The human kidney may have twenty renal pyramids, but usually they are not so numerous. The kidneys of marsupials, insectivoi's, rodents, and earnivors have usually only a sino;le renal pyramid. In the medullary zone between the renal pyramids are branehes of the renal artery and vein embedded in connective tissue; these interi)yramidal regions are known as the columns of JJertini. With the aid of a hand lens or low-power objective, an examination of the cortex reveals alternating light and dark radial striations. The lighter striations are called pars radiata. The darker striations are called i)ars convoluta and show a series of small dot-like struc- ^fSr ',» n> ',< V '" Pars convoluta Pars radiata ~-r~\ J'- \ \-- , , AN /^7 Papilla /'*'■'■ • — -^.-^v \ a / Pelvis Renal pyramid Interlobar artery Interlobar vein Primary calyx Secondary calyx Renal artery ^~-\C Renal vein- Fig. 125. — Diagram of a longitudinal section of a human kidney. tures, renal corpuscles, arranged along an imaginary radial axis. Before progressing further it is essential to understand in detail the structure and distribution of a nephridial tubule which is the microanatomical structural unit of the kidney. Each kidney possesses thousands of these nephridial tubules. The Nephridial Tubule.— Attention has already been called to the dot-like structures, the renal or Malpighian corpuscles, visible in the pars convoluta striations. Each nephridial tubule begins with one of these structures, as in the case of the mesone])hric tubule. Each renal corpuscle consists of a capillary complex, known as a glomerulus, which is surrounded by Bowman's capsule, a double- walled structure of squamous epithelium. An afferent arteriole carries blood into the glomerulus and an efferent arteriole carries blood away, the two arterioles being adjacent where they continue 200 THE EXCRETORY SYSTEM into the capillary tuft. The capillary tuft is itself closely invested with the visceral wall of Bowman's capsule, which fits in close around the arterioles as they join the capillaries of the glomerulus. This same inner, or visceral wall, of Bowman's capsule is reflected back as the outer, or parietal, wall. The ])arietal wall continues down around the inner wall and forms a narrow neck at the end of the renal corpuscle opposite the entrance of the arterioles. The neck continues into a portion of the nephridial tubule known as the proximal convoluted tubule. The wall of the neck portion consists first of low cuboidal and further on of cuboidal and columnar epithelial cells. The proximal con\oluted portion is very much contorted, extending somewhat toward the surface at first, then turning toward the medulla and finally continuing into the medulla as a very much narrower portion, known as the descending limb. This portion extends radially in the medulla for some distance, then forms a loop, known as Henle's loop, and turns back as the ascending limb running alongside the descending limb. The ascending limb is somewhat thicker than the descending limb. As it passes into the cortex, the tubule l^ecomes the much thicker and contorted distal convoluted tubule situated near the proximal convoluted portion. Toward the cortical surface it joins a collecting tubule by a short junctional tubule. These collecting tubules join the t)thers which in turn join larger tubules, until the largest collecting ducts (papillary ducts) are found in the papillse of the renal pyramids. These I)apillary ducts then join the calyces. (Fig. 126.) Let us return for a moment to the cortex. A pars convoluta con- sists chiefl>' of renal corpuscles, together with proximal and distal convoluted portions of the nephridial tu})ules. A pars radiata is formed mainly by collecting tubules. The medulla consists chiefly of collecting tubules, together with the descending and ascending limbs, and Henle's loop. Little has been said about the blood- vessels involved in all these regions, for the \ascular supply will be considered separately later. As indicated, a number of branchings, about seven, occur between a given papillary duct and the smallest collecting tubules draining into it. Each ])apillary duct is like a tree with many branches, the collecting tubules of different grades originating from the single trunk. All of these branches constitute parts of the collecting sys- tem and ha\'e the same origin as the calyces, pelvis, and ureter in the evagination of the mesone])hric duct. The ne])hridial tubule pr()i)er, which develoj^s from ne])hrogenic tissue and takes ])art in THE METANEPIIROS 201 the elaboration of urine, consists of those ])arts from Bowman's capsule through the distal convoluted portion and may he called the ne])hron. The main function of the excretory tubule is to conduct urine from the secretory tubule, or nephron, to the pelvis. (Fig. 12(i.) Connecting tubiiU Distal convoluted tubule Renal corpuscle Proximal convoluted tubule ijapsule Afferent arteriole Efferent arteriole Renal corpu,icle Interlobular vein Interlobular artery Arched arteri/ Arched vein ^^^_^ „ ,— , , . ^ Interlobar vein Descending limb II I \ 11 "AV)M\\\ Ai^r-;vlr I'/fi^ ^_ ^ ^^ Interlobar artery Ascending limb " ' " ' " ^^ > -> " n -^ , h , Collecting duct Loop Papilla Fig. 126. — Diagram of the iirinifproiis anfl vasfular system of a manmialian kidney. Blood Supply of the Kidney. — The renal artery and renal vein divide into trunks, of which the greater number pass over the front of the pelvis in man but ventrally in quadruped mammals. Branches of these, known as interlobar arteries and veins (cohnnns of Bertini) extend between adjacent pyramids to the boiuidary between cortex and medulla. There, as arcuate arteries and veins, they curve at right angles to lie between the cortex and medulla. From the arcuate arteries, small vessels (interlobular arteries) extend radially toward the cortex. From each interlobular artery a number of afferent glomerular arterioles originate, one to each renal cor- puscle. Each efferent glomerular arteriole forms a capillary net- work in the cortex surrounding the con^'oluted tubule of that 202 THE EXCRETORY SYSTEM nephron, and extends alongside the collecting tuliules of the cortex. This capillary network connects with the interlobnlar veins running alongside the interlobular arteries, and then these join the arcuate veins. The capillary system of the cortex joins with the capillary supply of the medulla. Here the capillary network surrounds the systems of collecting tu})ules and those portions of the nephridial tubules (nephrons) which are located in the medulla. The capillary network of the medulla connects with radially directed venulse rectse, which are small veins connecting with the arciform veins. The latter branches join to form interlobar veins, which occur alongside interlobar arteries and eventually join with the renal vein. Not only is the kidney well supplied with arteries, veins, and capillaries, but there is also a rich lym- ])hatic system. The nerves su])plying the kidney are branches of the sympathetic ])lexus. THE URETERS. Urine is conveyed from kidneys by tubes called ure- ters. The ureter of a pro- nephros is a pronephric duct and posteriorly this duct joins with the cloaca. The pos- terior portion of this same duct also serves as the ureter for the mesonephros, in which case it is known as the mesonephric, or Wolffian, duct. As an example of the structural character of this duct in the case of the Amphibia, the frog's ureter will serve. Mesonephric Duct. — Tn the frog, the ureter runs dorsally along the anterior two-thirds of the kidney, then turns to accompany the portal vein along the outer border. During its course along the kidney, the ureter is inclosed in the fibrous connective tissue of the kidney. In the remainder of its course, it lies free in the body cavity associated with the Miillerian duct. The mucous membrane of the ureter is longitudinally folded and varies from 1 to 3 cell layers in thickness. (Fig. 127.) In the anterior portion along the kidney, there may be a single layer of cuboidal or low cohnnnar Fig. 127. — Photograph of a cross-section of the ureter of Necturus in the region of the kidney. THE URETERS 203 cells, hut the mid-region along the kidney has a superficial layer of columnar cells and a basal layer of polyhedral cells. Through the main ])ortion of the ureter from the kidney posteriorly the superficial cells, which may have smaller cells interspersed, rest upon one or two layers of polyhedral cells. Immediately below the epithelium is a thin region of connective tissue surrounded by a circular band Nephridial tubules Mucosa Submucosa Musculans Fig. 128. — Diagram of ureter of the water snake and adjoining uriniferous tubules. -^ — Capillary Adveniitia Submucosa Circidar muscle Tratisitional epithelium Longitudinal muscle — Mesothelium Fig. 129. — Diagram of mammalian ureter (dog). of smooth muscle. A thin layer of connecti\-e tissue forms the most external coat. Metanephric Duct. — In reptiles, birds, and mammals where the metanephros is the functional kidney, the ureter occurs as a new structure developing from the basal portion of the old mesonephric duct, as already noted. (Fig. 128.) 204 THE EXCRETORY SYSTEM In the case of mammals, the ureter has three distinct coats. The Hning, Hke that of the kidney pelvis with which it is continuous, is composed of transitional epithelium. This with an underlying tunica propria of loose fibroelastic connective tissue constitutes the mucosa, (Fig. 129.) It has longitudinal folds, so that in cross- section the lumen apjDcars to be star-shaped. Outside the mucosa there is a muscularis coat which usually exliibits an inner layer of scattered longitudinally arranged strands of smooth muscle cells and an outer, thicker zone of circularly disposed smooth muscle cells. These two sets of muscle cells produce the peristaltic motion which continuously carries drops of urine away from the kidney. Covering the muscularis coat is the fibrosa which is composed of fibroelastic tissue and connects the ureter with adjacent structures. THE BLADDER. In most vertebrates there is some provision made for the tem- porary storage of the urine which is contimiously being excreted from the kidney. The urine is collected in sacs, called liladders, of which there are three types, namely, the tubal, the cloacal, and the specialized bladder of the Amniotes. The tubal bladder is found in many fishes where the posterior ends of the ureters (Wolffian ducts in this case) are enlarged for temporary urine storage. In some fishes there is a partial fusion of the two exjDanded ends, and in others there is a fusion of the two into a single large sac with which the ureters are connected. In the latter case there is a single exit located in a little eminence, called the urogenital papilla, which is in the wall of the cloaca and into which the genital ducts also open. A simple cloacal bladder is found in the lung fishes and amphib- ians. It is a thin-walled sac formed as an evagination from the ventral wall of the cloaca. In the frog, it is lined with a pseudo- stratified epithelium. A thin layer of connective tissue containing irregularly- arranged smooth muscle cells is subjacent to the epithelial covering. An outermost region of connective tissue similar to the peritoneum completes the structure of its wall. Such a bladder is anchored to the bod\' wall by a dorsal mesentery of peritoneum and similar attachments from the lateral faces to the l)0(ly wall. This type of bladder stores urine which collects in it from the closed cloaca. Stored urine is ejected by contractions of the body wall. The allantois of the amniotes also arises as an evagination of the cloacal wall and is homologous with the amphibian cloacal bladder. THE BLADDER 205 Thf hhiddcr occurriii^^ ain()ii' bladder the mucosa is wrinkled by folding of the underlying submucosa. In some bladders there are simple gland-like })ockets in the epithelial wall. TIkmt being THE URETHRA 207 no niiiscularis luiicosa, the suhiiiucosa of fihroclastic connective tissue unites with the tunica ])ro])ria undcrlx in<,^ the epithelial lining. A study of a cross-section of relaxed hladder wall shows a thick muscularis. The nniscnlaris coat is divided into a thick middle coat with circularly arranged cells, an innermost region with a thin layer of longitudinally arranged cells, and an outside region also with longitudinal layers of cells. The muscle tissue, although loosely arranged, is very strong. The fibrosa consists of fibroelastic connective tissue and merges with a ])erit()neal-like coating. At the junction of the hladder with the urethra there is a ring of cir- cularly disposed smooth nuiscle, the internal sphincter of the bladder. THE URETHRA. The urethra differs in the male and fenuile. In the male it forms the duct of the penis, and an account of its structure will l)e con- sidered with the male system of reproduction. The female urethra is short. (Fig. lol.) It extends from the bladder to the vestibule and opens into the vestibule along with the vaginal part of the female reproductive system. Internally it presents longitudinal folding of the mucosa. The epithelium of the mucosa is conunonly of the stratified squamous variety. There is no distinct subnuicosa; out- side the tunica ])ropria is a muscularis coat with an inner longi- tudinal coat and an outer circular coat. Outside the nniscnlaris is a fibrosa. Near the junction of the ureter with the ^•estibule there is a sphincter nniscle of the skeletal \'ariety outride the smooth muscle. REFERENCES. Chase, S. W. 1923. The mesonephros and urogenital ducts of Necturus maculosus (Rufinesque), Jour. Morph., 37, 457. Defrise, a. 1932. Cytophysiological studies of the nephrocytes of uni- segmental aglomerular and glomerular nej^hrons, Anat. Rec, 54, 185. Edwards, J. G. 1928. Studies on aglomerulai- and glomeiular kidneys. Am. Jour. Anat., 42, 75. 1933. The renal unit in the kidney of vertebrates. Am. Jour. Anat., 53, 55. Gersh, I. 1934. Histochemical studies on the mammalian kidney: II. The glomerular elimination of uric acid in the rabbit, Anat. Rec, 58, 369. Grafflin, a. L., and Moses, J. E. 1934. A microfluoroscopic study of tele- ostean kidneys, Anat. Rec, 59, 449. Gray, P. 1930. The development of the Amphibian kidney: Part I. The development of the mesonephros of Rana temporaria, Quart. Jour. Micr. Sci., 73, 508. 208 THE EXCRETORY SYSTEM Gray, P. 1932. The development of the kidney of Triton vulgaris and a comparison of this form with Rana temporaria, Quart. Jour. Micr. Sci., 75, 425. HoLTON, S. G., AND Bensley, R. R. 1931. The function of the diiTerentiated parts of the uriniferous tubule in the mammal. Am. Jour. Anat., 47, 241. Singer, E. 1934. Observation of the frog's kidney with the fluorescence microscope, Am. Jour. Anat., 53, 469. Steen, W. B. 1934. Special secretory cells in the transverse ducts of the frog's kidney, Anat. Rec, 61, 45. Stroer, W. F. H. 1932. The development of the pronephros in the common perch (Perca fluviatilis L.), Quart. Jour. Micr. Sci., 75, 557. See Appendix also. CHAPTER Xril. THE FEMALE llEPKODT (TIVE SYSTEM. A:mong the vertebrates, the sexual systems are generally separated, difi'erent infli\iduals developing into either male or female, but for a time during the early embryology the male and female sexual systems follow the same developmental plan and are indistinguish- able either anatomically or histologically. P^lements of both sys- tems are developed at this time, though genetically sex is determined at the time of fertilization. In early vertebrate embryos the uro- genital fold develops on either side of the dorsal mid-line. From the greater part of this fold the urinary system develops, as already described; but from the mesial ventral surface a longitudinal thick- ening arises to form the genital ridge. This ridge is covered with a cuboidal epithelium and separated from the developing kidney by mesenchyme. The simple epithelium of the genital ridge prolifer- ates into a stratified zone of polyhedral cells in which two types of cells soon make their appearance. Larger spherical cells, the pri- mordial sex cells, are scattered among the cuboidal or polyhedral indifferent cells, and the underlying mesenchyme forms string-like masses projecting into the epithelial mass. This mass represents the early indifferent stage in development of the sex glands, or gonads. The excretory ducts of the genital system' are also closely associated with the urinary system. Two longitudinal ducts are developed on each side from the mesoderm lining the ccielome, and these open caudally into the cloaca. The one duct is the Wolffian duct, already described in considering the excretory system, the other is the ]Mullerian duct, which becomes the oviduct of the female. With development, sexual differentiation occurs, and the system associated with the sex so determined progressively differentiates while the elements of the other system degenerate. THE OVARIES. The female organs of reproduction consist of the ovaries, in which the ova are produced, and a pair of ducts, the o\iducts, by which the ova are conducterl away. The ovaries of vertebrates are usiudly paired organs, but occasionally, as in elasmobranchs and birds, one 14 210 THE FEMALE REPRODUCTIVE SYSTEM ovary degenerates, or, as in certain teleosts, the two ovaries fuse. In the embryonic gonad, string-like continuations of the iniclerlying mesenchyme divide the ei)itheHal cells into cords and carry along developing blood vessels. Thus, each gland is composed of spherical cells supported by connective tissue, and the whole co\ered by an epithelial membrane continuous with the ])erit()neal lining of the coelome. The first indication of ovarian differentiation appears in the development of a layer of the cuboidal or polyhedral cells to form follicle or nurse cells about larger primordial sex cells. Gen- erally a single layer of cuboidal or columnar cells siu'rounds each maturing ovum and assists in the elaboration of the nutrition for its development. By the activity of these cells, yolk is added in the case of yolk-bearing eggs so that adequate nutrition is stored for future developmental demands to be met after fertilization. Not all the ova and their follicle cells complete their development. A number api)ear to reach various stages of differentiation and then disintegrate and are resorbed with an accompanying in\'asi()n of the surrounding connective tissue. The condition of the ovary shows considerable variation in the different grou])s of vertebrates and the de\'elopment of the OAum likewise varies. Among the oviparous forms the fertilized egg develops outside the body and must depend u])on its stored nutri- tion. In some viviparous forms it may likewise have to (lc])end entirely or in part u])on stored nutrition. In ])lacental mannnals, no yolk storage occurs and nutrition is furnished the embryo directly from the mother throughout its de\elopment. Fishes. — In myxinoid cyclostomes, an unpaired, elongate gonad is held l)y a median dorsal mesentery. Xo genital ducts are ])resent, and the ova and sperm are liberated into the !)0(1\ caAity from which they pass to the urogenital sinus by two pores. The anterior portion of this gonad ])ro(luces ova and the posterior portion ])ro- duces s])erm, but both are not functional at the same time. In this case an individual functions first as a male and then as a female. In some cyclostomes only one i)orti()n may l)e functional, though the other is ])resent. In elasmobranchs, such as the skate, the female has a single ovary; the right ovary degenerates. In the functional left ()\ary, ova periodical!}- matiu'e in round follicles on the ])crii)her\ and are liberated to be i)icke(l u]) by a pair of oN'iducts ()])ening behind the ])ericar(lium. The eggs of this grouj) are the larg(>st among the fishes and usualh' onl\' a small number mature each season. THE OVARIES 211 The teleost fishes ])r()(hice tlie smallest egimiing with an indiH'erent gonad in which ])i'im()r(lial sex cells are scattered among the indifferent cells, there is a i)rogressive difl'erentiation. The cuboidal epithelium TIIK OVMUES 215 C()\eriii!>.) Toward the uterus these foldings are not so extreme, and the tube wall is 77//'; oviDccrs 22.'^} !iiudi thicker in proportion to the lumen. Tlie ciha of the eohunnar e])ithelial cells beat away from the ostium, so tiiat the egj? and follicular fluid are propelled toward the uterus. The e])ithelial lining may show variations in the a])i)earance of the eohunnar cells at different periods of the ovarian cycle. The f7('/7/.s.— Regardless of its anatomical variations, whether sim])lex, hicornis, or dui)lcx, the uterine structure is the same fundamentally. At the end of the oxiduct the uterus begins as an abruptly exi)ande(l tube containing the same three coats of tissue. Fig. 139. — Photograi>h of a section through the oviduct of a kitten near the ovary. The mucosa is much folded. Ilowexer, the three divisions of adventitia, muscularis, and mucosa are much more developed than in the preceding portion of the duct. The adventitia is called the perimyometrium, the muscularis becomes the myometrium, and the mucosa is called the endo- metrium. (Figs. 140 and 141.) In the connective-tissue framework of the myometrium are many types of connective-tissue cells, including an embryonic variety capable of forming muscle cells in great numbers when pregnancy occurs. In ]:)regnancy the myometrium increases many times its size in the resting uterus, due to increase in cell numbers as well as to increase in cell size. The connective-tissue fibers also increase and there is a looser arrangement and more tissue juice. The uterus is interpreted usually as having no submucosa; the connective tissue of the mucosa continues into that of the subjacent 224 THE FEMALE REPRODUCTIVE SYSTEM miisciilaris region. The mucosa or endometrium undergoes remark- able modifications in cyclical, or seasonal, periods. (Fig. 141.) In humans, a series of changes are repeated each lunar month of the productive period if pregnancy does not occur. During each of these ^Ai " «. '5-v^ W^ ? ^ ^ ^z\^^ . . 1 ^ ■^i > l\ \^ ^ 1^ (f- w^ ^^fefejJS &«f ^ B w^ > Fig. 140.