The National Medical Series for Independent Study 2nd edition histology and cell biology Kurt E. Johnson, Ph.D. Professor af Anatomy George Washington University Medical Center Washington, D.C.Managing editor: Debra Dreger Project editor: Judd Howard Production: Keith LaSala, Laurie Forsyth, Judy Johnson Art direction and illustration: Wieslawa B. Langenfeld Composition and layout: Telecomposition, Inc. Williams & Wilkins Rose Tree Corporate Center, Building !I 1400 North Providence Road, Suite 5025 Media, PA 19063-2043 USA Library of Congress Cataloging-in-Publication Data Johnson, Kurt E. Histology and cell biology / Kurt E. Johnson. p. cm. — (The National medical series for independent study) (A Williams & Wilkins medical publication) Includes index. ISBN 0-683-06210-7 (pbk. : alk. paper) 1. Histology—Outlines, syllabi, etc. 2. Cytology—Outlines, syllabi, etc. |. Title. Il. Series. Ill. Series: A Williams & Wilkins medical publication. {[DNLM: 1. Cytology—examination questions. 2. Cytology— outlines. 3. Histology—examination questions. 4. Histology—outlines. QS 18 J67c] Ko QM553J624 $990 wes oN 018d be DNLM/DLC for Library of Congress 90-5226 CIP ISBN 0-683-06210-7 © 1991 by Williams & Wilkins, Baltimore, Maryland Printed in the United States of America. All rights reserved. Except as permitted under the Copyright Act of 1976, no part of this publication may be reproduced or distributed in any form or by any means or stored in a data base or retrieval system, without the prior written permission of the publisher. 10°98 7-65Dedication This book is dedicated to my squash and tennis partners, Steve Goldman, Ron Chander- bahn, Bryant Chomiak, Bob Walker, David Campbell, and Ralph Kramer. The many hours of fun and competition spent with them on the court have contributed immeasurably to my current psychological orientation. They get credit or are to blame, depending on your viewpoint. Whatever, | am grateful for their friendship. Thank you gentlemen! This book also is dedicated to the memory of my late colleague, Ernest N. Albert, Ph.D. He was a good friend and will be missed.nougthe it) hii) Gatti teu! ey Sn id inl rom ia 2 {mand cia ge ewitgea ch let bine shed va geet sella UE mt os CU el ee ati y aurwrem hist (ek nt eu Bet) et coMuay) p T J om nud ia Ss sift os tmp Lae BY Te LMedsrd | = HAE that i feNiim ee peek tL ik uy Ce Pe gy] ey + plait 4 err © efateat ds 7 TU sea dre [ot miRNA! detiarreeale ee OY vey tet mee a ota rp Te pe od a debate) alone cul best opted | ita =< Arc bee in cow, | | | | | | | | | |se © VON FS BT FF WN = —_ om Preface Acknowledgments To the Reader Methods Used in Histology and Cell Biology The Plasma Membrane and Cell Surface Cytoplasmic Organelles: Membranous Systems Cytoplasmic Organelles: Discrete Particulate Organelles The Cytoskeleton and Microtubule-Containing Organelles Nuclear Cell Biology The Extracellular Matrix Epithelial Tissue Connective Tissue Cartilage and Bone Peripheral Blood, Bone Marrow, and Hematopoiesis The Immune System vii Contents xl xili 25 25 45 55 65 79 93 107 123 13713 14 15 16 17 18 19 20 a1 22 23 24 25 26 af Muscular Tissue Nervous Tissue The Cardiovascular System The Respiratory System The Upper Gastrointestinal Tract The Lower Gastrointestinal Tract The Liver, Pancreas, and Gallbladder The Pituitary and Pineal Glands The Thyroid, Parathyroid, and Adrenal Glands The Placenta and Mammary Glands The Integumentary System The Female Reproductive System The Male Reproductive System The Urinary System The Eye and Ear Challenge Examination Index viii 155 169 183 197 211 223 239 251 259 271 281 295 309 319 333 347 393Preface NMS Histology and Cell Biology, 2nd edition, is an extensively revised version of NMS Histology and Embryology. There are four substantial changes in this new edition. First, material on embryology has been deleted and expanded into another NMS title, Human Developmental Anatomy. Second, the single chapter on cell biology contained in Histology and Embryology has been expanded into six new chapters that cover structural cell biology (Chapters 2—7). Third, there are numerous additions of material covering functional