— Photograph of a section through the resting uterus of the rabbit. The endometrium is thrown into several broad folds and many shallow tubular glands occur in it near the lumen. The dark band surrounding the endometrium is the myometrium. At the left the connective tissue and blood vessels of the mesentery are shown continuing with the perimyometrium. Fig. 141. — Uterus of the cat in heat, showing glandular endoiiietriiun and broad myometrium. THE OVIDUCTS 225 months an o\'inn is norniallydiscliarged from the o\ary and, pro\idcd no sperm are present in the oviduct to fertihze the ovum, the special l)reparation of tlie mucosa is not needed. A consequent loss of the major ])ortion of the nnicosa takes place under the name of menstrua- tion in humans, but in other forms degeneration and resorption takes place in such cases. Following fertilization, more profound changes occur in the body of the uterus if the embryo continues to develop, so a description of the histological structures of the uterus must also indicate the relation to ovidation and pregnancy. At certain periods of the lunar month in humans, and at other inter- cyclic periods for other animals, there is a condition which may be described as the resting condition of the uterus. The lining of such a uterus shows longitudinal ridges, due to the folding of the tunica propria beneath the superficial layer of ciliated columnar cells. Also in the mucosa are tubular glands extending more or less perpendicularly toward the myometrium. Some of the colum- nar cells forming these glands are also ciliated. Just beneath the superficial epithelium of the lumen and surrounding the gland cells is a rich reticular network with many mesenchyme-like cells and leukocytes (especially lymphocytes). In the underlying tunica I)ropria there is a rich capillary network derived from the vessels in the myometriimi. At its posterior extremity the uterus narrows to a short neck-like portion, the cervix, which joins the \'agina. In this region the mucosa has only the superficial co^'ering of columnar epithelium without any glands. The changes that occur in the uterus preliminary to and following menstruation and ])regnancy will be considered separately imder those headings. The Vcujina. This, is a tube-like portion continuous with the uterus and receives the copulatory organ, the penis, of the male. The same three coats are present in it as in the anterior portion of the ducts, an adventitia, a muscularis, and a mucosa. The adven- titia is similar to that of the uterus and Fallopian tubes. The muscularis is much thinner than that of the uterus, with an ex- ternal longitudinal and an internal circular region of muscle tissue. At the lower end of the vagina the muscularis forms a sphincter muscle. The mucosa consists of a coat of stratified squamous epithelium lining the lumen and a subjacent vascular region of connective tissue, the tunica propria, or corium, which may have projecting papilki- that throw the epithelium into ridges. Three zones are distinguishable in the epithelial lining: the one adjacent 15 226 THE FEMALE REPRODUCTIVE SYSTEM to the lumen is composed of several layers of fiat degenerating cells little, if at all, cornified; the middle zone has several layers of flat cells with a more granular content, suggesting the stratum granu- losum of the epidermis ; the deepest zone has a number of cell layers comparable to the stratum germanitivum of the epidermis. The basal layer of cells are short columnar and ])()lyhedral in shape and are active in giving rise to new cells. Between adjacent cells of the upper layers of the basal zone there may be intercellular bridges, giving the appearance of prickle cells found in the epidermis. In the tunica propria are many leukocytes, especially lymphocytes. Smears from the vagina of some rodents show a series of changes, and the type of cells cast into the lumen indicates the progress of ovulation. Some authors recognize a submucosa below the tunica propria as a connective tissue of looser texture between it and the muscularis. The connective tissue of the adventitia connects the vagina with surrounding structures. The Vestibule.— The vestibule is a shallow depression posterior to the vagina. Its surface is covered with stratified ei)ithelium, which rests on a tunica propria of fibroelastic connecti\e tissue. Below the tunica propria are muscles of the skeletal variety. A pair of vascular bodies form the clitoris adjacent to the entrance of the vagina. This is a structure similar to the corpora cavernosa of the penis and functions as erectile tissue in periods of sexual excitation. Several small glands are also present in the connective tissue around the opening to the vestibule, and their cohunnar cells secrete a mucous substance. The Bartholin glands which occur on either side of the vestibule may be homologous with the bulbo- urethral of the male. CESTRUS. In describing the ovary we followed the de^'elopment of ova from the primitive germ cells to the time of their liberation, noting that such ovarian activity varied considerably in different forms. Usually there is a regular cyclical appearance of ovulation; in some forms it occurs annually, but in others it occurs at shorter interxals. Among the lower aquatic vertebrates, as in the fish, the mature eggs are discharged into the water during a spawning season, at which time males and females congregate. (\)i)ulati()n d(M's not occur, but sperm are discharged into the water in the vicinity of the eggs so that fertilization and further de\eloi)ment are external. (ESTRUS 227 In other animals, as the frog, fertihzation is also external, hnt is aided by a closer proximity of the male and female in an embrace called am])lexns, a psendocopulation. When the females become turgid with mature eggs, the male clasps a female tightly and as the eggs are discharged sperm are poured over them. Among the reptiles, birds, and mammals, sperm are introduced into the female cloaca or vagina, and fertilization later occurs in the oviducts. Most vertebrates, including the lower animals, show mating interest at the time of ovulation, and their outward behavior is generally influenced by this internal condition. There are some accomjjany- ing changes in the oviducts and uterine regions. In the lower forms these changes involve an activity of the glands adding allnnnen and shells to the eggs, but in those forms where de\-elop- ment is internal there are additional preparatory changes in the uterus. In most of the lower mammals, ovulation occurs during the period known as heat, rut, or oestrus. In the rat, cycles of heat, or oestrus, occur e^•ery four days, in the guinea-pig every sixteen days, in the pig every twenty-one days unless fertilization occurs. In each of these periods there is some uterine preparation for ovulation and also changes in the vaginal condition. The oestrus cycles in these forms can be followed by changes in the vaginal cells to the time at which the follicle is mature, at which time the animal is said to be in heat. A desire for mating is manifested at this time, and at the time of or following copulation, ovulation occurs. Should copulation not be effected, ovulation usually occurs,, the egg degen- erates, and the cycle is begun anew with the maturing of other follicles. In man and the primates the main evidence of a regular oestrus cycle is the phenomenon known as menstruation, which occurs with the failure of fertilization. In these forms the uterine preparation for fertilization is much more extensive and involves a remarkable thickening and softening of the mucosa of the body of the uterus for reception of the fertilized egg. In case the egg is not fertilized l^efore it reaches the uterus such preparation is not utilized, and there follows a loss of the major portion of the uterine mucosa together with considerable blood. Menstruation represents a failure of the re])roductive process and is followed by regeneration and a return to the resting stage before there is a renewal of prepa- ration for the next o\'uIation. Maturation of follicles usually ceases with fertilization and is renewed after birth of the young. 228 THE FEMALE REPRODUCTIVE SYSTEM MENSTRUATION. In humans, ovulation occurs about every twenty-eight days. The changes undergone by the uterus may be indicated by separat- ing this hmar period into four parts, understanding, of course, that the tissue changes are gradual and grade into each other. Begin- ning to count the days with the onset of menstruation, there are four stages: Menstrual First to fourth day. Postmenstrual Fifth to tenth day. Intermenstrual Eleventh to twentieth day. Premenstrual Twenty-first to twenty-eighth day. The description already given for a resting uterus applies to the intermenstrual period. At the beginning of the premenstrual period the mucosa gradually increases in thickness until it is almost twice as thick as during the resting condition. (Figs. 142, 143, 144.) The glands become longer, assume a twisted course, and may branch externally. Droplets of glycogen and lipoid material develop in the cytoplasm of the gland cells, and the lumen of each gland becomes distended with secretion. The cells in the connective tissue immediately below the epithelium increase greatly in size. Capillaries in the tunica propria enlarge and are filled with blood to the point that their walls begin to give way and blood issues forth to collect in pools beneath the epitheliinn. The glands also begin to rupture laterally, and their secretion mingles with the escaped blood. The surface epithelium eventually can no longer withstand the distention, due to the continued collection of su})jacent fluids, and begins to give way at numerous points. With the ruptures in the epithelial covering, the underlying fluids pour into the lumen of the uterus, carrying along small masses of epithelial tissue, mouths of glands, and tunica propria. P^Nentually the entire spongy inter- nal lining of the uterine mucosa is sloughed off. Muscular contrac- tions in the uterine wall begin during this period and continue imtil material accumulated in the lumen is expelled. The postmenstrual period begins with the closure of the small blood vessels and cessation of loss of blood. E])ithelial cells from the broken ends of the uterine glands migrate out to the surface, where an epithelial lining is reformed. The glands are likewise re- formed and the connective tissue is restored to its former condition, so that by about the tenth day following the beginning of menstrua- tion the uterine mucosa is entire again. The uterus then enters MENSTRUATION 229 into the intermenstrual, or restiufj; {)erio(l, (lurinj^ wliieh time there are few changes until at the beginning of the premenstrual period, when renewed pre])arations are made for reception of the ferti- lized egg. Sometimes between two menstrual periods, generally Fig. 142 Fig. 143 Fig. 144 Figs. 142, 14.3 and 144. — Three diagrams illustrating changes in mucosa of the uterus during pre- menstrual period. Fig. 142. — Below is the myometrium. In the mucosa are simple tubular glands lined with simple columnar epithelium. The tunica propria is fibrous, with many connective-tissue cells and also leukocytes. The mucosa is also very vascular with many venous-like capillaries. Fig. 143. — The mucosa increases in thickness; the glands become elongated and more tortuous. Somewhat below the internal surface, connective-tissue cells at a certain level become much en- larged and are known as decidual cells. The capillaries increase in size. The epithelial cells in the zone of decidual cells enlarge and secrete the mucus collecting in the adjacent lumen. Fig. 144. — The mucosa is much thicker (possibly twice as thick as in Fig. 142.) The decidual cells are more prominent. Epithelial cells of the glands in the neighborhood of outpocketings filled with secretion give way; fluid seeps out from capillary spaces in this region — the capillary walls give way and the mucous secretions and blood mingle in pools. The process continues until there is a general sloughing ofT of the internal lining of the mucosa. Half way through this menstrual (slough- ing) period — the mucosa internal surface is lacking in epithelium. Later epithelial cells from ends of glands still left in deeper portions of the mucosa migrate out to the internal surface and organize a new lining and also outlets for the glands. It will be noted that the above mucosal changes do not involve the deeper portions of the glands as much as external portions adjacent to the limien of the uterus. 230 THE FEMALE REPRODUCTIVE SYSTEM accepted now to be about the tenth to the thirteenth day following the onset of menstruation, a follicle ruptures and discharges an ovum. Upon entering the o\iduct the ovum undergoes its final maturation division and is fertilized if sperm are present. PREGNANCY. Following fertilization, development begins and continues as the egg passes along the oviduct toward the uterus. During this passage, which takes about three days, a number of cells are formed. The embryo comes to rest in some pocket or fold of the uterine mucosa and continues its de\'elopment. While the uterus develops to a condition of the premenstrual period, the embryo develops the fetal membranes, and the chorion with tuft-like projections begins to form. This extremely small globe erodes its way into the uterine mucosa and, with development, establishes an intimate contact with the uterus through the placenta. To understand the relation- ship between the develo])ing embryo and the uterine wall, the student is referred to embryological texts for developmental details of the fetal membranes. Placenta. Among the marsupials, as in the opossum, gestation, or uterine development of the young is brief and the young are born relatively immature. In these cases the chorion is a smooth membrane in close contact with the vascular uterine mucosa. The outer face of the rudimentary yolk sac unites with the inner face of the chorion, and thus food compounds of the maternal blood vessels in the uterine wall are in juxtaposition with those in the membranes of the embryo, and these food compounds pass into the chorionic vessels to l)e transported via yolk stalk vessels to the embryo. Oxygen is ol)tained similarly. Excretions })ass from embryonic to maternal vessels. At the end of its uterine develo])- ment the membranes surrounding the embryo i)ull loose from the uterine mucosa and the young is born with little destruction of the uterine mucosa. In higher mammals the chorion develops l)ranching vascular villi that ])enetrate and erode the uterine nuicosa to establish varying degrees of intimate relati()nshi])s. Such associations of eml)ryonic and maternal tissues result in formation of an organ called the ])lacenta where nutritive, res])irat()ry, and excretory (exchanges are carried on. The uterine mucosa has in the meantime grown ovvv the entire surface of the chorion. Although the ])lacenta \aries PREGNANCY 231 ill t'oriii 111 (liferent iiiaiiiiiials, it iicxer imoKcs the entire outer surface of the chorion. As the embryo and its membranes grow, the amniotic sac and surrounding chorion covered with an over- growth of uterine mucosa bulge into the uterine cavity. That part or asjject which from the lieginning has been in closest relation with the uterine wall will be the site of the formation of the placenta. The placenta in humans is a disc-like plate, consisting of two com- ponents, one chorionic and one derived from the uterine mucosa. It is also termed the decidua basalis. The uterine mucosa which grows over the chorion of the embryonic mass, where it fills the uterine cavity, is known as the decidua capsularis and is continuous with the decidua basalis, or placenta. The remaining uterine lining also shows a mucosal thickening and is known as the decidua vera. Branching tuft-like outgrowths from the chorion invade the mucosa of the decidua capsularis and basalis. The chorionic villi of the decidua basalis are much more elaborate, and it is here that fetal nutrition, respiration, and excretion are effected. Chorionic F/Z/i. The epithelium covering of the chorion and its villi is a syncytium, and from the underlying mesenchyme connec- tive tissue develops carrying blood vessels. These villi become much extended until only a very thin layer of epithelium separates the fetal blood vessels from the blood in the sinuses of the uterine mucosa into which the villi project. The old superficial uterine wall epithelium disappears, although the superficial connective tissue is still quite firm. The deeper portions of the chorionic villi of the basalis lie in pools of blood in the deeper zone of the uterine mucosa, where the blood has escaped from the broken vessels, as described in the premenstrual condition of the uterus. The villi are covered with a syncytial epithelium which becomes thinner with greater ex^^ansion during embryonic growth. Within the epithelial covering of each villus is a connective-tissue core, con- taining two small arteries, veins, and capillaries. These vessels connect with those in the umbilical cord which arise from the center of the placenta opposite the surface associated with the uterine mucosa. The umbilical cord has a jelly-like connective tissue which encloses the two umbilical arteries, the umbilical vein, the allantoic stalk, and the rudimentary yolk stalk. Nutritive material and oxygen pass from the blood in the uterine ])ools through the epithelial membrane of the chorionic villi into the capillaries, which pass it through the umbilical vessels to the fetus. The fetus and its membranes grow rapidly until the uterine cavity 232 THE FEMALE REPRODUCTIVE SYSTEM is filled and the decidiia capsularis fuses with the decidua vera. At birth, the amnion sac is usually torn open, the contained liquid flows down into the vagina, and the young is expelled by the power- ful contractions of the hypertrophied myometrium of the uterine wall aided }\y contraction of the abdominal skeletal muscles. After the young emerges there is an after-birth. This consists in the exiDulsion of the amnion and the deciduse, together with a consider- able superficial portion of the hypertrophied uterine mucosa. This process involves considerable hemorrhage in case of intimate rela- tionship between the chorionic villi and the uterine mucosa. After the birth process is completed, the hemorrhage usually ceases and there is a slow and gradual regeneration of the uterine mucosa, returning to a condition similar to that found in an ordinary inter- menstrual, or resting period. REFERENCES. Allen, W. M. 1931. Cyclical alterations of the endometrium of the rat during the normal cycle, pseudopregnancy and pregnancy: II. Production of deciduomata during pregnancy, Anat. Rec, 48, 65. CoNEL, J. L. 1931. The genital system of the Myxinoidea: A study based on notes and drawings of these organs in Bdellostoma made by Bashford Dean, The Bashford Dean Memorial Volume, 67, 102; Archaic fishes, edited by E. W. Gudger. Crowell, p. S. 1932. The ciliation of the oviducts of reptiles, Proc. Natl. Sci., 18, 372. DE Beer, G. R. 1924. Note on hermaphroditic trout, Anat. Rec, 27, 61. Dederer, p. H. 1934. Polynuclear follicles in the cat, Anat. Rec, 60, 391. Evans, H. M., and Swezy, O. 1931. The uterus-ovary relationship and its bearing on the time of ovulation in the primates, Am. Jour. Physiol., 96, 628. ■ 1931. Ovogenesis and normal follicular cycle in adult mammalia, Mem. Univ. CaUf., 9, 117. FiTZPATRiCK, F. L. 1930. Bilateral ovaries in Cooper's hawk, with notes on kidney structure, Anat. Rec, 46, 381. Foster, M. A. 1934. The reproductive cycle in the female ground squirrel, Citellus tridecemlineatus (Mitchill), Am. Jour. Anat., 54, 487. Greulich, W. W. 1934. Artificially induced ovulation in the cat, Felix domesticus, Anat. Rec, 58, 217. Hamlett, G. W. D. 1935. Extra-ovarial sex cords on an armadillo ovary, Anat. Rec, 62, 195. Hargitt, G. T. 1930. The formation of sex glands and germ cells of mam- mals: III. The history of the female germ cells in the albino rat at the time of sexual maturity, Jour. Morph., 49, 277. 1930. The formation of the sex glands and germ cells of mammals: IV. Continuous origin and degeneration of germ cells in the female albino rat, Jour. Morph., 49, 333. 1930. The formation of the sex glands and germ cells of mammals: V. Germ cells in the ovaries of adult, pregnant and senile albino rats, Jour. Morph., 50, 453. REFERENCES 283 Hartman, C. B. 1929. How large is the mainmalian egg? Quart. Rev. Biol., 4, 373. LoEB, L. 1932. The parthenogenetic development of eggs in the ovary of the guinea-pig, Anat. Rec, 51, 373. Long, J. A., and Evans, H. McL. 1922. The crstrus cycle in the rat and its associated phenomena, Berkeley, Calif., Univ. Calif. Press, Mem. Univ. Calif., vol. 6. Maschkowzeff, a. 1934. Der Genitalapparat der Acipenserida;, Zool. Jahrb. Abt. Anat. u. Ont., 58, 397. Smith, B. G., and Brtjnner, E. K. 1934. The structure of human vaginal mucosa in relation to the menstrual cycle and pregnancy. Am. Jour. Anat., 54, 27. Swingle, W. W. 1926. The germ cells of anurans. Jour. Morph. and Physiol., 41, 441. Wolf, L. E. 1931. The history of the germ cells in the viviparous Teleost, Platyprocilus maculatus. Jour. Morph., 52, 115. See Appendix for general text references. CHAPTER XIV. THE ISIALE REPRODUCTIVE SYSTEM. In the male vertebrate the organs of reproduction generally consist of a pair of testes, in which spermatozoa are produced; ducts by which the sperm are carried away from the testes; and glands, whose secretions are added to the sperm passing through the ducts. DEVELOPMENT OF THE TESTES. As in the case of the developing ovary already described, there is an indift'erent stage of gonad development during which both testis and ovary cannot easily be distinguished. Relatively early. OVARY <: ^TESTIS Germinal epUhelium Egg cord Primary follicle Medullary cord Interstitial cell Rcfe ovarii Epoophoroii Germ inal epithelium ^i minifcrous tubule Inter.stili(d cell ft(./f/r.s//.s -Efferent duct Wolffian duct Fig. 145. — Diagram of the development of the testis and ovary. (Adapted from Kohn.) however, there is the bcgimiing of dllVereiitiai de\el()])iiu'iit leading to formation of either male or female organs of re])r()(hiction. (Fig. 145.) The (level()i)ing cords of cells, wliicli in the ovary broke into DEVELOPMEST OF THE TESTES 235 imiiieroiis follicles, continue unbroken in the testis to form semin- iferous cords in which a lumen later occurs and seminiferous tubules result. The mesenchyme between the tubules forms thin walls al)out them and also sei)ta se])aratino; the organ into com])artments called lobules. The septa connect with the sheath of connective tissue surroundino; the testis. (Fig. 14().) When the early ei)ithelial mass forms cords of cells, two types of cells become distinguishable. Of these, the greater number are small polyhedral or cuboidal, and among them are fewer larger, more or less spherical primitive sex cells with large nuclei. Some Fig. 146. — Diagram of the seminiferous tubules and rete testis of a mammal. observers correlate the small cells with the follicle cells surroimding the ova in a developing ovary and the large cells with the ova. When the testis is mature, two types of cells, namely, spermato- togonia and Sertoli cells, are the main components of the tubules. Spermatogonia, which will give rise to spermatozoa, are believed to be descendants of the large primitive sex cells, and the cells of Sertoli are thought to develo]) from the small cells corresponding to ovarian follicle cells. However, there are repeated cycles of cell degeneration and proliferation in these seminiferous tubules, and, according to some investigators, the early sex cells are lost in the process and the spermatogonial cells of the fimctional testis are derived from the original small follicle cells that survive. In either case, spermatogonial cells alternate in location with Sertoli cells 236 THE MALE REPRODUCTIVE SYSTEM in the walls of the mature testis tubules and are much more numer- ous. (Fig. 147.) The process of sperm cell formation from the spermatogonial cells is called spermatogenesis. Before maturity of the animal there is a series of abortive efforts to carry through development of sperm to completion, until finally the process is well established and fully differentiated and functional sperm are regularly produced. Testes, like ovaries, are not always active but show seasonal periods whose duration and occurrence varies with the species. Both testes and the sperm ducts show variations, depending on whether they are studied during or between breeding seasons. During the inactive stage following or preceding a breeding season, most of the spermatogonial cells have degenerated, the lumens of the tubules are lost, interstitial connective tissue between the adja- cent tubules increases in amount, and cords or spheres of large rest- ing spermatogenic and Sertoli cells are prominent. The conditions at these times resemble those of the immature or developing testis before spermatogenesis has begun. To study the acti\'e condition it is necessary to secure the testis of an animal during the breeding season. The vertebrate testis is usually a compound tubular gland, and the seminiferous tubules, where spermatozoa are formed, represent the secretory end-pieces. They are connected with a duct system by which the sperm pass to the outside. SPERMATOGENESIS. In general, spermatogenesis shows little variation among verte- brates, the series of divisions of cells and differentiation of the sperm presenting similar stages. The wall of each seminiferous tubule consists externally of circularly and longitudinally disposed col- lagenous and elastic fibers and connective-tissue cells. Between adjacent tubules there are regions filled wath interstitial tissue composed of loose fibroelastic connective tissue and certain special elements, called interstitial cells, thought to be secretory. Within the connective-tissue sheath of each tubule is a basement membrane on which rests the highly specialized stratified germinal ei)itlielium composed of Sertoli cells and spermatogonial cells. (Fig. 149.) Sertoli Cells.— These are long columnar-like cells with broad bases resting on the basement membrane, with the elongated jjortions of the cells extending like si)okes of a wheel part way toward the center SPERM ATOdEN ESI S 287 of the lumen. Thv nucleus is oxal and somewhat elouf^ated, Sertoli cells occur at ((uite rciijular intei'xals around the lumen of the tuhule and establish close relationshi]) with the more numerous and smaller developinij; s])ermato(jenic cells. 'rmatagonia SecoTidary spermatocyte Spcrmatazoa a, h, c = Stages in sperniiogeyiesis Fig. 147. — Diagram of a section through a seminiferous tubule, showing formation of spermatozoa from spermatogonia! cells. Spermatogenic Cells. — Beginning at the basement membrane, and extending in toward the lumen, spermatogenic cells form a stratified epithelial tissue several layers deep, interrupted at regular intervals by Sertoli cells. We have already studied the growth of stratified squamous epithelium, and the acti\ity of these spermatogenic cells presents something of the same sort, l)ut of a more highly specialized nature. ^Yhen acti\ity begins wliich will ultimately lead to the formation of sperm, a series of mitotic divisions take place, during which daughter cells of the dividing basal cells are constantlv ])ushed 238 THE MALE REPRODUCTIVE SYSTEM inward toward the lumen. A reduction of the chromosome number from diploid to haploid is effected, and then a differential process takes place as a result of which sperm are produced. The phases of development up to and including reduction of the chromosome number constitute spermatocytogenesis, and the following stage of differentiation of the more or less spherical cells with haploid chromo- some number into spermatozoa is called spermiogenesis. Not all the tubules or all portions of any one tubule are actively producing sperm at any one time. In an active tubule, there are rounded cuboidal spermatogonial cells resting on the basement membrane and undergoing mitosis. As each divides, one daughter cell is pushed toward the lumen and in turn it divides so that one of the daughter cells is again pushed further toward the lumen. The number of mitoses, or spermatogonial divisions, appears to show some variation in different animals, but the cells resulting from the last mitosis of this early proliferation period are known as primary spermatocytes, and they enter a resting phase during which they increase in size. Later they undergo a maturation process involving a reduction division and a following regular mitotic division which produces the spermatids, small spherical cells with a haploid chromosome number. After spermatids are formed they associate themselves closely with the tapering distal portions of the Sertoli cells. As a result of a transformation process, these small spherical cells become fully developed spermatozoa, with head, middle-piece, and tail. The head is composed of the condensed nuclear matter of the haploid number of chromosomes; the middle-piece immediately behind the head contains some of the cytoplasm and the centrioles; and the tail is a long flagellum-like process. IVIuch of the cytoplasm of the spermatid is sloughed off during differentiation of the sperm and discharged into the lumen where it may join the sperm in their passage toward the efferent ducts or be resorbed in part by the Sertoli cells. During the process of spermiogenesis the head remains buried in the Sertoli cell and the tail when formed extends into the lumen of the tubule. When fully formed the sperm become free of the Sertoli cells and pass into the lumen, but are themselves as yet quite inactive. The sperm show some variations with regard to size and shape, but the parts are fundamentally very similar. If a particular section of a seminiferous tubule shows activity. a number of cells appear to be in the same stage of development. If spermatids are present, a nutnber in the same stage ap])ear; similarl.N- only s])ennatocytes in the same ])liase, or only spermato- SPERM DUCTS 239 gonia may he ai)])ar(.'nt. If one ero]) of spermatids is {•()m])leting metamorphosis into sperm, then mitotic aeti\ ity may ah-eady have })eo;un in the spermatoii;onia initiating another ^rou]) of s])ermati{ls. In many mannnals it a])])ears tiiat spermatogenie activity passes along the seminiferous tuhiile in waves from their (Hstal ends toward the efferent ducts. Spermatogenesis has l)een intensively stiuHed in the rat, where it has been estimated that it takes about twenty days for a spermatogonia! cell to develop into mature sperm. This spermatogenic activity is limited to animals that have reached the age of maturity. Breeding seasons usually occur during this period of activity. Interstitial Cells. — Between the adjacent seminiferous tubules is a loose fibroelastic connective tissue similar to that forming the wall of the tubules. In this tissue region are the blood and lymph vessels, nerves, and so-called interstitial cells. (Fig. 149.) These cells appear to have about the same size as the granular leukocytes and show \ariation in shape. If they are connective- tissue cells they a])])ear to be a special variety and their acti^•ity is thought to be endocrine in nature. The spherical nucleus is relatively large, with coarse chromatin granules and one or two nucleoli; the cytoplasm shows variation in the degree of granulation of a fatty nature. Transitional forms indicate the origin of these cells from fibroblasts and their return to a fibroblast type imder conditions of inflammation. The true nature of these cells still remains imdetermined. Endocrine secretion on the part of the testis is evidenced by the effects following its removal and trans- plantation. For exami)le, if the testes are remo^•ed from an innna- ture animal, the secondary sexual characters of males do not de\elop; if this removal takes place after maturity there is a loss of sex emo- tions and reproductive activities, and the body accumulates con- siderable adipose tissue. The transplantation of a testis into the body of a female causes secondary sexual characters of the male to make their appearance in the absence of the ovaries. A number of such ex])eriments point to an endocrine activity on the part of the testis, luit do not indicate the tissue directly res])onsible. SPERM DUCTS. In studying the urinary system attention was called to the fact that in the male parts of the mesonephric tulniles became asso- ciated with the testis as eft'erent ducts for the sperm. At the same time the seminiferous tubules are developing in the testis, cells of 240 THE MALE REPRODUCTIVE SYSTEM the anterior mesonephric tubules proliferate to extend as cords into the region of the developing seminiferous tubules. Such meso- nephric derivatives unite with the cords forming in the testis, both develop lumens, and the numerous seminiferous tubules acquire an outlet through the modified mesonephric tubules, now called vasa efferentia, to the Wolffian duct. The whole anterior end of the Wolffian duct may become much convoluted to form the epididymis, a special part of the duct system, to be described later. In the amniotes the entire Wolffian duct is no longer urinary, but becomes the vas deferens, or main sperm duct, of the male. Variations exist through the ascending groups as regards the completeness of a separation of the two systems. In cyclostomes the sperm may have no duct system, but pass directly to the coelome and out through abdominal pores; in mammals the sperm pass through the converted mesonephric duct system independent of the urinary system until it joins the urethra, in the vicinity of the copulatory organ, the penis. In general, the sperm ducts are lined with a cuboidal epitheliiun where they unite with the seminiferous tubules, but approaching the posterior portions the epithelium changes to columnar, and in the vas deferens it may become stratified cohunnar. Ciliated cells often alternate with those of glandular types whose secretions enter the lumen and join the sperm. The epithelium rests on a connec- tive-tissue sheath encircled by scattered smooth muscle cells in the smallest ducts and a definite coat in the vas deferens. Surrounding the ducts peripherally is a vascular fibroelastic connective tissue which becomes more })rominent as the size of the duct increases. MALE REPRODUCTIVE SYSTEM OF FISHES. In elasmobranchs the testes are ovoid or cylindrical bodies. The mesonephros is the functional kidney, and in the male the anterior end of it degenerates so far as its urinary function is concerned, and its ducts establish connections with the tubules of the testis during development. Some of the embryonic mesonephric tubules of the anterior end form a much-coiled tube, the epididymis, through which sperm pass from the testis to the mesonephric, or Wolffian, duct, which serves to conduct both urine and sperm to the cloaca. In some cases the kidneys empty by special urinary ducts into a sort of urogenital sinus from which a papilla, the urogenital ])a])illa extends into the cloaca. The ])osterior ends of the two Wolffian ducts are enlarged and act as sperm reservoirs during the l)reeding season. MALE REPRODUCTIVE SYSTEM OF AMPHIBIA 241 MALE REPRODUCTIVE SYSTEM OF AMPHIBIA. The testes are more or less elongated, tubular bodies in the urodeles and lower forms, and ovoid in anurans. They are ovoid, yellowish-white bodies lying alongside or ventral to the kidneys and connected to the latter by a mesenteric sheet of tissue. Each testis is a mass of tubules fitting the general descri])tion given for the seminiferous tubules. The seminiferous tubules connect with the vasa efferentia, derived from embryonic mesonephric tubules and supported by mesentery. When the efferentia reach the functional kidney they pass into a longitudinal duct, Bidder's canal, which runs along the mesial border of the mesonephros. (Fig. 148.) Fig. 148. — Longitudinal section of testis of Necturus, showing seminiferous tubules radiating toward ducts in connective tissue below. During development of anurans, an anterior part of the testis, possibly derived from pronephric tissue, has large cells resembling oocytes or enlarged spermatocytes. This structure. Bidder's organ, may persist well into the mature stage of toads, but gradually degenerates. The remains of the ])ronephric ducts passing along the mesonephros form Bidder's canal. Its lumen is lined with columnar epithelium supported by a connective tissue and a few smooth muscle cells. The outermost region of the canal has con- nective tissue continuous with that of the covering of the kidney in which it is partially embedded. Bidder's canal communicates with the collecting tubules in the 16 242 THE MALE REPRODUCTIVE SYSTEM kidney and the ureter which runs along the outer margin of the kidney. Thus, in the breeding season the ureter, or Wolffian duct, acts as a sperm duct also. The testis shows seasonal variations in size, being largest during the breeding season. In many Amphibia, the Miillerian duct, which is the functional oviduct of the female, is still present in the male. It may be without a lumen in some, but in others it has the features of the resting oviduct of the female. MALE REPRODUCTIVE SYSTEM OF REPTILES. In reptiles and birds the mesonephros is an embryonic structure, but is replaced by the metanephros, which is the functional kidney of the adult, and ureters are new developments from the embryonic cloaca. Each testis has associated with it an epididymis, a coiled sperm-carrying duct, distally connected with the seminiferous tubules and proximally connected with the Wolffian duct. The Wolffian duct has become a vas deferens and is now solely a sperm-carrying duct. In some male reptiles the IVIiillerian duct still remains, and in Lacerta viridis is as large as in the female, where it is the oviduct. In the epididymal region the duct is lined with cuboidal or columnar epithelium, which may be ciliated; in the \'as deferens the epithelium is columnar or may api)ear stratified with regions of glandular cells alternating with ciliated cells. MALE REPRODUCTIVE SYSTEM OF MAMMALS. Among the mammals the testes are paired, o\o'u\, comi)act organs which show some variation in their location, not only in different species, but often in the same species at different periods. In such animals as elephants and whales the testes remain permanently within the body cavity, but in marsupials, rodents, bats, and some insectivora they pass out of the body into scrotal sacs during the breeding season. In primates, carnivora, and ruminants they remain permanently outside in scrotal sacs. In the testes that have descended during the breeding season, or in the case of primates wdiere they are permanently outside the body cavity, the scrotal sac adds several tissue sheaths about the testes. The scrotal sac, or scrotum, is formed by an evagination of the bod\- wall. Externally it has a covering of skin with its stratified squa- mous epithelium, glands, and hairs, su])])orted b\- layers of con- nective tissue, and muscle. Internally it is lined w itli a tissue simi- lar to the peritoneum and known as tunica Aaginalis parietalis. The same type of tissue is reflected over the anterior and lateral MALE REPRODUCTIVE SYSTEM OF MAMMALS 243 faces of the testes and somewhat \eiitrally and dorsally as the tunica vaginahs visceralis. Between these two tunics is a very loose fibro- elastic connective tissue and fluid, making possible a free movenuMit of the testis within the scrotum. AVithin the tunic vaginalis, a connective-tissue sheath, the tunica albuginea, extends perforated partitions, or septa, into the testis and forms compartments or lobules containing the seminiferous tubules. A mid-posterior region of this tissue located toward the surface of the testis is very vascular and called the mcdiastimmi. The lobules so set off in the testis Fig. 149. — Photograph of a cross-section of a seminiferous tubule of the woodchuck showing interstitial tissue between it and adjacent tubules. contain from several to many convoluted seminiferous tubules. (Fig. 149.) As the tubules approach the mediastinum they unite to form less-coiled tubules until a few much smaller straight tubules are formed which connect within the mediastinum into a network of small ducts called the rete testis. From the upper region of the rete testis a number of larger tubules compose the vasa efferentia, which are at first straight but become twisted and increase in diam- eter as they pass toward the upper posterior region of the testis. These join to form the epididymis, a much-coiled duct forming a mass divided into a head and tail portion at either end of the testis, and the body of the e]Mdidymis along the central portion. The tail portion of the epididymis straightens out to become the vas 244 THE MALE REPRODUCTIVE SYSTEM deferens and passes upward through the inguinal canal into the body cavity with the spermatic cord. The spermatic cord is com- posed of connective tissue containing the vas deferens, the spermatic artery, spermatic vein, the ner\'e trunk, and a plexus of veins, called the pampiniform plexus. After entering the body cavity, the vas deferens continues anteriorly for a distance, then tin-ns down around the ureter to join the urethra somewhere along its passage from the bladder to the exterior. Each vas deferens has an enlarge- ment, called an ampulla, near its terminal region in the case of primates, shrews, bears, dogs, and most rodents. In some, the walls of this region have mucoid glands, (Fig. 150.) Epuliih/mis Vas deff reus Tubuli coniorti Seminiferoii iutnde Septa Fig. 150. — Diagram of the tubular system of a mammalian testis. Connected with each vas deferens in the ami)ullar, or terminal, region is a saccular organ, the seminal vesicle. These occur in most mammals, but not in marsupials and carnivora. The neck of each ampulla and each seminal vesicle unite to form the ejacukiiory diiri. There is a large glolnilar mass known as the prostate (/land, which surrounds the urethra near the bladder, and each ejaculatory duct traverses the prostate to join the urethra. The i)rostate contains a number of small glands and occurs in most mammals, but not in marsu])ials. A short distance along the urethra toward the exterior MALE REPRODUCTIVE SYSTEM OF MAMMALS 245 is a pair of small sac-like structures, known as the bulbo-urethral, or Cowper's glands, which are present in almost all mammals, but not in (logs and bears. They are large in rodents and pigs. IIa^•ing already considered the histology of the seminiferous tubules, let us now study the other parts of the conducting system. Tubuli Recti.— Where the seminiferous tubule transforms into a straight tul)ule, the spermatogonial cells are lacking, and the straight tubules are lined with columnar cells resembling the Sertoli cells of tlu' tubules. Rete Testis.— The irregular meshwork of these spaces is lined with short cuboidal or squamous epithelium. The free surface of the cells possesses a flagellum. In between the rete testis is connective tissue, nerves, and vessels of the mediastinum. Vasa Efferentia (Tubuli Contorti), — The cells lining the ducts rest upon a basement membrane and vary in size. A clump of tall, ciliated columnar cells may ha\'e adjacent to them progressively shorter cells. Thus, little pockets are formed in the wall of the efferent ducts. At the free surface of the short type of cell is a flagellum. The cells of these tubes produce a secretion which is added to the mass of sperm moving through the ducts to the epi- didymis. In the connective tissue outside the })asement membrane there is a small amount of smooth muscle. Epididymis.