cell biology in Chapters 8—27. And finally, many new questions, answers, and explanations have been added. It is hoped that these changes will make the book more useful to students in the classroom and in preparing for licensure examinations. Kurt E. Johnsongynte “I wvie® In igi dew mn ytpan age 5 | puoife: Dey sacle 2 eat smi crgtinh ahabd Hath se abies vee att em ue te eta fd Se ole? eachoevti ad Tom -aggtia mermebh nit) Myo aden ote bile oo Dine bs af certian etitorntiva oo islaaagn Pe aT tt Demet hits die | Mi ds fed deeie orl atest ortberlA, Fett i. igpat det fim) oy eae 8 ert aed ween 4i> Gan) Gerth [ee med ant yyy debe, basa. seer Hee ses teen bi nothiiea sure say uirsci) inured 0-8 Pranjuits | = =!) svi Sow Pr Tenienaifa win rnin oy! & inbaA, Soa rien fe packet! “4H! trl ¢ extadM tel lu! =. scum mr tut orathen firey emmys | gigas pert fewyeed a |) irichiloe: steal wrcatnermyimoes a) exysordl coi caetrvttssrey tye Theth ciscinebends Siertilof ) hdAcknowledgments | would like to thank my colleagues, past and present, for their help with providing photographic material for this book: Dr. M.K. Reedy and Dr. J.D. Robertson (Department of Anatomy, Duke University); Dr. E.N. Albert, Dr. D.P. DeSimone, Dr. M.J. Keoring, Dr. J.M. Rosenstein, Dr. F.J. Slaby, Dr. R.J. Walsh, and Mr, Fred Lightfoot (Department of Anatomy, George Washington University); Dr. B. Gulyas (NICHD); Dr. J.A. Long (Department of Anat- omy, UCLA); and Dr. H.A. Padykula (Department of Anatomy, University of Massachusetts). The editorial assistance of Debra Dreger also is gratefully acknowledged. Her dedication to excellence and sense of humor are greatly appreciated. | am grateful for all of these contributions and acknowledge that any errors contained herein are my own. xiainsmngbalwumloA We eT agi Tt hee ot a A td red mitt of Linfeumurr andqpeypenarigy DOT eee SR fetter id 8 Ell Seta alii narieruahy quit), yercbeash | PNM ON Ye, Mert) lipethherl nt htt nat TR ta lee (7 LCL ea inenaue ate PEP re Te eA DET eteeling? a fo aegis 1 omy hs fd sarpost Mewar et rt etd tt carver iia ier Ay oy ty AY elie fe eS ie a ine er oct yen om te oye p aig CLI, ay ET eer Me sal dade a te leita oyu whet me ele i, ene Let geile al] “Tem iT rt tear! Depriteie eMyV Uh Ue | tea mE Ne tyre! order ite i} | ee quill act SeaecrS eu, ee enti hm defi ol Sel biuowr \ < -s =iteeeas a a OI me ae =To the Reader Since 1984, the National Medical Series for Independent Study has been helping medical students meet the challenge of education and clinical training. In today’s climate of burgeoning information and complex clinical issues, a medical career is more demanding than ever. Increasingly, medical training must prepare physicians to seek and synthesize necessary information and to apply that information successfully. The National Medical Series is designed to provide a logical framework for organizing, learning, reviewing, and applying the conceptual and factual information covered in basic and clinical sciences. Each book includes a comprehensive outline of the essential content of a discipline, with up to 500 study questions. The combination of an outlined text and tools for self-evaluation allows easy retrieval of salient information. All study questions are accompanied by the correct answer, a paragraph-length explana- tion, and specific reference to the text where the topic is discussed. Study questions that follow each chapter use the current National Board format to reinforce the chapter content. Study questions appearing at the end of the text in the Challenge Exam vary in format depending on the book. Wherever possible, Challenge Exam questions are presented as clinical cases or scenarios intended to simulate real-life application of medical knowledge. The goal of this exam is to challenge the student to draw from information presented throughout the book. All of the books in the National Medical Series are constantly being updated and revised. The authors and editors devote considerable time and effort to ensure that the information required by all medical school curricula is included. Strict editorial attention is given to accuracy, organization, and consistency. Further shaping of the series occurs in response to biannual discussions held with a panel of medical student advisors drawn from schools throughout the United States. At these meetings, the editorial staff considers the needs of medical students to learn how the National Medical Series can better serve them. In this regard, the Harwal staff welcomes all comments and suggestions. xiii1ob6e8 ont of See bh obs ine fete aly id tym East a aT WE ae | 34M tee ite tes Sate ERP: agit $342) oie Waray iebieat at ganar uli tu aethrkc AY) actly att ina ldots eerie cme cr me oan Wen et ae aH! | bial Darts races oni TID Tete 9 ds ar eens hy sete dedi goriwhtd heattrent sitar! mmpnl 26 oy near : Yee hae! ny eeliaeerl [+l aljar at bri fae murniaial ape 1 wy MPSA BO RRL Se! plier teri nim LL ooh) Nore aif Tl HOOT Ly Leet rey (| peste ‘etn ee ey Srp a ie ras eG eomiy Ly aires al A rag tie Te Merges enim |e) ohmll il ue) eu ne Hany» bens Mg ds tee ibis. et ndine meat! ae PSmirge woe afd avs ppt ripe arVeiby kg kee creme Tanti: vneilGa dives iy ips eal saipehe alge yet: | ie AY se ee en Siete Tite oett ont heyucomw ond, tl Hillmen alrcte A restate beets oe ty en «ee ee ye og eee, iets bith eer ecm tA tecemdtiar fac fore baal] [pasereg «LLU a ay tig Fe avedleit eset cre tritex | mmmmlh cecal 1 res i sf? wy je, bel thei cn 2nioie ui do a eihtiiderig Ah rites Py pga! tint & (Ml a ee Weiler qn bitetat mgt ate= vil nathan HOU [pte yf 71+ Gl) ee corte me sic ‘rauntis TOS) ep mT ptt mrt Jae ae ge ea ames -eictt In hier acl? | ater! “wii a ia | } u murano om, reais je keen eet vie | bom atta ete ult he jk | (i, more sti! gt ype tes sete Ered cel tev i Uitte ara mundi pn / | wt RMN ee Pot sytem ra lannce le wun they yet Teele ag j yey = ee ee a lees te fh el ‘(em ALLEL yee ] Mimeethe e edit decree race le ibys onl leuny y pttise: Hee 2 my oT, publ penal = ee sr! oc iseruaelteth fin utes bh gn) erect leat) A Jeep beste ai, |= gies | SU Chl aie! re eet th eae’ Re rnedé he mite oy) wed prin Miah le othe: | te petty ape iioe eon ee te ee (VE *limigg 1) Le lhe aH and atten be | i} 1 J 1 | | i ah a a i i tiie 1 \ Vy —— DE ) =a eecinnenllee1 Methods Used in Histology and Cell Biology I. INTRODUCTION. Histology and cell biology researchers use a variety of methods of extending visual perception to study the relationship between structure and function in human cells and tissues. A. Microscopes allow researchers to see cellular and macromolecular details that are not visible to the naked eye. 1. Light microscopes employ transillumination and are used to examine living and prepared specimens and specimens with inherent or applied fluorescent properties. 2. Electron microscopes illuminate specimens with an electron beam. They have 1000 times the resolving power of light microscopes and provide resolution to the threshold of atomic detail. a. Transmission electron microscopy (TEM) uses thin specimen sections and reveals details of the cell's interior. b. Scanning electron microscopy (SEM) provides a high resolution view of the cell surface and environment. B. Supplemental study methods. Often, microscopy is used in conjunction with one or more of the following techniques to study details of cellular anatomy and physiology. 1. Staining is used in light and electron microscopy to increase the contrast of specimen structures. Some stains also convey information about chemical composition. 2. Autoradiography uses radioactive isotopes to localize substances within cells. 3. Differential centrifugation uses centrifugal force to separate cellular organelles and inclusions, thereby permitting biochemical analysis of subcellular fractions. 