— The hunen is larger and circular in cross-section. Adjacent to the basement membrane are small cells and internal to them are large columnar cells of varying heights. At the free surface of each of the latter cells is a filamentous projection that is non-motile Init from which secretion originating from the cytoplasm of the cells is discharged into the lumen. As the epididymis ap- proaches the vas deferens the columnar cells are not so tall. In the connective tissue outside the basement membrane there are smooth muscle cells, which constrict the tube and serve to propel sperm and fluid onward toward the vas deferens. The rete testis, tubuli contorti, and epididymis are portions of the old mesonephric tubules used here exclusi^'ely to carry sperm. Vas Deferens. (Fig. lol.) This is a larger tube than the epi- didymis and its lumen is also greater. The epithelium of its mucous membrane is of the stratified columnar variety. The tunica propria has long folds which make the lumen irregular in shape. There is no definite submucosa, but outside the tunica propria is a muscle coat which consists of a thin innermost coat in which the smooth muscle cells are arranged lengthwise of the tube, a relatively thick middle circular coat of muscle, and an outer longitudinal coat which 246 THE MALE REPRODUCTIVE SYSTEM is thinner than the circular coat. Outside the muscle coat there is an adventitia of fibrous tissue in which there are groups of smooth muscle cells. As the vas deferens continues into the body cavity, a connective-tissue extension of the adventitia supports the sper- matic artery, spermatic vein, nerves, and a plexus of veins, called Muscularis ^W^mm -Miicom Fig. 151. — Diagram of the vas defereiLS of a mammal. Fig. 1.52. — Photograph of a section through the spmiual vesicle of the rat. the pampiniform plexus, the whole being called a spermatic cord. The ampulla at the distal end of the vas deferens has large ridges in tlic tunica ])roi)ria, and in its walls are glandular outpocketings. Seminal Vesicles. Each seminal vesicle is an elongated, lol)uIated, twisted irregular sac, with an adxciititia coutiuuous witli that of MALE REPRODUCTIVE SYSTEM OF MAMMALS 247 the vas (IctVrcns. Within the iuhentitia is a tliiii niuseularis coat with an outer loiijiitudinal sheath. The tunica i)r()j)ria of loose connective tissue su])i)orts an e})ithehuni of siin])le eohunnar epithe- hum or of cells of the same tyj)e as the vas deferens, i. e., stratified columnar. Any cross-section reveals many com))li('ated ])assages and s])aces set off from each other. (Fig. 152.) This is because the mucous membrane is elal)orately folded and in i)laces the folds have fused to form a complex system of labyrinthine passages within the lumen. The seminal ^'esicles probably do not store sperm but su])])ly a secretion which is added to the sperm fluid proper after the latter enters the urethra at the time of discharge. The lumen is usually filled with an acidophilic colloidal material. Fig. 153. — Photograph of a cross-section of the dog's prostate, showing .5 glandular masses opening into the urethra by excretory du.f'ts. The Prostate. — The prostate is a glandular mass, roughly spherical in shajie, which surroimds the male lU'ethra just distal to its origin from the bladder. (Fig. loo.) Within the ])rostatic mass are a number of small glands of the compound tubular and alveolar type. There are a number of ducts from these glands entering the urethra. Around the whole ])r()static mass is a capsule of fibroelastic connec- tive tissue which has extensions within the mass between adjacent glandular structures. The epithelium lining the glands varies from cuboidal to columnar. In the connective tissue between the glandular ])ortions are patches of smooth muscle cells indicating that the mass may contract and force its secretions into the urethra. The secretion is a thin, whitish fluid and forms the bulk of the spermatic fluid. Sometimes spherical masses of ^'ar^•ing size with concentric lamelhie occur in the lumens of the glands. 248 THE MALE REPRODUCTIVE SYSTEM Cowper's Glands. — Cowper's glands are also known as bulbo- urethral glands and are a pair of small masses located a little further along the urethra from the prostate. They consist of connective tissue, smooth muscle cells, and small branching com- pound tubulo-alveolar glands. The walls of the glands have small pockets and simple epithelium of different types lines them ; in some places the cells are squamous, in others columnar. The main ducts are lined with transitional or stratified columnar epithelium. Other small glands occur along the course of the urethra. COPULATORY ORGANS. Lower Vertebrates. — In forms where fertilization occurs before the eggs leave the body of the female there is a structural modification in the male to facilitate the introduction of sperm into the female reproductive system. In the male elasmobranch, each pelvic fin adjacent to the cloacal aperture has a long finger-like process called a clasper. These two structures are inserted into the cloaca of the female, and the sper- matic fluid runs along the groove formed between the clasper s. In some species of elasmobranchs, curved spines may l)e present at the outer end of the claspers. In most fishes, fertilization is external. In the frog, the male embraces the female and, as the latter dis- charges eggs into the water, spermatic fluid is discharged from the male into the water about the eggs. A unique method is found in some urodeles, such as Amblystoma and Triton, where males dis- charge packets of sperm, called spermatophores, formed by the addition of secretions from glands in the cloacal wall. After these have been discharged, the female crawls o\-er the sperm-])acket until the swollen lips of the cloaca seize it and withdraw it into the cloaca. In reptiles, a copulatory organ is present. On each side of the transverse cloacal aperture, among the snakes and lizards, there is a pocket in which an eversible sac lies. When these two sacs are inflated and everted they form a copulatory organ, called an hemi- penis. On the surface of each sac is a groove, along which si)erniatic fluid passes when copulation with a female takes place. In croco- diles and turtles, there is a cloacal i)enis formed from the ventral wall of the cloaca. Under the epithelium of this region there is loose fibroelastic connective tissue containing large vasculiir spaces; roPVLATORY ORdAN^ 249 a subjacent region is composed of denser connective tissue and skeletal muscle. During the breeding season, when co])ulati()n is to take place, the vascular spaces become engorged with l)lood and convert it into erectile tissue that is thrust out from the cloacal opening. Between breeding seasons this structure lies colla])sed within the cloaca. The monotreme has a cloacal penis somewhat like that of re])tiles and birds, except that the urinary passage, the urethra, runs through its center. This penis lies retracted in a cloacal sheath except during the breeding season. Penis of Mammals. -In the body, or shaft, of the penis, masses of mesenchyme tissue give rise to corpora cavernosa or erectile tissue and also the cartilage or bone which forms in some species. The urethra passes only a short distance from the bladder before becoming associated with the ducts and glands of the genital system. Xear the bladder it has a transitional epithelial lining, but this often changes to stratified columnar further along and then to strati- fied squamous at the o])ening. Three regions of the urethra ha^-e been separated. The prostatic portion extends from the neck of the bladder through the prostate gland, where ejaculatory ducts usually join it. The membranous, or middle part, is short, extend- ing from the i)rostate region to the beginning of the corpora caver- nosa of the penis. The cavernosa portion extends through the length of the penis. Along the course of the urethra are glandular out- pocketings of the dorsal and lateral walls, already described, embed- ded in the fibroelastic connective tissue, with varying amounts of smooth muscle also represented. A cross-section through the body of the penis shows that the major portion of it is formed by the corpora cavernosa. In man, two corpora cavernosa penis occupy the upper portion, but in other mammals they usually fuse to form an unpaired U-shaped mass. In man a third, corpus cavernosum urethra, surrounds the urethra and is located ^•entral to the other two (corpora cavernosa penis). In many mammals the urethral ca\'ernosa tissue is poorly represented. (Fig. 154.) These corpora constitute the erectile tissue of the penis. Among the rodents, the vas deferens may not join the urethra until they reach the tij) of the penis. The corpora cavernosa are composed of a vascular sponge-like network with irregular venous sinuses capable of being filled with blood and distended under considerable pressure. Each of these bodies is surrounded by a thick fibrous membrane, the tunica 250 THE MALE REPRODUCTIVE SYSTEM albuginea, with outer fibers mainly longitudinal and inner fibers circular. Between the two corpora this sheath forms a more or less complete septum, but toward the glans both corpora may communicate. The corpus cavernosum urethra is siu'roimded by a less dense tunica albuginea, in which circular smooth muscle may appear toward the urethral wall. The venous sinuses are more imiform and more elastic fibers are present. When the os priapi, or penis Fig. 154. — Photograph of a cross-section of the penis of a chipmunk near the tip. It is externally surrounded by the skin, within which is connective tissue. The corpus cavernosum penis is a large bean-shaped mass. The V-shaped opening is the urethra, below which is the vas deferens; both of these ducts are surrounded by scanty corpus cavernosum urethra tissue. bone, is present, it forms just ab()\e the urethra from mesenchyme of the developing ])enis. Surrounding the C()r])ora and forming the general groundwork of the penis is a vascular fibroelastic connective tissue which is covered externally by the skin in whose subcutaneous tissue some skeletal muscle may be found. The outer knob-like end of the penis, the glans, is composed of a mass of dense vascular connective tissue covered by a reflected fold of skin, the pre])uce. At times of sexual excitement, erection of the penis follows reflexes conducted to the muscular wall of the arteries leading into REFERENCES 251 the cavernous tissue so that a relaxation occurs resulting in the supplying of an unusual amount of blood to the venous sinuses. The spaces of the cavernosa become engorged, so that the veins emptying them are compressed to the point of interfering with the outward flow of blood. Continued flow of blood into the sinuses enlarges the corpora under pressure and results in making the penis (juite rigid. After completion of spermatic discharge or passage of excitement, the arterial walls regain their former tone, become constricted, and the inflow of blood diminishes. The emptying of cavernosa spaces is slowly effected; the j^enis slowly contracts and becomes relaxed. REFERENCES. Adams, A. E. 1930. Studies on sexual conditions in Tiiturus viridescens, Jour. Exp. Zo()l., 55, (3.3. Chase, S. W. 1923. The mesonephros and urogenital ducts of Necturus maculosus. Jour. Morph., 37, 457. Cheng, T. H. 1929. A new case of intersexuality in Rana cantabrigensis, Biol. Bull., 57, 412. Evans, T. C. 1930. Sex reversal in Rana pipiens, Anat. Rec, 48, 47. Hann, H. W. 1930. Variation in spermiogene.sis in the Teleost family, Cottidse, Jour. Morph., 50, 393. Johnson, F. P. 1934. Dissection of human seminiferous tubules, Anat. Rec, 59, 187. JuHN, M., AND Gu.ST.AVsoN, R. G. 1932. The response of a vestigial Mlillorian duct to the female hormone and the persistence of such rudiments in the male fowl, Anat. Rec, 52, 299. McCuRDY, H. M. 1931. Development of the sex organs in Ti-iturus torosus. Am. Jour. Anat., 47, 387. Moore, C. R. 1926. The biology of the mammalian testis and scrotum. Quart. Rev. Biol, 1, 4. Reese, A. M. 1924. The structure and development of the intromittent organ of the Crocodilia, Jour. Morph., 38, 301. Swift, C. H. 1916. Origin of the sex-cords and definitive spermatogonia in the male chick, Am. Jour. Anat., 20, 375. TuRMER, C. L. 1931. An ovo-testis in yellow perch (Perca flavescens). Science, 74, 370. TooTHiLL, M. C, AND YouNG, W. C. 1931. The time consumed by sperma- tozoa in passing through the ductus epididymis of the guinea-pig as deter- mined by means of India ink injections, Anat. Rec, 50, 95. See Appendix for general text references. CHAPTER XV. THE ENDOCRINE GLANDS. It is known that the nervous system controls integration of bodily acti\ities so that there is a harmonious functioning of the organ systems making possible successful adjustments to changes in the external world. In addition, an important part of the uni- fying and coordinating function is effected by the system of chemical coordinators, the endocrine gland secretions. The secretion products of exocrine glands, as already described, collect in ducts. Endocrine glands, on the contrary, have no excre- tory duct systems. Their secretions pass into the blood or lymph vessels and thus enter the circulation stream for distribution to various organs and svstems. Pineal gland Adrenal Gonad Pitiiilary Pancreai Fig. 155. — Diagram showing the location of ondoorine glands. An endocrine gland may be defined as one that secretes into the })]o()d stream some hormone or chemical sul)stance which stimulates or depresses the physiological activity of other groups of cells, thus affecting growth, development, and the general condition of the body as a whole. Secretion of one gland may affect the activity or effect of the secretion of some other endocrine gland, i. e., they are physiologically interrelated. THE PITUITARY GLAND 253 Observations indicate that certain i,Man blood. The nornud seci-etion is essential to normal calcium metabolism, uictabolisui of sugar. THE ADh'EXAL C LANDS 259 iiiaintenaiicc of the iiitroi^cn fciuilihriuin, and I'oniiatioii of hone and dentin. Proper com})ination of parathyroid secretion and cal- cium salts is essential to normal muscle contraction and administra- tion of parathyroid extract relieves tetany dxw to faulty function of the ])arathyroids. Entire removal of ])arath\r()i(ls results in death. 3 Cords of cells separated by capillaries Fig. 160. — Diagram of iiianimalian parathyroid, showing capillaries between the cords of cells. THE ADRENAL GLANDS. In quadru])ed manunals, one of these glands is located at the anterior end of each kidney. In man they are called suprarenals, because they are located at the up])er end of the kidneys. They have no known functional connection with the kidneys. In man they are somewhat flattened, small bodies, the right one being tri- angular in form, the left somewhat crescent-shaped. In other mammals they are bean-sha])ed masses, embedded in adi])ose tissue which tends to form near the kidneys. The adrenals are exceedingly well sui)i)lied with blood vessels and it has been estimated that blood to the amount of about five times their weight circulates through them every minute. They are surrounded by a firm capsule of connective tissue, extensions of which pass into the interior of the gland. Cross-sections of fresh adrenal glands made with a sharj) knife reveals an outer cortical zone, light yellow in color and of firm textiu"e, siu'rounding a central medullary region of looser 260 THE ENDOCRINE GLANDS texture, and dark red in color. The two zones ha\e an entirely different embryonic origin. (Fig. 161.) The cortex develops as a series of buds from the anterior third of the Wolffian body. The cells of the medulla originate from cells related to those of the coeliac plexus of the sympathetic system and form epithelioid groups. As development proceeds, the latter mass of cells becomes invested with those of the cortical region. In fishes the medullary cords exist as small masses, each adjacent to a sympathetic ganglion and arranged segmentally alongside the Fig. 161. — Photograph of a longitudinal section through the adrenal of the wood- chuck. A connective-tissue sheath surrounds the entire organ. The darker cortex surrounds the central lighter medulla, except at the hilus on the right of the figure. Below are sections of blood vessels. elongated kidney, while in between the kidneys are two long masses of tissue homologous with the mammalian cortex. In am])hibians there is a closer association between the cortex portion and the medulla. In reptiles the two are in contact, in birds they are inter- mixed, and in mammals the cortex surrounds the medulla. Mammalian Adrenal.— Each gland in the case of mammals is surrounded by a connective tissue capsule, which supports arteries, veins, capillaries, and lymphatics. Strands of connective tissue extend in between the epithelioid cells of the cortex into the medulla, supporting a reticular tissue network and ca])illaries. The Cortex.-— Three zones of e])ithell()id cells may be distinguished in the cortex. (Fig. 162.) Immediately beneath the capsul(> is a THE ADRENAL (ELANDS 261 narrow zone, the zona glonierulosa, wliere the polyhedral-shaped cells are arranged in oval groups in the form of flattened or incomplete vesicles. The nuclei stain deeply and the cytoplasm has an affinity for basic dyes. Internal to this is the zona fascicu- lata, with long double rows of larger polyhedral or somewhat cuboidal-shaped cells, often binucleate, radiating in toward the medulla. In the outer portion of these colunms the cells have fat droplets and cholesterol, but usually the technique dissolves the Z. reticularis Fig. 162. — Diagram of a section through the adrenal of a mammal. lipoid substance, so that the cells may have a spongy appearance. Internal to the zona fasciculata is the narrow zona reticularis with cells forming a meshwork of cords one cell in width. These cells contain a brown pigment which increases toward the boundary of this zone with the medulla and becomes more e\ident in glands of old animals. A capillary net in\'ests the cell groups in the glonier- ulosa, and from it capillaries extend between double rows of cells of the fasciculata to become a meshwork again in the reticularis, where they connect with capillaries of the medulla. New cells may form by mitosis in the glomerulosa and outer medulla of a mature gland, 262 THE ENDOCRINE GLANDS and cells degenerating there migrate into the reticularis, where they are removed by macrophages. The M edulla . ^This region consists of an irregularly arranged meshwork of polyhedral cells separated by sinusoidal capillaries. The medulla cells are also roughly rectangular in section, with the longer sides in contact with adjacent cells and with a sinusoidal capillary adjacent to the short sides. When fixed in chromic acid the cytoplasm of the cells is dark brown in color, and if potassium dichromate is used their cytoplasm shows fine brown granules. Because of this affinity for chromium they are referred to as chromaf- fin cells. In ferric chloride the medulla stains green and in iodine yellow. The fine brown granules are secretory products which reduce dichromate of potassium to chromium dioxide. The number of granules present indicates the degree of secretory acti^^ity. There is a plexus of fine nerve fibers in the cortex, especially in the zona reticularis. Axons of nerve cells are in close relation with the chromaffin cells of the medulla. Here also are a few ganglion cells, or even small ganglia near the central vein. Since the medulla is derived from the same source as the sympathetic nervous system, this central portion of the adrenal is comparable to paraganglia to be described presently. Secretion of the cortical and medullary portions appear to have dift'erent functional effects. The cortex is said to produce a special hormone, now called "cortin." Lack of cortin in man, due to ]:)athological lesions of the cortex, results in Addison's disease. Animals deprived of adrenals die, but life is prolonged if extract of cortical tissue is administered. Epinephrin, or adrenalin, or adrenin, is secreted by cells of the medulla. Circulating in the blood stream, it maintains tone in the small arteries, or arterioles, and so assists in regulating blood- pressure. Local injections of adrenalin cause temporary constric- tion in small ^'essels, and in this manner it is used for preventing hemorrhage. Lijections of it into a heart that has ceased beating for a short period in some cases results in a resum])tion of rhythmical contractions. THE CAROTID GLANDS. There is a small mass of glandular tissue similar to that of the adrenal medulla at the forking of each carotid artery into its external and internal branch. This mass, known as the carotid gland, has a connective-tissue sheath enclosing a mass of epithelioitl cells well supplied with a ca])illary network. Small sympathetic ganglia are associated with these glands, and the resemblance of the glandular THE PIXKAL BODY 2()3 cells to those of the adreiuil inedulhi suggests an eiidocrhie fuiictiuii, thouijh experimental evidence is lacking. PARAGANGLIA. In \ari()us organs of the body, occasional syini)atlietic ganglia are fonnd associated with a small mass of chromaffin cells resembl- ing those of the carotid gland, or adrenal medulla. These glandular masses, known as i)araganglia, have a histological structure suggest- ing an endocrine function, but as yet there is no exi^erimental evidence as to their function. THE PINEAL BODY. The pineal body received its name because of its resemblance to a pine cone. It is also known as the epiphysis cerebri, and is located in the dorsal region of the brain between the cerebrum and cere- bellum, where it appears as a small red body. It is attached to the third ventricle by a stalk and is surrounded by the pia mater. Islands of epithelioid cells, separated and surrounded by capillaries, are embedded in a connective-tissue stroma. Some of the cells resemble cytons of multipolar nerve cells without any processes. A network of fibers from the sympathetic system pass through the connective tissue. It has been assumed that this glandular tissue has an endocrine function, because it has been observed that a tumorous growth of the pineal gland appears to be associated with an unusually early maturing of the sex glands. That is, it has been assumed that the normal pineal secretes a hornione that delays the too-rapid development of sex organs. However, exjjeriments give conflicting results. Other Endocrine Glands Already Considered.