4, Freeze-fracture-etch is used in conjunction with electron microscopy. This technique creates a metallic replica of a specimen and is used to study the details of membrane structure and intercellular junctions. Il. SPECIMEN PREPARATION varies for light and electron microscopy, although the steps are similar in both formats. Sections Il A and B describe the basic steps in preparing specimens for light microscopy and TEM. Section II D describes specimen preparation for SEM. A. Fixation and sectioning 1. Fixation is the first step in specimen preparation for light microscopy or TEM. Fixation preserves cell structure while introducing a minimal number of artifacts (artificial structures produced during fixation that may appear to be true histologic features). a. Small tissue or organ fragments are immersed in a buffer solution containing a fixative such as glutaraldehyde. b. Electron microscopy specimens typically are immersed in a buffered osmium tetroxide (OsQ,) solution after fixation with glutaraldehyde. Osmium tetroxide has a high affinity for cell components that contain lipids (e.g., plasma membrane, nuclear envelope, membra- nous components of the mitochondria and Golgi apparatus).Histology and Cell Biology 2. Dehydration. After fixation, specimens for light microscopy and TEM are dehydrated by im- mersion in a series of solutions containing increasing concentrations of ethanol. Dehydration is necessary because most embedding media (e.g., paraffin, plastic monomers) are immiscible in water. 3. Embedding. After dehydration, the specimen is treated with a liquid embedding medium that infiltrates and hardens the specimen so that it can be sliced into thin sections suitable for staining and microscopic examination. a. Light microscopy often uses paraffin, which hardens the specimen as it cools, as an em- bedding medium. (1) Paraffin is easy to work with, speeds specimen preparation, and stains reliably. (2) However, paraffin has low tensile strength and, therefore, cannot be cut into very thin sections. b. TEM uses plastic monomers as an embedding media. (1) Specimens are infiltrated with propylene oxide and then with unpolymerized mixtures of plastic monomers, cross-linking agents, and catalysts. (2) Specimens are then heated in an oven which polymerizes the plastic and hardens the specimen. (3) These plastics yield very hard specimens that can be cut into ultra-thin sections (20-100 nm thick). 4. Sectioning a. Light microscopic studies typically use 5—10 ppm thick paraffin-embedded sections. (1) Sections are cut with a rotary microtome, a machine that moves the specimen across a sharp metal blade, advancing the specimen the desired thickness (e.g., 10 xm) after each pass. (2) Successive sections come off the microtome in a ribbon. Sections are cut from this ribbon and mounted on glass slides. b. TEM studies typically use 0.02—1.0 4m thick plastic-embedded sections. (1) A special microtome containing an extremely sharp glass or diamond blade is used to cut thin, smooth sections. (2) Sections are floated off the blade in a trough of water and then collected on small metal grids. c. Section thickness affects image resolution. (1) In thick sections, structures existing at different levels in the section overlap and interfere with each other in the image, thus reducing resolution. (2) Thin sections eliminate the overlapping problem and allow higher resolution views of specimens. B. Staining. Light microscopy sections usually are stained with dyes or fluorescent tags. TEM staining involves treating the section with heavy metal salts. 1. Acidophilia and basophilia a. Acidophilia. Tissue components that bind acidic dyes (e.g., eosin, orange G) are acidophilic. Acidophilic substances bear a net positive charge and bind negatively charged (i.e., acidic, anionic) dyes. Acidophilic structures include: (1) Erythrocyte cytoplasm (due to its high concentration of hemoglobin) (2) Collagen fibers (3) Mitochondria (4) Lysosomes b. Basophilia. Tissue components that bind basic dyes (e.g., hematoxylin, methylene blue, toluidine blue) are basophilic. Basophilic substances bear a net negative charge and bind positively charged (i.e., basic, cationic) dyes. Basophilic structures include: (1) Nuclei (2) Aggregates of rough endoplasmic reticulum, such as those found in cells that secrete large amounts of protein (e.g., plasma cells, which secrete large amounts of immuno- globulins) (3) Extracellular matrix 2. Metachromasia a. Metachromasia, which literally means a change in color, is a property of certain dyes (usually basic dyes). Structures that stain