— The islands of Langerhans in the pancreas secrete insulin controlling metabolism of carbohydrates. The endocrine nature of the ovary and testis has also been indicated. The liver has also had attributed to it an endocrine activity. Possibly all cells have an endocrine activity to some extent, but as yet only the more conspicuous organizations have been demonstrated experimentally. REFERENCES. Adams, H. E., Kuder, A., and Richards, L. 1932. The endocrine glands and molting in Triturus viridescens, Jour. Exp. ZooL, 63, 1. Addison, W. H. F., and Richter, M. N. 1932. A note on the thyroid gland of the swordfish, Biol. Bull., 63, 472. Allen, E. 1932. Se.x and Internal Secretions, Baltimore, Williams & Wilkins Company. 264 THE ENDOCRINE GLANDS Cameron, A. T. 1935. Recent Advances in Endocrinology, Philadelphia, P. Blakiston's Son & Co. Chandler, 8. B. 1932. The relation of parathyroidectomy to estrus, preg- nancy and lactation in the albino rat, Anat. Rec, 53, 105. Charipper, H. a., and Haterius, H. O. 1932. The histology of the anterior pituitaiy of the albino rat in relation to the oestrus cycle, Anat. Rec, 54, 15. Gilbert, M. S. 1934. The development of the hypophysis: Factors influ- encing the formation of the pars neuralis in the cat. Am. Jour. Anat., 54, 287. Harman, M. T., and Derbyshire, R. C. 1932. The development of the suprarenal gland in the guinea-pig (Cavia cobaya), Am. Jour. Anat., 49, 335. Herrell, W. E. 1934. The growth and regeneration of tissue in frog tadpoles following the administration of an extract of the anterior pituitary gland, Anat. Rec, 59, 47. HoERR, N. 1931. The cells of the suprarenal cortex in the guinea-pig: Their reaction to injury and their replacement. Am. Jour. Anat., 48, 139. Johnson, G. E. 1931. Hibernation in mammals. Quart. Rev. Biol., 6, 439. Lowe, E. 1930. Seasonal and sexual variation in the thyroid glands of the cat. Quart. Jour. Micr. Sci., 73, 577. Modell, W. 1933. Observations on the structure of the blood vessels within the thyioid gland of the dog, Anat. Rec, 55, 251. Raymond, N. 1932. The occurrence of parafollicular cells in the thyroid of the rabbit, Anat. Rec, 53, 355. Sauer, F. C, and Latimer, H. B. 1931. Sex differences in the proportion of the cortex and the medulla in the chicken suprarenal, Anat. Rec, 50, 289. Sharpey-Schafer, E. 1926. The Endocrine Organs, Longmans, Green & Co., New York. Singer, E. and Zwemer, R. L. 1934. Microscopic observations of structural changes in the adrenal of the living frog under experimental conditions, Anat. Rec, 60, 183. Smith, C. 1924. The origin and development of the carotid body, Am. Jour. Anat., 34, 87. Zalesky, M. 1934. A study of the seasonal changes in the adrenal gland of the thirteen-lined ground squirrel (CHtellis tridecemlineatus), with particular reference to its sexual cycle, Anat. Rec, 60, 291. See Appendix for general text references. CHAPTER XVI. TFA'USiqVK. The destructive reactions within living cells are counterbalanced by synthesis of elements for repair and continued life. When cells die, syntheses cease and the enzymes involved in vital processes become acti\e in breaking down the products they formerly helped to build. Such disintegration processes working within the dead or dying cell constitute autolysis. In addition, bacteria present, or soon entering dead and dying tissues, rapidly add to the disinte- gration processes and the accompanying distortion or loss of char- acteristic cellular structure. To prevent these changes, and also to make cells and intercellular substance insoluble and stainable, tissues from freshly killed animals are immersed in fluids called fixatives. Such fixing fluids shoidd accomplish certain definite things in addition to preventing autolytic and bacterial decomposition. They should penetrate tissues rapidly and coagulate or otherwise preserve protoplasmic substances, leav- ing intra- and extracellular structures preserved in the same relative spatial relations as during life. They should prevent sub- sequent changes, such as shrinkage, hardening, and dissolution, and should not hinder later staining. With these requirements for a fixati\'e, let us consider the properties of some chemicals in common use as components of various fixing fluids. FIXATION. Fixing Reagents. — .4 ce^ic Acid.— This precipitates nucleoproteins and, therefore, fixes chromosomes very well, but does not affect the proteins or fats. It causes permanent swelling and distortion of collagenous fibers of connective tissue. Due presumably to its failure to precipitate or otherwise change most of the substance of protoplasm so as to retard its penetration, it diffuses throughout tissues very rapidly. It causes some swelling and leaves the tissue soft. Picric Acid. — Sa,tuTSited aqueous solutions of this chemical precipitate all proteins by forming protein picrates, and its pene- 266 TECHNIQUE tration is less rapid than acetic acid. It causes some shrinkage but does not harden tissues very noticeably. ForiJialin .—Weak solutions (10 per cent) of formalin in water are used as fixing agents. They cause changes in protein by form- ing additive com}:)oinids. The action is slow and the tissues are hardened due to eflfects on the cell membrane. Shrinkage is not an immediate result but may follow during later stages in technique. Alcohol.— This precipitates proteins, the precipitates of nucleo- proteins being soluble in water. It dissolves lipoids but precipitates glycogen. Tissues treated with alcohol shrink and harden and later staining is difficult. Osmium 7V^/-o.i-i(^g.— Solutions of this chemical in water are called osmic acid. This fixes and blackens fat, chondriosomes, and the Golgi apparatus. Its effect upon the remainder of the proto- plasm is one of fixation without precipitation. Shrinkage is slight, penetration is slow and variable, and staining is made difficult. Chromic ^cid. — Solutions of this reagent act as oxidizing agents. It precipitates proteins presumably by forming compounds with them, but leaves fat and lipoids unaflfected. It should be washed out with water, so that interfering precipitates are not formed, as they would be if washed with alcohol. The chromic acid solution causes slight shrinkage and hardening but makes staining with basic dyes easier. Potassium Z)w7?/'o?/?a/f. — Unacidified solutions of this reagent render albumin insoluble but do not precipitate it. It is similar to osmium tetroxide in that it renders proteins insoluble without precipitating them. Chromatin is dissolved, so that it is a poor chromatin fixative. Its action is slow, and tissues should be washed in water after its use. When made acidic, its solutions fix in the manner of chromic acid. Mercuric Chloride. — Satursited solutions of this reagent in water act as precipitants of proteins, have no effect on lipoids, and do not destroy chondriosomes. The solution penetrates quickly and causes some shrinkage. It is necessary to wash it out after short fixation. Crystals tend to form and should be dissolved later by washing in alcohol to which iodine has been added. These are but a few of the numerous reagents in use, and only a brief note is made of their action. For example, it was just stated that acetic acid and picric acid are both useful, but that the first causes swelling of tissue and the second causes shrinkage. By a proper combination these effects may be counterbalanced. A review FIXATION 2()7 of the actions of the reagents noted above incUcates a reason for the protoplasmic conditions observed in fixed tissues. We will now indicate a few fixing fiiiids found useful in general work. Fixing Fluids.— Bouin's Fluid. — The fornuila is as follows: 75 parts saturated aqueous solution of picric acid; 25 parts formalin (40 per cent formaldehyde); 5 parts glacial acetic acid. This is one of the best general fixatives, and in it the effects of the three chemi- cals are well balanced to give a fair preservation of cellular structure. Chromosomes are especially well fixed. Glycogen, fat, chondrio- somes, and the Golgi apparatus are not usually preser\ed. Tissues may be left for long periods in it without harm, but twelve to twenty-four hours is ordinarily an adequate time. There is little or no hardening or shrinkage, and tissues will later stain well. After fixation the tissue may be washed in 50 or 70 per cent alcohol to get rid of the free fixati\e remaining. Flemmimfs Fluid.— The formula is 1 per cent aqueous solution of chromic acid, 15 parts; 2 per cent aqueous solution of osmium tetroxide, 4 parts; glacial acetic acid, 1 cc, or even less, to be added just before using the fixati\'e. This solution is useful for fixing chromosomes, chondriosomes, and fat. The amoimt of acetic acid should be reduced to a few drops for better results with chondrio- somes. The osmic acid fixes the fats and chondriosomes. The acetic acid fixes the chromosomes and aids in preventing shrinkage. The chromic acid fixes proteins and chromosomes. However, fixation may be une\'en, part of the tissue being overfixed, part properly fixed, and the inner portion of the tissue being underfixed. Fixation should extend over a twenty-foin--hour period when acetic acid is present to the extent of 1 part, but the period should be ex- tended to four flays when the acetic acid is reduced and when it is desired to retain chondriosomes. After fixation the tissues should be washed for twelve hours in running water to remove all the free fixative present before going ahead with the technique. Zenker s Fluid.— The formula is 2 grams potassium dichromate; 1 gram sodium sulphate; 5 grams mercuric chloride; 100 cc. water; 5 cc. glacial acetic acid. The acetic acid should be added just before using the fluid. Tissue should be fixed for twelve hours. Long fixation causes formation of crystals in the tissue. After fixation the tissues should be washed in rimning water for several hours, then transferred into 70 per cent alcohol to which iodine has been added. The latter becomes clear, due to the extraction of mercury salts from the tissue. Fresh iodized alcohol should be 268 TECHNIQUE used again until the retention of the iodine color indicates that all free mercury salt has been removed from the tissues. The tissue then may be stored in 70 per cent alcohol. A modification of Zenker's fluid is called Hellys fluid. It has the same formula as the above, but substitutes 5 cc. of formalin for the acetic acid. It gives an excellent picture of protoplasmic structure, but care must be taken in later steps in technique so that distortion through shrinkage does not take place. Chromosomes are not so distinct as with Zenker's fluid, but cytoplasmic structures are well preserved and can be demonstrated by various stains. Helly's fluid must be thoroughly washed out in running water after twelve hours' fixation, and the tissues must also be later immersed in 70 per cent alcohol to which iodine has been added. Formalin Solutions.— A 10 per cent solution serves as a good pre- servative and fixative in the case of delicate tissues that will stand hardening. It has been used as a preliminary reagent in a number of techniques employed for nervous tissue. Tissues fixed in it should be transferred to 70 per cent alcohol after twelve hours' treatment with the formalin solution. Shrinkage often occurs later during paraffin embedding. Carnoys Fluid.— The formula is: 75 cc. of 100 per cent alcohol and 25 cc. of glacial acetic acid. This fixing fluid fixes chromosomes well, precipitates glv'cogen, but usually dissolves chondriosomes and the Golgi apparatus. It penetrates quickly, so that an hour serves to fix soft pieces of 1 cm. in thickness. It combines the effects of two chemicals, the alcohol causing precipitation of the proteins and glycogen of the cytoplasm; the acetic acid fixing the nucleo- proteins and preventing some of the shrinkage and hardening of the alcohol. Tissues should be washed in absolute alcohol after fixation before proceeding to embed. For additional information concerning fixatives the student is referred to the texts cited at the end of this chapter, but for a general treatment of fixation reactions it is recommended that J. K. Baker's monograph on " ( \vtological Technique" be studied. Containers. As containers for the fixative and excised material, it will be found advantageous to use wide-mouthed bottles of 1- or 2-()imce caj^acity i)rovided with cork stoppers. In order that the fixative may reach all ])arts of the surface of the material, it is well to place a small bit of absc^-bent cotton in the bottom of each l>ottle to prevent the tissue from adhering to the glass, and so keep- ing one surface from free contact with the fixing fluid. The bottle HANDLING THE MATERIAL 2()9 should be well filled with the fixative so that there will be many times the volume of the tissue to be fixed. The small amount of water from the tissues should not dilute to any a])])reciable dej^ree the concentration of the fixing fiuid. HANDLING THE MATERIAL. Dissection. — Pieces of tissue should be small. This is important for good fixation. Generally pieces, 0.5 cm. to 1 cm. in thickness, can be used. The length and breadth of the piece should likewise be kept to the smallest practical size. The pieces should be handled as little as possible with dissecting instruments and care should be taken not to compress them during dissection. The operator should keep in mind that he is later going to make sections of these pieces, and should remove material from the desired portion of the organ in the proper manner for longitudinal, cross-, or tangential sections. The selected pieces can be transferred from the animal to the bottles of fixing fluid with a spatula, or lifterl lightly with forceps, without permitting the instriuuents to enter the fluid. If dissection is pro- longed, it is advisable to keep the organs of the freshly killed animal moistened with the proper physiological saline solution (0.85 ])er cent sodium chloride for mammals; 0.7 per cent for amphibians). Foreign Matter. — In fixing parts of the alimentary canal, it is necessary to remove the contents of the lumen. This may be done by washing out with ])hysiological saline before cutting out pieces for the fixing fluid. Or fixing fluid may be forced under gentle pressure into the limien of the canal with a pipette. If the bladder is dilated with urine, the neck of the bladder can be ligated, and then the entire bladder removed and placed in a beaker of fixing fluid for a few minutes to stiffen the wall in the extended position. The bladder then can be opened, emptied, and so cut that fixative can freely come in contact with the internal surface. The stomach can be handled in the same fashion. Or a small strip of stomach wall can be placed on a strip of stitt' paper and thus inunersed in the fixative for a few minutes, and then, when stiffened in the extended condition, it can be removed from the paper and entirely immersed in fixative. Pieces of artery or ner\'e can likewise be placed extended on a paper strip, and this placed in fixative until the pieces are stiff enough to be removed from the paper for further fixation. Heat. — Fixatives are ordinarily used at room temperatures, but the effect is hastened by increasing the temperature. Heat itself 270 TECHNIQUE is a coagulant of protoplasm, and its effect nuist be considered in addition to the action of the fixative itself. It will be found that the final picture obtained by different tem])eratures of the same fixative and the same material will show certain differences, and these modifications should l)e checked carefully. Records. — One of the most important things to remember is to keep a careful record of the material, the organ or part of the organ removed, the age and sex of the animal, the fixative used, the time of fixation, and any modifying conditions, such as health of the animal and method of killing. These details may })e kept in a note-book and corresponding code numbers placed with the fixed material, or the entire information may be recorded on the slip that accompanies the fixed material. The writing on these slips should be done with a soft pencil. To avoid confusion, do not place many tissues together in a bottle. It is wiser to place not more than three small pieces in each bottle so that no difficult^' later arises in identifying them for further steps in the technique. PARAFFIN EMBEDDING. We have indicated the })roper length of time needed for fixation with each of the fluids descril)ed abo^'e. It has also been shown that the fixed tissue should be thoroughly washed free of all fluid fixati^■e remaining. Usually the tissue pieces are preserved in 70 per cent (or NO per cent) alcohol. The next problem is to prepare the material for cutting sections which after proper staining can be studied microscoi)ically for their structural organization. Prior to the development of modern technical processes, the early workers at first held pieces of tissue or organ in one hand and cut thin slices of it free hand with a sharp knife or razor. However, a piece of fresh or fixed tissue "gi\'es" when sections are cut in this manner, and the material is more or less crushed. This is avoided if the tissue is infiltrated with paraffin. Paraffin is solid at room tem- peratures, but a simple yet somewhat time-consuming technique enables one to satisfactorily impregnate the tissue piece with ])araffin. This is accomplished as follows: Paraffin is not miscible with water, so that all water nnist be removed from the tissues. To accomplish this, the 70 per cent alcohol in which the piece of tissue has been ke])t is discarded and SO ])er cent alcohol replaces it. After an hour or so, the SO per cent is discarded and replaced with 95 i)er cent alcohol. An hour PARAFFIN EMBEDDING 271 or so \atvv this is (•lianii;e(l to 100 per criit alcohol. This has no water in it, and when (hfi'used throujijh the tissue, water will have been removed. This process of dehydration must not be too rapid or distortion, caused by too rapid withdrawal of water, may occur. The alcohol may also exert an effect on sonic of the protoplasmic substances which have been preserved but not precipitated or coagulated by the fixati\es. Tissues should not remain too long in the higher alcohols, /'. e., 95 and 100 ])er cent. Paraffin does not mix with alcohol; therefore, some other common chemical must be employed that is miscible with alcohol and paraffin which can be used to transfer the object from alcohol to paraffin. Xylol or benzene can be used for this purpose. Xylol is preferable, since it is not so inflammable as benzene, although benzene causes less shrinkage than xylol. Therefore, transfer the tissue from 100 per cent alcohol to a mixture of equal parts of 100 per cent and xylol for a certain period (one hour) ; then change to pin-e xylol for one to two hours; then pure xylol and paraffin for a similar period. If the paraffin used has a melting-point of 50'' to 52° C, then the mixture of xylol and paraffin must be kept at a temperature not far below this to keep the mixture in solution and so enable it to dift'use all through the tissue. It should be remembered that there must be no trace of water left in the tissue. So far it is infiltrated with a mixture of one-half xylol and one-half paraffin. This mixture is discarded and the bottle is filled with pure melted paraflSn at a temperature just above its melting-point, i. e., 55° C. In order to maintain the paraffin in a liquid condition, the supply of melted paraffin and the specimen bottles containing tissues being embedded are kept in a constant temperature oven, sometimes called a paraffin oven. After a certain length of time (one hour) the first supply of paraflin is discarded and rei)laced with fresh paraffin and ])laced l)ack in the oven for another hour. It is essential to remove all traces of xylol and to have the pure paraffin diffuse all through the tissue. It is even advisable to change the paraffin once more for another hoiu- in the oven. A pa])er boat or a glass dish (Syracuse dish) coated with glycerin is now filled with pure melted paraffin, and the tissue piece poured with its paraffin or transferred to this dish with warmed forceps or spatula. The tissue piece is quickly and properly oriented with a view to the type of sections to be made later. Then a surface film of solid paraffin is produced by blowing across the top of the dish, and the container is gently immersed in cold water for a time, until 272 TECHNIQUE the paraffin hardens thronghont homogeneously and quickly. In a few minutes the hardened paraffin block can be removed and stored indefinitely in this form or prepared for sectioning. If the weather is very warm, or if the technique is carried out in a warm room, one would use a higher melting-point paraffin, while a lower melting- point ])araffin is suitable for use in winter or under colder conditions. If very thin sections are required, one must use a harfler (higher melting-point) paraffin than if thicker sections were to be made. Ordinarily it is wise to avoid heating the tissue any higher than absolutely necessary in order to avoid the hardening and shrinking effect of heat, so that 58° C. represents a maximum temperature for the paraffin bath. In the foregoing account of dehydration we passed from 95 per cent alcohol to 100 per cent alcohol and from this to xylol. Since 100 per cent alcohol is expensive, and since it tends to harden tissues, it can be dispensed with by the use of aniline oil. This is cheaper and does not harden or shrink the tissues. The following tables exi^lain the two procedures: It is understood that the tissue has been properly fixed, washed free of fixative, and has been stored in 70 per cent alcohol. Procedure. Absolute Alcohol Method. Aniline Oil Method. 80 per cent alcohol, one hour. §—80 per cent + i aniline, one hour. 95 per cent alcohol, one hour. 3—95 per cent + f aniline oil, one hour. 100 per cent alcohol, one hour. Aniline oil (until translucent), one hour. ^ — 100 per cent + J xylol, one hour. | aniline + i xylol, one hour. Xylol, one hour. Xylol, one hour. Fresh xylol, one hour. Fresh xylol, one hour. I xylol + 5 melted paraffin, one hour and kept in warm place so that the paraffin remains melted. Pure melted paraffin (about 52° C. m.p.) in the paraffin oven, one hour. Change the paraffin and keep specimen in oven, one hour. Change the paraffin again and keep specimen in oven for about one hour. Embed as directed above. Toluol is an excellent substitute for xylol and tissues can be left in it for longer periods. Furthermore, it is more volatile and therefore more readily lost during paraffin transfers. CELLOIDIN EMBEDDING. For material which is tougher, or which demands a technique which does not involve heating, a slower method of embedding in celloidin instead of paraffin has been devised. It has the advan- tage of causing less shrinkage and distortion but does not permit such thin sections to be easily cut. Only single sections are made at a time, and so the handling of large numbers of sections is more CELLOIDIN EMBEDDING 273 tiine-consuniing tliaii is the case witli paraffin sections. Solutions of celloidin in an cthcr-alcohol mixture are utilized in celloidin infiltration. Celloidin is furnished in small solid i)ieces. Remove all the pieces from a 1-ounce bottle, dry them thoroughly, and place in a glass-stoppered bottle, adding about 150 cc. of absolute ethyl alcohol and 150 cc. of sulphuric ether. Stopper the bottle and keep the stopper free of the solution at all times. It will take several days before the celloidin has completely dissolved. This will make a stock solution of concentrated celloidin, which we will call the No. 1 solution. When it is in complete solution, make stock solution No. 2 l)y diluting part of No. 1 with about three times as much of equal parts of absolute alcohol and ether. Also make stock solution No. o by diluting solution No. 1 with about ten times as much of equal parts of al)Solute alcohol and ether. Keep a supply of each of these three solutions on hand, taking care that each is kept in a tightly stoppered bottle, as evaporation of the ether- alcohol soon changes a thin solution to a thicker one. Tissue in Bouin's or Zenker's fluid can be embedded in celloidin as well as those especially designed for nerve study. It is well to use thin pieces, i. e., about 0.3 cm. thick, because infiltration pro- ceeds slowly and with difficulty. Each successive fluid presently named must penetrate the tissue piece, and as the size is increased the time needed must be extended. It is suggested that the student at first use a piece of soft tissue, such as the liver, about 0.5 by 0.5 by 0.3 cm., and follow the longer time period given in the following table. We will suppose that the tissue has been fixed in Bouin's fluid, washed, and taken from 70 per cent alcohol, where it has been preserved. The process takes place at room temperature and in bottles that can be tightly stoppered. The time can be shortened by keeping tightly covered bottles at a temperature of 40" C. Celloidin Embeddixc; Method. From 70 per cent alcohol to 95 per cent, for two hours. From 95 per cent to fresh 95 per cent alcohol, for one hour. From 95 per cent alcohol to 100 ])er cent alcohol, for two hours. From 100 per cent alcohol to fresh 100 per cent, for tweh'e to twenty-four hours. From 100 per cent alcohol to one-half absolute alcohol + one- half ether, for twelve to twent\'-four hours. IS 274 TECHNIQUE From ether-alcohol mixture to celloidin No. 3 (thin), for three to four days. From celloidin No. 3 to No. 2 celloidin (medium), for four to six days. From celloidin No. 2 to No. 1 celloidin (thick), for fi^'e to eight days. At the end of this time the tissue should be completely saturated with celloidin. We can proceed from this point in two ways: Fashion a little paper receptacle and fill it with celloidin No. 1. Add but a few drops at first and wait a moment until they thicken, add a few more drops, and then fill the receptacle. The tissue piece is then placed in the box and arranged properly, with a view to plane of sections to be cut later. The surface of the celloidin rapidly forms a film when exposed to the air. Evaporation of the solvent (ether-alcohol), with consequent hardening, should be per- mitted to proceed slowly until the celloidin mass is like a heavy gum in consistency. The paper receptacle is now carefully trans- ferred to a covered glass dish containing chloroform, where hardening is completed. The chloroform causes the celloidin to harden suffi- ciently in one to two hours, when the paper box and excess celloidin can be cut away and the embedded block can be placed in a bottle of one-half 95 per cent alcohol + one-half glycerin until ready for cutting. The other method both embeds and mounts the tissue preparatory to sectioning. Vulcanized fiber blocks formed to fit the clamp of the microtome should be used. Do not use wood blocks. The solvent in the celloidin mixture extracts oils from wood and these discolor the preparation. A strip of stiff paper is fastened about one end of the fiber block so that a receptacle is formed at this end. Celloidin No. 1 is added, drop by drop, to this receptacle, covering the bottom of it with a relatively solid coat which is allowed to thicken before filling the receptacle with celloidin from the specimen bottle. The tissue is transferred also and oriented in the celloidin which covers it on all sides. The celloidin is then permitted to harden somewhat in the air, as in the first case, and then the block Avith the celloidin-filled receptacle is transferred to a covered dish of chloroform. After hardening in chloroform the block is stored, as in the first procedure. If the first method was used it is necessary to fasten the celloidin block to a fiber block before cutting sections. To do this thick celloidin (No. 1) is poured over one end of the fiber block and over SECTIONING 275 one end (bottom) of the celloidin block. The two are pressed together; the celloidin at the junction is hardened in the air until it is thick, then in the chlorofonn as before. SECTIONING. As soon as material is properly embedded one may proceed directly to cutting sections for staining. ^Machines, called micro- tomes (from the Greek, "to cut small"), have been devised and can be regulated to cut sections of desired thickness easily and uni- formly. Two general type^ of microtomes are in common use: one, working on a rotary principle, is called a rotary microtome (Fig. 163), and the other, cutting by sliding mo^'ements, is known as the sliding microtome. The rotary type is suited for cutting Fig. 163.— Illustration of the rotary microtome. (Courtesy of Spencer Lens Co.) paraffin embedded material, while celloidin material is sectioned with the sliding microtome, although the latter may be used for paraffin material also. Rotary Microtomes and Paraffin Sections. — The illustration presents the princij^al features of this tyi)e of microtome. The following information should be sujjplemented by classroom demonstration in the use of the machine and also by considerable actual experience in section cutting by each student. Small metal discs or wooden blocks are furnished to which the i)araffin block may be attached. These supports can be then fastened into a holding device in the microtome. We will suppose that we ha^•e a disc of paraffin containing three pieces of embedded tissue. One of these is cut out of the disc bv 276 TECHNIQUE means of a warm scalpel blade, leaving plenty of paraffin around the tissue. To fasten this paraffin block to metal disc or wood block, an old scalpel blade is heated so that it will easily melt paraffin. The blade is then held against the metal disc or paraffined end of a wood block, and then the paraffin piece is pressed against the other surface of the blade. The blade is withdrawn and the melted paraffin thus produced hardens and seals the paraffin block to the metal or wood holder. It is advisable to place mounted tissue in cold water for a minute or two to completely harden the paraffin. The next step is to trim the paraffin block containing the tissue. Some of the extra paraffin about the object is cut away, so that the piece is in the shape of a truncated pyramid, with the base toward the wood block or metal. The paraffin at the free end is trimmed down until little is left between the end face and the tissue. The free face should be rectangular or square in form, and the angles between adjacent edges should be right angles. (Fig. 164.) It is an easy matter to prepare a considerable number of small wood blocks, and these can be furnished to all the students who can proceed to mount embedded material and have it ready for sectioning. Or if one has a good many paraffin blocks to section, it is more efficient to mount all on wood blocks at one time and store them in boxes properly- labelled for sectioning later. Storing paraffin blocks in water a week or more before cutting makes cutting easier and prevents brittleness and cracking of the tissue. The clamp holding the mounted ])araffin-wood block is attached to a movable part of the microtome that is connected with a finely threaded screw. The turn of the wheel tiu-ns the screw and ad- vances the part holding the paraffin block. It will be noted that in this ])rocess the paraffin block rises and falls. Each machine has a setting de\'ice by which the thickness of the sections can be Fig. 164. — Diagram illustrat- ing paraffin block ready for sec- tioning. SECTIONING 277 arranged. The settinji; device has a rather wide range of adjust- ment. Sections 10 microns thick are made in general work in microscopic anatomy. The special microtome knife is held in a clamp that can be moved by hand, but must be locked rigidly in place while sectioning. Suppose the setting device is adjusted for sections 10 microns thick, and the wheel is turned. Between the time that the paraffin block passes above the knife edge and then descends to it on the down stroke, the paraffin block has advanced forward exactly 10 microns, but does not advance after the tissue passes below the knife edge. So a section 10 microns thick is cut from the anterior face of the paraffin block each time on the down stroke. However, the ])osition of the paraffin l)lock must be correct. The trimmed end should have the form of a rectangle or square. When the knife is placed in the knife-holder, the back face of the knife should make a slightly acute angle with the plane of the table. The lower edge of the face of the paraffin block should be exactly parallel with the edge of the knife. The knife-holder should be advanced so that the edge of the knife is nearly in contact with the descending block of tissue and then locked. The wheel of the machine is then turned and sections begin to be cut. If conditions are correct, each section will remain with its upper edge just along the front cutting edge of the knife. When the next section is cut, its lower edge will fuse with the upper edge of the previous section because the friction of cutting melts the paraffin along this line. Thus a ribbon of sections is formed. One could cut a ribbon many feet in length. However, it is more convenient to make one about 10 inches in length and then transfer this to a shallow paper box. To do this, the outer end is supported with a section-lifter held in the left hand while cutting. When the desired length is cut, the end next to the knife is carefully lifted off with a scalpel or section- lifter and the length of ribbon placed carefully in the paper box. When the next ribbon is cut, it is laid alongside the first in the box and so on, until the desired number of sections are obtained. If one desires to study all parts of an organ, such as the submaxillary gland, it is necessary to save all the ribbon sections and all the sections can be mounted in regular order, thus making a complete series of slides covering the entire microanatomy of that organ. Ribbons of sections stored away in shallow drawers or paper boxes can be kept indefinitely provided they are stored in a cool, dust- proof place. If kept in a warm room or one which becomes very warm on hot summer days, there is a tendency for the thin paraffin 278 TECHNIQUE to melt, and the ribbons may become attached to the Iwttom of the receptacle in which they are stored. The Sliding Microtome and Celloidin Sections. The accompanying illustration presents an idea of a present-day example of the sliding microtome. (Fig. 165.) The knife is mounted in a holder that fits over the smooth surface of the sliding device. The position of the knife may be fixed by the screws that clamp down on the handle. The fiber block with the attached celloidin block con- taining the embedded tissue is fastened into the clamp at one Fig. 165.— Illustration of a sliding microtome. (Courtesy of Spencer Lens Co.) side of the slide over which the knife is carried. The clamp holding the embedded block is connected with a device which is essentially a fine-threaded screw to which a micrometer is attached. When the micrometer disc is properly turned a certain distance, the screw lifts up the embedded block a certain definite distance above the cutting edge of the knife. The knife must be arranged at an oblique angle with the line of the slide carriage. Both knife and embedded block are kept moistened with 70 per cent alcohol through the sectioning process. The knife is drawn past the celloidin block with the right hand. One can determine by means of the micro- meter disc how far to turn the screw, and so determine the thickness SECTIONING 279 of the sections to be cut. The strokes of the knife should be smooth and rai)id. The knife should have been correctly sharpened to secure f^ood sections. Sections of from 10 to 30 microns are usually cut, but with practice thinner ones may be obtained. Single sections are obtained by this method, and these must be transferred with a camel's-hair brush as they are cut to a dish containing 70 per cent alcohol. Mounting Paraffin Sections. After paraffin sections have been cut and transferred to a paper box, the next step is to attach them to slides. The sections as they come from the microtome are usually slightly compressed or wrinkled, due to the pressure as they cross the knife. This must be remedied when the sections are attached to the slides. In the first place the slides must be thor- oughly clean so that no dirt or grease particles are on their surfaces. They can be cleaned by boiling in soap and water, thoroughly rinsed in water, then rinsed in 95 per cent alcohol, and dried with a clean cloth free from lint or dust. Next, the sections must be made to adhere to the slide to permit the subsequent steps of the staining technique. A solution of equal parts of carefidly strained egg- white and glycerin is used as an adhesive. A small amount of thymol or methyl salicylate added to the adhesive mixture prevents disintegration by bacterial action. A common method of applying the egg-albumen adhesive is as follows: A small drop about the size of the head of a pin is placed in the center of one surface of the slide, and this drop is carefully smeared over the surface with the ball of the little finger that has been thoroughly washed. A drop or two of distilled water is then added to the surface. Then a small piece of ril)bon containing one or two sec- tions of the tissue is cut from the long ribbon with a scalpel and transferred to the water over the egg albumen on the slide. It is advisable to use water that has been boiled (and cooled) to avoid bubbles gathering between the slide and the sections. The ribbon piece should be arranged parallel with the length of the slide. Two or even three small ribbon pieces may be arranged alongside each other. The same surface that is upward when the ribbon comes from the microtome should be upward on the slide. The under side is more shiny than the upper. After the ribbon pieces have been arranged, the slide can then be transferred to a warming table (Fig. 166), the surface of which is kept at a temj^erature of about 45° C. or a little less. More water can be added to float the sections but not enough to run off the slide. The gentle heat warms the 280 TECHNIQUE paraffin enough so that the surface tension of the water will pull the sections out flat. Sometimes sections are badly folded, and in that case one can carefully pull them out flat when the slides are on the warming table. However, such sections are never as satisfac- tory as those which are comparatively flat when received from the microtome. The slides can be left on the warming table until all the water has evaporated or the excess water carefully- removed after the tissue is expanded. Care should be taken that no air bubbles are left under the sections, since the sections are free from the glass at such points and may later fall ofi' the slides or make Fig. 166.— Illustration of a warming tabic. (Courtesy of E. Lcitz & Co.) une^'en places in the stained and mounted sections. The heat used in flattening the section should not be great enough to liquefy the paraffin of the ribbon pieces. A second scheme for fastening sections to slides is to make a solution of 1 ]jart of egg-albumen fixative to 100 parts of cool boiled water. This thin adhesive is added directly to a clean slide, with a pipette, and the ribbon pieces floated on it; the remainder of the process is similar to the foregoing. A third method is to fill a large shallow vessel with water which has been warmed to about 45° C. Small pieces of ribbon are floated on the water. It will be noted that they flatten out immediately. Then a clean slide smeared with egg-albumen fixative can be lowered under such a ribbon piece to remove it. After draining away the excess water and arranging the section, the slide can be set aside until all the water SECTIONING 281 has cNuporated. (Jood results arc ohtaliied from badly wrinkled sections hy this method. In all three cases, the slides should be set aside in a dry place until all the water has evaporated l)efore staining; usually this should be al)out twent\-four hours. Submaxillary Gland - Cat Longitudinal Section Series A-11 Bouin 3/30/24 H. & E. # 4= # ¥ «# '#>" # Fig. 167. — -Diagram of a iircparation of serial sections. Serial Sections. After becoming proficient in cutting sections, the student may wish to cut an entire organ into sections and mount these in order. When finally ])repared one can study the micro- anatomy of the organ from one end to the other. Usually trans- verse or longitudinal sections are made. If there is a right and left gland, cross-sections can be made from one and longitudinal from the other. The attempt should l)e given up if the tissue does not section well. We will suppose that it has been possible to make 282 TECHNIQUE complete ribbon cross-sections of the entire organ, and these have been placed in order in the paper boxes. If many sections are to be mounted, the student can use larger slides. After the slide is labelled and the albumen added, then, beginning at the front end of the series of sections, one removes a strip less than 2 inches (1| inches) in length and carefully places this along the edge of the upper surface of the slide, as indicated in the diagram. (Fig. 167). Then adjacent to this, the next strip, and so on. Leave an empty space, about 0.5 cm., along each lateral margin. Place the slide on the warming table, add water, orient, and dry as in single moimts. The number of sections one can get on a slide depends on the size of each section. After completing the mounting of Slide 1, proceed with 2 and remember to label it. Proceed until all the sections are mounted. STAINING OF PARAFFIN SECTIONS. Thin sections of fresh living material examined with the aid of a microscope appear homogeneous, since there is little optical differ- entiation of cellular structures. Similar examination of fixed tissues which have been cleared reveal more details of structure and organization. These are brought out most distinctly and definitely, however, by the proper treatment of the sections with various dye solutions. The color of the dye or stain is adsorbed, absorbed, or chemically held l)y the different elements of the cell protoplasm in a characteristic manner. Early studies revealed the fact that the nucleus is acid in reaction and has an affinity for basic dyes, while the cytoplasm is basic and has an affinity for acid dyes. When two dyes, one acid and one basic, are used on tissues, the nuclei and the cytoplasm are differently colored, so that a differential staining effect is obtained. Differentiation of various protoplasmic ele- ments has been the aim of munerous staining techniques, so that it is now possible to employ special processes to demonstrate such cellular structures as centrioles, cell mem})ranes, chondriosomes, Golgi apparatus, secretion granules, fat droplets, nucleoli, chromo- somes, chromidia, glycogen, elastic fibers, Xissl bodies, and many others. These s})ecial techniques can be experimented with after the student has become familiar with the more conunon general methods used to demonstrate tissue organizations. For this purpose we will describe in some detail a few general techniques. Progressive Staining. Tii ])rogressive staining the object is to treat the sections with the stains until the tissue has the ])ro])er STAINING OF PARAFFIN SECTIONS 2S3 color. This is deteriniuecl by obser\'ing the progress of the staining under the microscope at short intervals during the procedure. It is not advisable to stain too deeply, since later removal is never alto- gether satisfactory, although it is possible. In the following method we will employ Harris's hematoxylin as a nuclear stain and eosin as a cytoplasmic stain. After preparing the nuclear and cytoplasmic stains, it is necessary to assemble a series of jars just large enough to hold the slides and in sufficient numbers to contain all the reagents needed. Shell vials, a little over 1 inch in diameter and about 4 inches long, are useful for the beginner. The Coplin jar is a more useful vessel, since it can accommodate at least fi^'e slides at one time. Fourteen of these jars will be foimd adequate. The fluid in each should extend about 2 inches from the bottom, so that the upper third of the slide will be free of fluid. Following is a list of jars and the fluid in each: (1) Xylol; (2) aniline oil; (3) 95 per cent alcohol; (4) 70 per cent alcohol; (5) 50 per cent alcohol; (6) distilled or tap