with these dyes (e.g., mast cell cytoplasmic granules) are metachromatic structures.Chapter 1 b. Methods Used in Histology and Cell Biology 3 (1) When a dye such as toluidine blue binds to tissue substances that occur in low concentrations, the substance stains orthochromatically (blue). (2) When the same dye binds to tissue substances that occur in high concentrations, the substance stains metachromatically (purple). Cartilage staining is an example that clearly illustrates metachromasia. (1) Areas of cartilage extracellular matrix containing high concentrations of chondroitin sulfate, which contains numerous sulfate (SO,,7~) groups and consequently has a strong negative net charge, are metachromatic. (2) Cartilage cell nuclei contain lower concentrations of negatively charged moieties (e.g., DNA) and are, therefore, orthochromatic. 3. Periodic acid-Schiff (PAS) reaction a. The PAS reaction is used to identify carbohydrates by exposing tissue sections to periodic acid oxidation and then reacting them with Schiff’s reagent (essentially leukofuchsin in solution). b. For example, the PAS reaction can be used to identify glycogen (a glucose polymer stored in the cytoplasm of various cells) in liver cells. (1) During periodate oxidation, hydroxyl groups on the glucose oxidize into aldehyde. (2) The free aldehydes react strongly with bisulfite groups on leukofuchsin and yield a magenta condensation product. c. Other PAS-positive cell structures include the following. (1) The glycocalyx, the carbohydrate-rich coat surrounding many cells, is strongly PAS- positive because it contains an abundance of glycoproteins. (2) Most epithelia rest on a basement membrane that is strongly PAS-positive because it contains numerous glycoconjugate-rich macromolecules. d. Researchers use histochemical reactions in conjunction with PAS reaction to identify specific cellular constituents. (1) Glycogen, for example, can be distinguished from other PAS-positive cell substances by preincubating slides with a-amylase, an enzyme that destroys only glycogen. (Cell structures that contain glycogen are PAS-positive and a-amylase sensitive.) (2) Mild acid hydrolysis of DNA creates Schiff reagent reactive groups in the deoxyribose of DNA. This histochemical method of staining is a specific, sensitive test for DNA and is the basis for the Feulgen reaction. C. Special stains 1. Staining elastic fibers and reticular fibers a. Elastic fibers contain elastin (a protein) and stain heavily with orcein dye. Also, Weigert’s elastic stain selectively reveals elastic fibers. b. Reticular fibers (connective tissue fibers that exist in many organs) are a special kind of collagen fiber that has a rich coat of glycoproteins. They are especially prevalent in the liver, spleen, and lymph nodes. c. Reticular fibers are argyrophilic structures; their glycoproteins can reduce silver salts to silver metals. This reduction process deposits a black stain around these structures. 2. Fluorescent tags. Fluorescent molecules are used to tag antibodies to specific tissue antigens so they can be localized and examined in a fluorescence microscope. b. Fluorescent tags can be very specific for some intracellular or extracellular components. For example, antibodies can be prepared for specific proteins such as actin (intracellular) or collagen (extracellular). The antibodies are covalently coupled to fluorochromes (fluorescent chemicals), such as fluorescein isothiocyanate (FITC), and then bound to cell sections or whole cells where the tagged antibodies bind with specific antigens. 3. Histochemistry is used to localize enzymes within tissues. a. b. Typically, frozen sections are incubated in reaction mixtures that create reaction products from enzymatic activities in the specimen. Then, reaction products are precipitated in a way that minimizes diffusion away from the production site. For example, alkaline phosphatase activity in the renal brush border is demonstrated by incubating frozen sections with phosphorylated substrates in alkaline buffers and then precipitating the phosphates with lead salts, which appear black or brown in the sections.