water; (7) hematoxylin stain (one- half stock solution, one-half water); (8) water; (9) 50 per cent alcohol; (10) 70 per cent alcohol; (11) eosin in 70 per cent alcohol; (12) 95 per cent alcohol; (13) aniline oil; (14) xylol. When using paraffin sections the paraffin must be removed before proceeding, since paraffin does not mix with either alcohol or water, and the dyes are dissolved in the latter. The slide is placed in the xylol jar for about three minutes, during which the paraffin will dissolve. After this step the sections should never be permitted to remain out of the fluids until permanently prepared. After the xylol, the slide is drained of free xylol and placed in the aniline oil jar for two minutes, then drained and placed in the 95 per cent alco- hol for two minutes, and similarly in the 70 per cent, 50 per cent, and water for two minutes each. The slide is placed in the hematoxylin stain for three to five minutes, then rinsed in water and examined under the microscope. The section should have a rich purple or blue appearance. Micro- scopic examination should show a purplish chromatin network in the nuclei of the preparation. If the general appearance is a light lavender, the section is imderstained. It should then be placed in the stain for a moment or so more and reexamined. If the section appears dark purple it is probably overstained. In progressive staining it is desirable to stain until just the right effect has been produced and then to stop further staining by placing the section 284 TECHNIQUE in water. If overstained, the excess can be extracted by placing the sHde in 70 per cent alcohol to which a drop or so of hydrochloric acid has been added. However, this is not satisfactory with this type of stain. We will suppose that the correct staining effect has been attained without extraction. After rinsing in running tap water for fifteen minutes the slide is placed in 50 per cent alcohol for a minute or so, then in 70 per cent alcohol for two minutes. It is next placed in the cytoplasmic stain, such as 70 per cent alcohol and eosin, for thirty to fifty seconds. It should be removed and rinsed in 95 per cent alcohol and examined for the cytoplasmic stain effect which should not be too deep in color. Then the slide is placed in aniline oil for two minutes, drained, and placed in xylol for two minutes, and placed in the first xylol jar for two minutes more. The purpose of the two xylol baths is to prevent carrying over any aniline which would stain the final preparation. The student should have at hand a bottle of Canada balsam, or gum damar, dissolved in xylol to a relatively thick viscous consistency but thin enough to flow easily. Clean and thoroughly dry cover-glasses should also be ready. A pair of fine forceps will also be needed. The slide is then taken from the second xylol jar, the undersurface wiped dry, placed on filter paper on the table and 1 or 2 drops of the balsam placed over the section. A cover-glass is lifted with the forceps and carefully lowered over the section. It is well to let one edge of the cover-glass rest at the side of the slide and then to let it fall gently by its own weight over the section, thus preventing air bubbles from collecting under the cover. After the section has thus been covered, it should be placed on the warming table or in a warm oven for some hours in order that the xylol in the balsam mixture may evaporate and thus harden the gum. The hard gum has the same optical refraction as the slide and cover, and so allows light to pass through without refraction and also seals the cover to the slide. After the gum has hardened the slide can be cleaned by di])i)ing in toluol, draining, and permitting to dry. However, the cover should not be rubbed until the gum has thoroughly hardened. Regressive Staining. In this technique the sections are far over- stainetl at first and then excess stain is extracted until just the right effect is produced. An excellent example of a well-tried stain for tiiis ])urj)()se is the so-called Heideiihain's henuitoxylin. It is highly recommended for differentiation of mitotic figures and is, therefore, indicated for studies in cell division, spermatogenesis, and oogenesis. Two solutions are used, a mordant and the stain. STAINING OF PARAFFIN SECTIONS 285 To proceed with staining, the slides are phieed first in xylol and brought down, as before, through aniline oil to water. The slides are then placed in a 4 per cent iron alum solution for at least an hour for thorough mordanting. Then the slides are washed thoroughly in water and placed in the hematoxylin stain for one to several hours. They will appear deep black if the staining has been effec- tive and no details are at all evident upon microscopic examination. After rinsing in water they are ])laced in a 2 per cent iron alimi solution for extracting excess stain, and after a minute or so exam- ined under the microscope. It is an advantage to have the extrac- tion take ])lace slowly. They should he rinsed, examined, and returned if the stain is still too dee]). The de-staining may also be carried out under the microscope in a glass Petri dish containing the 2 per cent alum. The chromatic network in the nucleus should appear sharply outlined. If one has a metaphase stage in mitosis, the chromosomes should ajipear clear-cut. When the extraction has gone far enough, it can l)e stopped by washing the slides in water to remove e\'ery trace of the free alum solution. Then the slides are "run up" through oO i)er cent alcohol to 70 ])er cent alcohol. They can be counterstained in a solution of orange G or eosin in 70 per cent alcohol for thirty to fifty seconds, as in the preceding techniques. They are then transferred to 95 per cent alcohol, aniline oil, and xylol, as before, and later mounted. If hematoxylin is not used in a slightly alkaline condition it acts partly as an acid dye, and the cytoplasm is stained reddish and the nuclei are poorly stained. The Mallory Connective-tissue Stain. — This stain gives a colorful effect with sections that contain considerable connecti\'e tissue, muscle, and epithelium. Two solutions are needed. Solution A is a 0.5 per cent solution of acid fuchsin in distilled water. Solu- tion B is 0.5 gram aniline blue (water solution); 2 grams orange G; 100 cc. of 1 per cent aqueous solution of phosphomolybdic acid. The stain works better with tissues that have been fixed in Zenker's fluid. x\fter the sections have been brought down, as before, from xylol to water, they are placed in Solution A (acid fuchsin) for about four minutes. Then this is drained off" and the slides are placed at once in Solution B for about eight minutes. They are then rinsed several times in 95 per cent alcohol; it is better to use more than one jar of 95 per cent. Then proceed to aniline oil, xylol, and mount as before. Connective tissue, muscle, and epithelium stain differentially, the prominent colors being red, blue, and yellow. 286 TECHNIQUE Staining With Silver Nitrate.— The mesothelial covering of pieces of fresh mesentery can be stained in bright simhght with a 1 per cent solution of silver nitrate in distilled water as follows. Remove pieces of the fresh mesentery and spread carefully on a glass slide or stiff moist paper, then place in the bottom of a shallow glass dish. Wash in distilled water. Then bathe the pieces of tissue in the silver nitrate solution for about five minutes. Wash all free silver nitrate away with water and place the tissue in the water in brilliant sunshine until the membrane turns a brown color. If it is desired to stain the nuclei, this can be done by treating with Harris's hema- toxylin. After passing through the various grades of alcohol, aniline oil, xylol, the tissue can be mounted on a slide in balsam. Care should be taken that the tissue does not wrinkle or fold. STAINING CELLOIDIN SECTIONS. We will suppose that a piece of tissue fixed in Bouin's or Zenker's solution has been embedded in celloidin and that sections of such a piece have been cut with a sliding microtome and collected in a dish in 70 per cent alcohol. We can stain these sections in the Harris hematoxylin and eosin combination as follows: Discard the 70 per cent for 50 per cent alcohol and change this to water, each bath lasting a few minutes. After the sections have been washed in water, the water can be discarded and the dish filled with hematoxy- lin stain. A section can be lifted out occasionally to determine the degree of staining. When the stain has taken sufficiently, it is replaced with water. From this they pass to 50 per cent alcohol; to 70 per cent alcohol; to eosin or orange G for counterstaining ; to 95 per cent alcohol; to aniline oil; to cedar oil or oil of origanum. We merely decant off the liquid in the dish each time and then replace it with the next liquid. At the last step transfer a stained and cleared section to a slide with a spatula and, having added 1 or 2 drops of Canada balsam, apply a cover-glass. PREPARATION OF STAINS. Harris's Hematoxylin.— To prepare this stain make U]) two solutions independently : A. Add 1 gram of hematoxylin crystals to 10 cc. of 100 i)er cent ethyl alcohol. B. Add 20 grams of potassium or aiiimoniuin alum to 200 cc. of distilled water. To get the alum into sohitiou it is necessary to heat to boiling. STAINING CELLO I DIN SECTIONS 287 When the ahim sohition is at the l)oihii^f-point add the hema- toxyhn sohition. Satisfactory staining sohitions have been obtained by continuing to heat the sohition for about fifteen minutes after so mixing, then setting stain aside for a week to age in a loosely stoppered container before adding a few thymol crystals to act as preservative. A second method is used to make the dye ready for immediate use. In this case, 0.5 gram of yellow oxide of mercury is added as soon as the hematoxylin is added to the alum, and the solution remo^•ed from the heat. The flask is immersed in a larger vessel and cooled as quickly as possible. Before use, in either method of preparation, the solution should be filtered and a few crystals of thymol be added. This is the stock solution and may be kept for long periods in the well-stoppered bottle and improves after several weeks' aging. The yellow oxide of mercury employed to ripen the stain does not keep well when dried, so it has to be made at the time of prepa- ration of the solution. To prepare it, make a saturated solution of mercuric chloride by adding about 8 grams of the salt to 100 cc. of water and boil. Add to the saturated sohition thus formed, about 40 grams of potassium hydroxide. A heavy precipitate is formed. Filter the solution and retain the precipitate, then wash the precipitate with distilled water until no chlorine can be detected when silver nitrate is added to the water that has passed over the mercury precipitate. This yellow paste may be kept if the filter paper upon which it is deposited is kept immersed in water. When added to the hot hematoxylin mixture it is necessary to merely approximate the weight of the paste but excess should be avoided. The stock solution may be diluted with equal or twice its volume of water before use, depending upon the time sections are to be left to stain. Heidenhaiiis Hematoxylin . — In this method of staining, the mordant and dye are kept separate, not mixed, as in the case of Harris's hematoxylin. Two solutions are prepared. Solution A is made by dissohing 4 grams of crystals of ferric alum (iron ammo- nium sulphate) in 100 cc. of distilled water. Solution B is prepared by dissolving 10 grams of certified hematoxylin crystals in 100 cc. of 100 per cent ethyl alcohol and then allowing to age, for several weeks at least, in a loosely stoppered container. This stock solution of hematoxylin should be diluted with water to 0.5 per cent for use in staining. Allowing the dilute solution to stand before using increases its eftectiveness. For de-staining the stained sections a 2 per cent solution of the mordant may be used. 288 TECHNIQUE Eosin.— To prepare this cytoplasmic stain, 1 gram of eosin powder is added to 100 cc. of 70 per cent alcohol. This solution may be diluted by an equal or greater volume of the 70 per cent alcohol, if it is desirable to have the staining effected more slowly. Orange G and Erythrosin. — Both of these dyes may be prepared in the same manner as eosin. A very satisfactory combination of their effects may be obtained by mixing about one-third of erythrosin with two-thirds of an orange G solution. So combined, sections containing connective tissue and muscle have the former stained orange and the latter a reddish color. REFERENCES. Anderson, J. 1933. How to Stain the Nervous System: A Laboratory Handbook for Students and Technicians, Edinburgh, E. and S. Livingstone. Baker, J. R. 1933. Cytological Technique, London, Methuen & Co. Carleton, H. M. 1926. Histological Technique, London, Oxford Univ. Press. Conn, H. J. 1925. Biological Stains, Geneva, N. Y. Gage, H. S. 1925. The Microscope, Ithaca, N. Y., Cornstock Publ. Company. Galigher, a. E. 1934. The Essentials of Practical Microtechnique, Berkeley, Calif., A. E. Galigher, Inc. Lab. of Microtechnique. Gatenby, J. B., and Cowdry, E. V. 1928. Lee's Microtomist's Vade- mecum, 9th ed., London, J. and A. Churchill. Guyer, M. F. 1927. Animal Micrology, Chicago, 111., University of Chicago Press. McClung, C. E. 1929. Handbook of Microscopical Technique, New York, Paul B. Hoeber, Inc. APPENDIX. Current References.— The student should refer to the available current issues of the following journals to follow the results of the more recent contributions to microscopic anatomy and the nearly related subjects. American Journal of Anatomy. Anatomical Record. Biological . 1 hstracts. Biological Bulletin. Journal of Morphology. Science. Stain Technology. Quarterly Remeui of Biology. Zoologica I Record. Anatomical Texts.— The following are a few of the texts which will be found useful for reference: Adams, A. L. 1933. An Introduction to the Vertebrates, New York, John Wiley & Sons. Daniel, J. F. 1934. The Elasmobranch Fishes, 3d ed., Berkeley, Calif., Univ. California Press. Borland, W. A. N. 1932. The American Illustrated Medical Dictionary, Philadelphia, W. B. Saunders Company. EcKER, A. 1889. The Anatomy of the Frog, Oxford, England, Clarendon Press. Goodrich, E. S. 1930. Structure and Development of Vertebrates, New York, The Macmillan Company. Hyman, L. H. 1922. A Laboratory Manual of Comparative Vertebrate Anatomy, Chicago, Univ. Cliicago Press. KiNGSLEY, J. S. 1926. Outline of Comparative Anatomy of Vertebrates, Philadelphia, P. Blakiston's Son & Co. Noble, K. G. 1931. Biology of the Amphibia, New York, McGraw-Hill Company. Walter, H. E. 1928. Biology of the Vertebrates, New York, The Mac- millan Company. Histological Texts. — The following textbooks of histology will be found useful for additional reference and illustrations: Addison, W. H. F. 1927. Piersol's Normal Histology, Philadelphia, .1. B. Lippincott Company. Bremer, J. L. 1927. A Textbook of Histology, Philadelphia, P. Blakis- ton's Son & Co. 19 290 APPENDIX CowDRY, E. V. 1928. Special Cytology, New York, Paul B. Hoeber, Inc., 2 vols. 1934. A Textbook of Histology, Philadelphia, Lea & Febiger. Dahlgren, U., and Kepner, W. A. 1928. Principles of Animal His- tology, New York, The Macmillan Company. Elwyn, a., and Strong, O. S. 1932. Bailey's Textbook of Histology, 8th ed., Baltimore, William Wood & Co. Jordan, H. E. 1931. A Textbook of Histology, 5th ed.. New York, D. Appleton & Co. Lambert, H. E. 1930. Guide to Study of Histology and Microscopic Anatomy, Philadelphia, P. Blakiston's Son & Co. Maximow, a. a., and Bloom, W. 1934. A Textbook of Histology, 2d ed., Philadelphia, W. B. Saunders Company. Ramon-Cajal, S. 1933. Histology, Baltimore, William Wood & Co. Sabotta, J. 1930. Textbook Vol. 1 and Atlas Vol. 2 of Human Histology and Microscopic Anatomy, trans, by W. H. Piersol, New York, G. E. Stechert & Co. Schaper, E. S. 1934. Essentials of Histology, 13th ed., Philadelphia, Lea & Febiger. PREPARED SLIDES. For the introductory work in cytology and embryology, appro- priate preparations are utilized. The following list of slides are available for our students either in individual kits gi^'en to them for use throughout the term or in accessory kits for use in the laboratory: Embryos of the frog, several sections of late stages. Embryo of the chick, transverse sections of seventeen-segment stage in various regions. Fetus of a bat, longitudinal sections. Fetus of a pig, cross-sections. Simple squamous epithelium, mesothelium of mesentery treated with silver and hematoxylin. Retina of the eye, transverse section for pigmented epithelium. Connective tissues: Umbilical cord of the i)ig at birth, transverse section. Umbilical cord of the fetal cat, transverse sections. Reticular tissue in sections of lymph nodes from which lympho- cytes were partially washed. Reticular tissue in the liver or spleen im])regnated with silver. Subcutaneous tissue spread of the cat and rabbit fixed in Bouin, stained in iron-hematoxylin and eosin. Subcutaneous tissue spread of the cat and rabbit stained for elastic fibers. Chromatophores in section of cleared and sectioned frog lung. Adipose tissue in the fat body of a frog. APPENDIX 291 Cornea of the eye, ral)bit. Tendon of a frog and cat, lonjjitndinal sections. ]\Iembranons bone of a small fetal cat. Developing femur of a new-born rabbit. Groimd bone, cross- and longitudinal sections. Bone-marrow of the cat. Blood: Smears stained with Wright's stain. Blood of a fish, the black bass. Blood of Amblystoma. Blood of a turtle. Blood of a bird, starling and grouse. Blood of various mammals. Muscle tissues: Smooth muscle of the frog's intestine, dissociated and routinely stained. Skeletal muscle and Achilles' tendon junction of the frog with chondroid tissue associated with the tendon. Longitudinal sections of Bouin fixed and iron-hematoxylin and orange G stained material. Skeletal muscle and tendon junction of the mouse, longitudinal sections. Cardiac muscle of the frog, dissociated and routinely stained. Cardiac muscle in the heart of a shark, Bouin fixed, stained with iron-hematoxylin and eosin. Cardiac muscle of the cat. Nerve tissues: Cerebral cortex of the cat, Golgi preparation. Cerebellar cortex of the cat, Golgi preparation. Cerebellum of the squirrel, Bielschowsky method and thionin stain. Diencephalon and pituitary of a fetal cat. Spinal cord of the cat, fixed in alcohol and stained with meth- ylene blue (Xissl method). Spinal cord of the cat, Golgi preparation. Spinal cord of the frog. Nerve fibers, routinely fixed and stained but teased, not em- bedded or sectioned. Nerve trunks, cross- and longitudinal section, of the cat. Teased nerve fibers of the frog's sciatic nerve treated with osmium tetroxide. 292 APPENDIX Blood vessels: Small artery of the cat and rat. Aorta of the dog. Mesenteric vein of the cat. Jugular vein of a bird, a hawk. Lymph organs: Tonsil of the dog. Tonsil of human. Mesenteric lymph gland of the squirrel and cat. Cervical lymph gland of the cat and dog. Hemolymph gland of the rat. Spleen of the bullfrog. Spleen of Necturus, Zenker fixed and Mallory stained. Spleen of the water snake. Spleen and duodenum of a hawk. Spleen of a cat and dog. Thymus of a cat. Integument: Integument of a bass (fish with ctenoid scales), celloidin em- bedded. Integument of Necturus. Integument of a frog. Wing of new-born sparrow, longitudinal section, showing skin and developing feathers together with developing bone. Footpad of a squirrel, embedded in celloidin. Human scalp, embedded in celloidin. Ear of a rabbit, cross-sections. Mammary gland of the cat, longitudinal section through the nipple. Resj)iratory system: Gill of a dogfish, longitudinal section through the septa. Gill of Necturus, longitudinal section. Lung of Necturus, cross-section. Glottis and openings into the lung of the frog, longitudinal section of pharyngeal region. Lung of a toad and frog, cross-section. Trachea and lung of the water snake (Natrix), cross-sections. Trachea and esophagus of a bird (a grouse), cross-section. Lung of a hawk, cross-section. Larynx of the cat, cross-section. APPENDIX 293 Trachea of the rat in a cross-section of the throat. Trachea of the cat and dog, cross-sections. Lung of the dog, longitudinal section of a lobule. Digestive system: Lip of the dog. Tongue of a fish (bass) and toad, longitudinal section. Tongue of the frog, cross-sections. Tongue of a rat, cross-section of the tip region. Tongue of the cat, longitudinal section. Tongue of the rabbit, cross-section of portion with foliate papillae. Tongue of the cat in the region of the vallate papilla-. Parotid and submaxillary glands of the woodchuck and cat. Esophagus of a frog, cross-section. Esophagus of a turtle, cross-section. Esophagus in a cross-section of the neck of an embryo cat. Esophagus of a dog, cross-section of middle third. Esophagus and stomach junction of the dog, longitudinal sec- tion. Stomach of the dogfish, cross-section. Stomach of a bullfrog, cross-section. Stomach of a turtle, cross-section. Stomach of a cat and dog, fundus region. Stomach and duodenal junction of the dog, longitudinal section. Small intestine of Xecturus and frog, cross-sections. Small intestine and pancreas of the water snake (Xatrix). Duodenum, jejunum, and ileum of the chipmlmk, cross-sections. Duodenum of the bat, cat, and dog, cross-sections. Ileocecal valve of the cat and dog, longitudinal sections. Appendix of human, cross-section. Spiral valve of the dogfish, cross-sections. Rectum of a frog, cross-section. Rectum of a dog, woodchuck, and guinea-pig, cross-sections. Cross-section of a newt (Triton) in the cloacal region, celloidin embedded. Rectum and anal junction of the dog, longitudinal section. Pancreas of a frog. Pancreas of the cat, rat, and dog. Liver of a fish (black bass). Liver of Xecturus. >94 APPENDIX Liver of the alligator. Liver of pig, Mallory stained. Liver of the cat and woodchiick. Liver of the rabbit with injected bile capillaries. Gall-bladder of an alligator. Excretory system: Kidney of a fish (the black bass), and ureter. Kidney and testis of Necturus, Zenker fixed and IVIallory stained. Kidney of a toad, and ureter, also adrenal. Kidney of the water snake (Natrix), and ureter. Kidney of a hawk. Kidney of a fetal cat showing developing nietanephros and remnants of mesonephros. Kidney of a cat and rat. Ureter of an alligator. Ureter of a dog, cross-section. Bladder of a frog. Bladder of a turtle. Bladder of a dog, collapsed condition. Bladder of a mouse, collapsed and distended conditions. Urethra of female woodchuck, cross-section. Female reprodiictiw system: Ovary of a fish (the black bass), cross-section. Ovary of Amblystoma and Necturus. Ovary of the water snake (Xatrix). Ovary of immature dog and cat. Ovary of adult cat, rabbit, and chipmunk, showing corpora lutea, etc. 0\iduct of a skate (an elasmobranch). Oviduct of the frog, cross-sections of se\eral levels. Oviduct of a turtle. Oviduct of a cat, pig, and rabbit. Uterus of a rabbit, resting condition. Litems of a cat in heat. Vagina of a guinea-pig, cross-section. Fetus and attached placenta of the mouse. Placenta of a rat. Mate reproihirtlre system: Testis of Amblyostoma, cross-sections. Testis of a toad. APPENDIX 295 Testis, kidney, and adrenal of an alligator (young). Testis of the kitten, longitudinal section, showing epididymis. Testis of the rat, cross-section. Stained with iron-heinatoxylin. vSeniinal vesicle of the guinea-pig, cross-sections. Vas deferens of the dog. Vas deferens and rudimentary Miillerian duct of the wood chuck. Prostate of the dog, cross-section. Penis of a chipmunk and squirrel in different regions. Penis of a dog, cross-section of distal third. Endocrine glands: Pituitary of the calf, longitudinal section. Pituitary of a kitten, longitudinal section. Thyroid of frog. Thyroid of a turtle. Thyroid and ])arathyroid of a dog. Adrenal of frog. Adrenal of a dog, rabbit, and cat. Carotid of a cat. The slifles listed above are grouped into systems; those for the tissues are supplemented by types of tissues represented in the slides listed under various systems. These slides should also be supi)lemented hx study of fresh preparations of living tissues whercN'er practical and also by specially prepared demonstration slides. The majority of the slides enumerated above have been pre])are(l in our own laboratory, but there are several supply houses from which a great Nariety of slides appropriate to this work may be obtained. INDEX. (References to illustrations given in boldface type.) Absorption in the intestine, 176 of water, 157, 180 Acetic acid in fixation, 265 Acid stains, 282 Acidophil cells, pituitary, 254 Acinus, 43 Acromegaly, 255 Addison's disease, 262 Adipoblast, 55 Adipose tissue, 55, 56 Adrenal (suprarenal) gland, 259, 260 amphibian, 260 bird, 260 blood supply, 260 capsule, 259 chromaffin cells, 262 cortex, 261 fish, 260 mammalian, 260, 261 medulla, 262 Adventitia, alimentary canal, 167 arteries, 117, 118 After-birth, 232 Agranulocytes, 74 Alcohol in fixation, 266 Alimentary canal, 166 Alveolar ducts, 154 sacs, 154 Alveoli, glandular, 43 pulmonary, 154, 155 Ameboid wandering cells, 51 Amitosis, 20 Amphiaster (achromatic spindle), 19 Anaphase, 19 Anatomy, 13 Aniline oil in embedding, 272 in staining, 283 Animal pole, 22 Anisotropic band in muscle, 91 Anus, 181 Aorta, 117 Appendix, vermiform, 180 Arachnoid membrane, 109 Archenteron, 22, 23 Areolar connective tissue, 50 Argyi-ophil fibers (reticular tissue), 49 Arrector pili muscle, 143 Artefacts, 14 Arteries, 115 Arteries, function of, 118 large (elastic), 117 medium (muscular), 116 small, 116, 117 Arterioles, 115, 116 Astrocytes, 106 Astrosphere, 17 Atresia of follicles, 216 Auricular-ventricular node, 88 Autolysis, 20, 265 Axon, 95, 97 B B.\RTHOLiN glands, 226 Basement membrane, 38 Basic chi'omatin, 18 stains, 282 Basophils, 75 Benzene, 271 Bidder's canal, 241 organ, 241 Bile, 189 capillaries, 188 ducts, 189 Bladder, allantoic, 205 cloacal, 204 development of mammalian, 205 gall, 190 mammalian, 205, 206 swim, 148 tubal, 204 Blastocyst, 25 Blastoderm, 24, 25 Blastomere, 22 Blastopore, 23 Blastula, 22 I Amphibia, 23 I Amphioxus, 22 Sauropsida, 24 Block, fiber, in celloidin embedding, 274 wood, in paraffin embedding, 276 ' Blood, 70 capillaries, 113, 114 association with tissue juice, 114 circulation in, 115 cell formation (hemopoiesis), 76 cells, 70, 71, 73 plasma, 70 Blood vessels. *S'ee Arteries, Capillaries, Veins. 298 INDEX Blood, origin of, 113 Body cavity (coelome), 23 Bone, 61 canaliculi, 61, 68 cells, 61 endochondral, 63, 64 intramembranous (membranous) , 61, 62 lacunae, 61 lamellae, 62 marrow, 62, 68, 78, 79 blood cell formation in, 78 red, 66, 78 structure of, 79 yellow, 68 microanatomy of a long bone, 66 spongy, 62, 65 trabeculse, 62 Bouin's fluid, 267 Brain, 109 Bronchi, 152 Bronchial arteries, 155 tree, 153 veins, 155 Bronchioles, 153 respiratory, 153, 154 terminal, 153, 154 Brush border, 37 Bulbourethral glands, 248 Calcium metabolism, 258 Calyces, 198, 199 Canada balsam, 284 Capillaries. See Blood, Lymph and Bile. Capsule, 127, 198, 201, 260 Carbohydrate metabolism in liver, 190 Cardiac muscle, 84, 85, 86 Carnoy's fluid, 268 Carotid gland, 262 Cartilage, 58 elastic, 60 fibrous, 60 hyaline, 59 in bone formation, 61, 63 origin, 58 Cecum, 180 Cell, 14, 16 acidophil, 254 amoeboid wandering, 51 argentophil, 177 basophil, 254 bipolar, 99 bone, 61 centro-acinar, 182 chief, 172 chromaffin, 262 chromophobe, 254 Cell, ciliated, 37 differentiation, 13 endothelial, 31 fat, 53 flagellated, 37 follicle (nurse), 210 ganglion, 96, 101 goblet, 32, 34, 42 inclusion, 17 interstitial, of ovary, 214 of testis, 239 KupfTer, 188 hver (hepatic), 184, 185, 186 lutein, 218 mast, 52 membrane, 17 mesenchyme, 53 metabolism, 20 mucous, 45 neurolemma, 101 olfactory, 151 oogenic, 211 pancreatic, 182 Paneth, 177 parietal (oxyntic), 172 pigment, 52 plasma, 75 prickle, 141 primordial sex, 209 re])roduction, 18 satellite, 96, 101 secretion, 40 serous, 45 Sertoli, 236 spermatogenic, 237 spermatogonial, 235 undifferentiated (mesenchymal) , 53 unipolar, 99 Celloidin, 272 embedding, 272 method, 273 sections, 278 Centriole, 19 Centrosome, 16 Centrosphere, 17 Cervix, 225 Chloroform, 274 Chondrin, 59 Chondriosomes, 15 Chondi'oclasts, 63 Chondrocytes, 59 Chondroid tissue, 58 Chorionic villi, 231 Choroid jjlexus, 106 Chromatin, 17 Chromatophore, 52 Chromic acid, 266 Chromosomes, 17 Cilia, 37 Ciliary movement, 38 Clasmatocytes, 51 INDEX 299 Cleavage, 22 Amphibia, 23 Amphioxus, 22 Mammalia, 25 iSauropsida, 24 Clitoris, 226 Cloaca, 181 Coelente rates, 13 Ccrlome, 23 Ccelomic })ouch, 23 Cohnheim's areas, 91 Collagenous fibers, 53 relation to argyrophil fibers, 49 Color change, 52 Columns of Bertini, 201 Connective tissue, 47 adipose, 55, 56 cells, 50 chondroid, 58 classification of, 47 dense fibrous and elastic, 56 elastic membranes, 54 function of, 54 ligamentous, 57 loose fibroelastic (areolar), 50, 54 mesenchyme, 47, 48 mucous, 48 reticular, 49 serous, 55 tendinous, 57 Containers, 268 Coplin jar, 283 Copulatory organs, 248 lower vertebrates, 248 mammalian, 249 Corium (dermis), 134 amphibian, 137 fish, 135 mammalian, 139 reptile, 138 Corpora cavernosum ])enis, 249, 250 urethra, 250 Corpus albicans, 218 cavernosum, 249 luteum, 211, 217 spurium, 218 verum, 218, 219 Cortin, 262 Cover glass, 284 Cowper's gland, 248 Crenation, 72 Cretinism, 257 Cumulus oophorus (germ hill), 214, 217 Cuticle, 38 Cytocentrum, 19 Cytology, 14 Cytomorphosis, 20 Cyton, 95 Cytoplasm, 15 Cytosome, 15 Decidtja, 231 Dendrites, 97 Dentine, 135 Dermis (corium), 134 Destruction of blood cells, 80 Development of bone, 61, 63 of blood cells, 76 of blood-vessels, 113 of Graafian folUcles, 216 of lymphatics, 121 Diaphysis, 65, 66 Digestive system, 157 Diplosome, 20 Discs, intercalated, 86 Dissection, 269 Ducts, collecting, 35, 195, 201 ejaculatory, 244 intercalated, 183 papillary, 200 secretory, 164, 165 sperm, 239 Dura mater, 109 Dyes, acid, 282 basic, 282 eosin, 288 erythrosin, 288 hematoxylin, 286 Janus green, 16 Mallory's, 285 Orange G, 288 Sudan III for fat, 56 Wright's, 74 Dwarfism, 255 E Ear, elastic cartilage in, 60 Ectoderm, 23, 24 Effectors, 103 Egg albumin, 279 Elastic membranes, 117, 119 Elastin, 54 Eleidin, 142 Embryology, 14, 22 Embryonic connective tissue, 47 Enamel, 38, 159 Endocrine glands, 45, 252 Endoderm, 23, 24 Endometrium, 223 Endomysium, 90 Endoneurium, 102, 103 Endosteum, 65, 67 Endothelium, 31 Enteroceptors, 104 Eosin, 288 Eosinophil, 75 Ependyma cells, 106 Epidermis, 134 amphibian, 136, 137 300 INDEX Epidermis, bird, 139 fish, 135 mammal, 139, 140 reptile, 138 Epididymis, derivation of, 240 mammal, 244, 245 reptile, 242 Epinephrin, 262 Epineurium, 103 Epiphysis, 65, 67 Epithelium, 27 ciliated, 33, 36, 37 classification of, 27 function of, 27 germinal, 32 glandular, 40 intercellular bridges of, 39 neuro-epithelium, 34 pigmented, 37 pseudostratified, 33 regeneration of, 39 simple, 27, 28 columnar, 32 cuboidal, 31 squamous, 30 stratified, 34 columnar, 35, 36 cuboidal, 35 squamous, 34, 35 surface modifications of, 37 transitional, 36 Erythrocytes, 71 degeneration, 72 formation of, 78 Erythrocytoblast, 78 Esophagus, 167 amphibian, 36, 167 mammalian, 35, 168, 169 reptilian, 167, 168 Ether-alcohol, 273 Excretory system, 192 Exocrine glands, 41 Exteroceptors, 104 Fallopian tube, 222 Fasciculi, 89, 90, 103 Fat cells, 53 in fibroblasts, 53 in hibernation, 56 Feathers, 139 Female reproductive system, 209. Reproductive system. Ferric alum, 285 Fibers, argyrophil, 49 collagenous, 53 elastic, 54 muscle, 88 nerve, 101 Fibers of Remak, 102 Purkinje, 88 Sharpey's, 64 Fibrils, collagenous, 53 muscle, 81 nerve, 97 Fibroblasts, 51 Fibrocytes, 50, 51 Fixation, 265 Fixing fluids, 267 Bouin's, 267 Carnoy's, 268 Flemming's, 267 formaUn, 268 Kelly's, 268 Zenker's, 267 reagents, 265 acetic acid, 265 alcohol, 266 chromic acid, 266 formalin, 266 mercuric chloride, 266 osmium tetroxide, 266 picric acid, 265 potassium dichromate, 266 Flagellum, 37 Follicle, Graafian, 216 hair, 142, 143 ovarian, 210 primary, 214, 216 Foramen, 67 Foreign matter, 269 Formalin, 266 G GALL-bladder, 190 Ganglia, 107 cells, 96 spinal, 107 sympathetic, 108 Ganoid fishes, 77 Gastric pits, 172 Gastrula, 22 Amphibia, 23 Amphioxus, 22 Sauropsida, 24 Gelatin, 53 Germinal center, 122 disc, 24 Gigantism, 255 Gill slits, 148 Gills, amphibian, 148, 149 See \ fish, 148 Glands, adrenal (suprarenal), 259, 260 alveolar, 43 apocrine, 41 Bartholin, 226 Brunner's, 178 buccal, 158 bulbourethral (Cowper's), 248 INDEX 301 Glands, cardiac, 172 carotitl, 262 ceruminous, 145 classification, 41 compound, 43 alveolar, 44, 45 tubular, 4;j, 44 endocrine, 45, 252 exocrine, 41 fundic, 172 holocrine, 41 labial, 158 Lieberkuhn's, 177 mammary, 145 Meibomian, 144 merocrine, 41 mixed, 45 mucous, 45 parathyroid, 258, 259 parotid, 164, 165 pituitary, 253 poison, 138, 166 prostate, 247 pyloric, 173 rectal, 179 salivary, 164 sebaceous, 143, 144 serous, 45 shell, 220, 221 simple alveolar, 43, 44 tubular, 42, 43 sublingual, 166 submaxillarv, 165 sweat, 140, 142 thyroid, 255 unicellular, 41 uterine, 224, 225, 229 von Ebener's, 164 Glandular pockets, 42 Globin, 71 Glomerulus, renal, 30, 193, 195 Glottis of frog, 149 Glycerine, 271 Glycogen, 186 Golgi apparatus, 16, 97 Granulocytes (granular leukocytes), 75 basophil, 75 eosinophils, 75 formation of, 78 neutrophils, 75 Gray matter, 109 Gum, 159 damar, 284 H Haversian systems, 64, 67, 68 Heart beat, myogenic and neurogenic theory of, 87 Heat and (rstrus, 227 in fixation, 269 Hematoxylin, Harris', 286 Heidenhain's, 287 Hemochromogen, 71 Hemocytoblast, 78 Hemoglobin, 71 Hemolymph gland (node), 127 Hemolysis, 72 Hemopoiesis, 76 Hemopoietic centers, 76-79 Henle's loop, 200, 201 Heparin, 192 Hibernation, 56 Hilus (hilum), 124, 198 Histiocytes, 51 Histology, 13, 14 Hydrochloric acid, production of, 173 ; Hyperthyroidism, 257 Hypertonic, 72 I Hypophysis cerebri, 253 I Hypotonic, 72 Ileocecal junction, 180 Ileum, 122, 180 Infundibulum, 253 Insulin, 184, 190, 263 Integument, 134 amphibian, 136, 137 bird, 139 fish, 135 mammal, 139 reptile, 138 Intercalated discs of cardiac muscle, 86 Internode, 102 Interstitial cells of ovary, 214 of testis, 239 lamellae, 67 Intestine. See Small and Large. Iron alum, 285 Islands of Langerhans, 183 Isotonic, 72 Isotropic ("J" brand), 91 Isthmus of thyroid, 256 "J" DISC in muscle, 91 Janus green in vital staining of mito- chondria, 16 Junction of muscle and tendon, 93 Hagfish, 77 Hair, 142, 143 Hassall's corpuscles, 132 Haversian canals, 64 lamellae, 62 Keratin, 142 KeratohyaUne, 141 Kidney, 192 302 INDEX Kidney, amphibian, 19-1 cortex, 198, 199 development of, 192 frog, 194, 195 mammalian, 197, 198, 199 medulla, 198, 199 mesonephros, 193 metanephros, 196 pronephros, 193 Kinocilia, 38 Kupffer cells, 188 Lacteal, 177 Lacunae, 59, 67, 68 LamelUe, endosteal, 65, 67 fibrillar arrangement in, 67, 68 Haversian, 64 interstitial, 67 periosteal, 62, 67 Large intestine, 178 amphibian, 179 fish, 178 mammal, 179, 180 reptile, 179 Larynx, 151 Leukocytes, 73 Ligaments, 57 Lip, 158 Liquor folliculi, 216 Liver, 184 amphibian, 184, 185 bile capillaries of, 188 blood cell formation in, 185 supply of, 186, 187 endocrine activitv of, 190 fish, 184 function of, 189 glycogen, 186 lobules, 186 mammalian, 185, 186 reptiUan, 185 sinusoids, 185, 187 Lumen, 42 Lungs, 149 amphibian, 149, 150 mammalian, 152, 155 reptilian, 150 Lymph nodes, 124, 125, 126 nodules, 122, 123 vessels, 119, 121 Lymphatic capillaries, 121 system, 121 Lymphocytes, 74 M "M" BAND in striated nuiscle, 91 Macro])hages, 51 Male reproductive system, 234. See Reproductive system. Mallory connective tissue stain, 285 Malpighian corpuscles, kidney, 199 spleen, 127 layer of epidermis, 134 Mammary gland, 145 Marrow cavity, 68 red, 66, 78 yellow, 68 Mast cells, 52 Mediastinum, 243 Megakaryocytes, 79 Melanin, 53 Melanophores, 53 Menstruation, 228, 229 Mercuric chloride, 266 oxide, 287 Merocrine glands, 41 Mesenchyme, 47, 48 Mesoderm, 23, 26 somatic, 24 splanchnic, 24 Mesonephric tubule, 194 Mesonephros (Wolffian body), 30, 193 of frog, 194, 195 Mesothellum, 31 Metanephros, 196 blood supply of, 201 description of tubule, 199 development of, 196 Metaphase, 19 Methyl salicylate, 279 Microdissection api:)aratus, 21 Miciogliocytes, 106 Mi('i()sc()])ic anatomy, 13 Miciotome, rotary, 275 sliding, 278 Mitochondria (chondriosomes), 15, 97 Mitosis, 18 Monocjrtes, 74 Mordant, 285 Motor end plate, 105 endings, 105 neurons of sjiinal cord, 100, 101 Mucin, 41 Mucous membrane, 157 Mullerian duct, female, 209, 219 male, 242 Multipolar cells, 100 Muscle cardiac, 84, 85, 86 skeletal, 88 development of, 89 variation in size of, 92 smooth, 81, 82, 84 contraction of, 84 organization of, 83 tissue, 81 Muscularis mucosa-, 158, 167 Myelin, 101 Myelinated fibers, 101, 102 INDEX 303 Myelocytes, 79 Myeloid tissue, 77 Myoblasts, SI Myofihrilhe, 81 Myogenic theory, 88 Myoid cells of thynuis, 132 Myometrium, 22.'i Myxedema, 257 N Nails, 146 Necrosis, 20 Nephridial tubule, 199 Nephron, 201 Nephrostome, 192 Nephrotonie, 192 Nerve cells, 95, 98-101 degeneration, 108 endings, 10-4 fibers, 101 histogenesis, 94 IHMii.iicnd, 102, 103 regeneration, 108 tissue, 94 Nervi nervorum, 103 Neiu-al crest, 95 groove, 25 plate, 94 tube, 95 Neurofibrils, 97 Neurogenic theory of heart beat, 87 Neuroglia, 106 Neurolemma, 102 Neuron, 95 bipolar, 99 development of, 94 ganglion 96, 101 motor, 101 multipolar, 99, 100 processes of, 97, 100 types of, 98 unipolar, 99 Neutrophils (heterophils), 75 Nissl bodies, 96 Nodes of Ranvier, 102 Non-myelinated fibers, 102 Normoiolasts, 78 Notochord, 24, 68 Nuclear membrane, 17 Nucleic acid, 17 Nucleolus, 18 Nucleoproteins, 17 Nucleus, 17 CEsoPHAGUs, 167. Sec Esophagus. CEstrus, 226 Olfactory area, 151 Oocyte, 215 Oral cavity, 159 Oi'ange G, 288 Oiganogeny, 26 Os piiapi, 250 Osmium tetroxide (osmic acid), 2()6 Osmotic equilibrium, 72 Ossification, eiidochondi-al, 6;5, 64, 65 intramembranous, 61, 62 Ostein, 61 Osteoblasts, 62 Osteoclasts, 63 Osteocytes, 62 Ova, 209 amphibian, 211, 212 fish, 211 mammalian, 213, 217 origin of, 209 reptile, 212, 213 Ovary, 209 amphibian, 211, 212 fish, 210 frog, description of, 211, 212 early development, 209 endocrine activity, 214 mammalian, 213 early development, 214 structure of, 214, 215 reptile, 212 descrij^tion of lizard ovary, 213 Oviducts, 219 amphibian, 220 mammalian, 221, 223 reptiUan, 221 Ovulation, 216 Oxychromatin, 18 Oxyhemoglobin, 72 Pacinian corpuscles, 105 Pancreas, 181, 182, 183 lower vertebrates, 184 Paper boat for embedding, 271 Papilhie, 160, 161 circumvallate, 163 filiform, 162 folliate, 162, 163 fungiform, 1()2 Paraffin embedding, 270 oven, 270 procedure in, 272 sections, mounting of, 279 serial, 281 Paraganglia, 263 Parathyroids, 258, 259 Parotid glands, 164, 165 Pars convoluta, 199, 200 distalis, 254 intermedia, 255 304 INDEX Pars neiiralis, 255 radiata, 199, 200 tuberalis, 254 Pelvis, 198, 199 Penicilli, 128, 129 Penis, 249, 250 erection of, 250 Perichondrium, 59 Perimysium, 89 external (epimysium), 89 internal, 89, 90 Perineuriiun, 103 Periosteum, 62, 66 Peritoneum, 55 Pever's patches, 122 Pfluger's cords, 214, 215 Pharyngeal pouch, 256, 258 Pharynx, 166 Pia mater, 109 Picric acid, 265 Pigment cells, 37, 52, 185 granules, 17 melanin, 53 Pineal body, 263 Pituitary, 253, 254 divisions of, 253 function of, 255 infundibulum, 253 intermedia, 255 pars distalis, 254 intermedia, 255 neuralis, 255 tuberalis, 254 Pituitrin, 255 Placenta, 230 Plasma, 70 cells, 75 Platelets, 76 Pleura^, 55 Plexus, choroid, 106 myentei-ic (Auerbach's), 167 pampiniform, 244 submucosal (Meissner's), 167 Portal canal, 189 Potassium dichromate, 266 Pregnancy, 230 Primitive groove, 24, 25 streak, 24, 25 Progressive staining, 283 Pronephros, 193 Prophase, 19 Proprioceptors, 104 Prostate gland, 247 Protoplasm, 15 Protoplasmic bridges (intercellular bridges), 38, 39 Protozoa, 13 Pulmonary alveoli, 154, 155 artery, 155 unit, 155 vein, 155 Purkinje cells, 99, 100 fibers, 88 Pyramidal cells, 99, 100 Pyramids, renal, 199 Q "Q" BANDS in muscle, 91 R Rathke's pouch, 253 Receptors, 103 Records in fixation, 270 Rectum, 181 Red blood cells, 71 Reflex arc, 97, 98 Regeneration, epithelia, 39 muscle, 84, 87, 93 nerve, 108 Regressive staining, 284 Renal corpuscles, 30, 199, 201 pyramid, 198, 199 Rennin, 173 Reproductive system, 209 female, 209 amphibian, 211 fish, 210 mammalian, 213 reptile, 212 general development of, 209 male, 234 amphibian, 241 development of, 234 fish, 240 mammalian, 242 reptihan, 242 Respiratory system, 148 amphibian, 148 fish, 148 mammalian, 150 reptilian, 150 Rete testis, 243, 244, 245 Reticular cells, 49 fibers, 49 Reticulo-endothelial system, 188 Retina, 37 Ribbon of sections (paraffin), 277, 281 Rouleaux, 71 RugiT-, 171 Rut, 227 Saliva, 164 Sarcolemma, 91, 92 Sarcomere, 92 Sarcoplasm, 81, 92 Sarcostyles, 91 INDEX 305 Satellite cells, 96, 101 Scales, ctenoid, 136 placoid, 135 reptile, 138 Scrotum, 242 Sebaceous glands, 143, 144 Sebum, 144 Secreting areas, 42 Secretion, 40 granules, 40 Sectioning, 275 celloidin, 278 paraffin, 275 Segmentation cavity, 22, 23 Seminal vesicles, 244, 246 Seminiferous tubule, 234, 235, 243 Sensory endings, 104 Serous cells, compared with mucous, 45 membrane (serosa), 55, 169, 174 Sertoli cells, 236 Sharpey's fibers, 64 Sheath of Henle, 102 of Schwann, 101 Silver nitrate, 286 Sinusoids, liver, 185, 187 spleen, 128, 129, 130 Skin, 134. »SVe Integument. Small intestine, 173 amphibian, 32, 174, 175 fish, 174 mammalian, 175, 177 reptilian, 175 Smooth muscle, 81 Sole, 140, 141 Somatopleure, 23 Sperm, 238 Spermatids, 238 Spermatocytes, 23S Spermatocytogenesis, 238 Spermatogenesis, 236, 237 Spermatogonia, 238 Spermiogenesis, 238 Spicules, bone, 61 Spinal canal, 110 cord, 110 ganglia, 107 Spiral valve, 178 Splanchnopleure, 23 Spleen, 127-130 blood circulation in, 128, 129 development of, 77 function of, 130 lower vertebrates, 130 mammalian, 128, 129 sinusoids, 128, 129, 130 Staining, celloidin section, 286 parafhn sections, 282 progressive, 283 regressive, 284 Stereocilia, 38 Stomach, 169 20 Stomach, amphibian, 170, 171 fish, 169, 170 mammalian, 171, 172 reptilian, 171 Stratum, coi-neum, 134, 140, 1 12 germinitivuiu, 134, 140, 143 granulosuni, 141, 143 lucidum, 141, 143 Striated bordei', 37 Stroma, 71 Subcutaneous tissue, 50, 51 Subcutis, 135 Sublingual gland, 16() Submaxillary gland, 165 Submucosa, 167, 168 Sweat gland, 43, 140, 142 Swim bladder, 148 Sympathetic ganglia, 108 Synapse, 98 S>Ticytium, 17 Taste buds, 163 Technique, laboratorv, 265 Teeth, 159 Telophase, 19 Tendons, 57 Testes, 234 amphibian, 241 development of, 234 endocrine activity, 239 interstitial tissue (cells), 239 mammalian, 242 reptilian, 242 Theca folliculi, 214, 216 externa, 216 interna, 216 Thrombocytes (spindle cells j, 76 Thymocytes, 13!^ Thymol,' 279 Thymus, 131, 132 involution, 133 Thyroid, 255, 257, 258 anlage, 256 cells, 256 follicles, 256 function of, 257 Tigroid bodies. See Xissl bodies. Tissue cultiu-e, 21 fluid (juice), 50, 54 Toluol in embedding, 272 Tongue, 159 amphibian, 160 fish, 159 mammalian, 161-162 reptilian, 160 Tonsils, 122, 123 Trachea, 151, 152 Traumatic degeneration of ner\'e hbers, 108 306 INDEX Trophectoderm, 25 TubuU recti, 244, 245 Tunica adventitia, 115, 118 albuginea of ovary, 215 of testis, 243 intima, 115, 118 media, 115, 118 propria, 158 vaginalis parietalis, 242 visceralis, 243 Umbilical cord, 231 Ureters, 202 amphibian, 202 mesonephric, 202 metanephric, 203 mammalian, 203 reptilian, 203 pronephric, 202 Urethra, 206, 207 Uriniferous (nephridial) tubule, 199 collecting, 31, 198, 201 distal convoluted, 195, 200, 201 function of, 196 glomerulus, 30, 194, 199, 201 neck, 195, 200 proximal convoluted, 195, 200 Uterus, 222 bicornis, 222 duplex, 222 in heat, 224 resting, 223, 224 simplex, 222 Vagina, 225 Valves, lymphatics, 121 veins, 119 Vas deferens, 245, 246 Vasa vasorum, 119 Vascular system, 113 Vasi nervorum, 103 Vegetal pole, 22 Veins, 118 Vestibule, 226 Vim, 176, 177 Vocal cords, 149, 151 Volkmann's canals, 64 W Wallerian degeneration of nerve fibers, 109 Wastes of metabolism, 54 White matter, 109 pulp, 127 Xylol, 271 Yellow fibers (elastic), 54 Yolk, 17, 22 plug, 23 "Z" band in muscle, 91, 92 Zenker's fluid, 267 Zona fasciculata, 261 glomerulosa, 261 pellucida, 216 1 radiata, 216, 217 reticularis, 261 Zygote, 22 Zymogen granules, 172, 182