HISTOLOGY, CYTOLOGY AND EMBRYOLOGY (a course of ..., Assignments of Histology

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HISTOLOGY, CYTOLOGY AND EMBRYOLOGY (a course of lectures)

• Lecture 1. Origin and Subiect Matter of Histology (V.L. Goryachkina) CONTENTS:
• Lecture 2. Membranous and Nonmembranous Organelles (V.L .Goryachkina)
• Lecture 3. Nucleus and Cell Cycle (V.L. Goryachkina)
  • Embryonic Development (T.V. Boronikhina) Lecture 4. Introduction to Tissues and Tissue Development. Initial Stages of
• Lecture 5. Epithelial Tissue (S.L. Kuznetsov)
• Lecture 6. Blood and Lymph (T.V. Boronikhina)
• Lecture 7. Connective Tissues (V.L. Goryachkina)
• Lecture 8. Cartilage and Bone (T.V. Boronikhina)
• Lecture 9. Muscle Tissues (V.L. Goryachkina)
• Lecture 10. Nervous Tissue – I (S.L. Kuznetsov)
• Lecture 11. Nervous Tissue – II (S.L. Kuznetsov)
• Lecture 12. Nervous System – I (S.L. Kuznetsov)
• Lecture 13. Nervous System – II (S.L. Kuznetsov)
• Lecture 14. Primary Sentient Sense Organs: The Eye and the Organ of Smell (T.V. Boronikhina)
• Lecture 15. Secondary Sentient Sense Organs: The Ear and the Organ of Taste (T.V. Boronikhina
• Lecture 16. Cardiovascular System – I (V.L. Goryachkina)
• Lecture 17. Cardiovascular System – II (V.L. Goryachkina)
• Lecture 18. Central Organs of Hemopoiesis: Red Bone Marrow and Thymus(V.L. Goryachkina)
• Lecture 19. Peripheral Organs of Hemopoiesis and Immunogenesis (V.L. Goryachkina)
• Lecture 20. Endocrine System – I (S.L. Kuznetsov)
• Lecture 21. Endocrine System – II (S.L. Kuznetsov)
• Lecture 22. Gastrointestinal Tract – I (S.L. Kuznetsov)
• Lecture 23. Gastrointestinal Tract – II (S.L. Kuznetsov)
• Lecture 24. Gastrointestinal Tract – III (S.L. Kuznetsov)
• Lecture 25. Respiratory System (V.L. Goryachkina)
• Lecture 26. Integumentary System: Skin and Its Appendages (V.L. Goryachkina)
• Lecture 27. Urinary System (S.L. Kuznetsov)
• Lecture 28. Male Reproductive System (T.V. Boronikhina)
• Lecture 29. Female Reproductive System – I (T.V. Boronikhina)
• Lecture 30. Female Reproductive System – II (T.V. Boronikhina)
• Lecture 31. Human Embryology – I (T.V. Boronikhina)
• Lecture 32. Human Embryology – II (T.V. Boronikhina)

Lecture 1 The Origin and the Subiect Matter of Histology (V.L. Goryachkina)

From its derivation, the word “histology” (the Greek histos – tissue, logia – study or science of) means the science of tissues. But what is a tissue? This was derived from the French tissu, which means a weave or texture. It was introduced in the language of biology by Bichat (1771-1802). He wrote a book on the tissues of the body, in which he named more than 20 tissues. However, he did not use the microscope to classify them. Seventeen years after Bichat’s death the term “histology” was coined by the microscopist Meier, who used microscopy for describing tissues. It was established that there were only four basic tissues: epithelium, connective tissue, muscle tissue, and nervous tissue. In the 17th century Robert Hooke built a compound microscope. He examined a thin slice of cork and observed that it was composed of tiny empty compartments. He named the small compartmenTS-cells. Subsequently, other biologists studied plant tissue with a microscope, and it became obvious that in living plants the small compartments contained a little jelly-like body. Furthermore, as animal tissues were studied by microscopy, it became obvious that they were composed of tiny jelly-like bodies. So by 1839, the cell doctrine was postulated independently by Schleiden and Schwann. The microscope revealed the cell to have two main parts. Living cells have a more or less central part. This central part was named its nucleus (the Latin nux - nut), because it reminded of a nut lying in the center of its shell. For the same reason the Greek prefix “ kary ” (the Greek karyon - nut) may also be used in words with reference to the nucleus. For example, the dissolution of the nucleus as a cell dies is called karyolysis (the Greek lysis denotes dissolution). A membrane was seen to enclose the nucleus, and it was named the nuclear membrane or envelope. One or more rounded, dark-staining bodies in the interior of the nucleus were each called a nucleolus. Tiny granules or clumps of dark-staining material scattered about within the nucleus were called chromatin (the Greek chrom – color) because of their affinity for certain dyes. The outer and generally larger part of the cell was called cytoplasm (the Greek “kytos” denotes something that is hollow or that covers; plasma, something molded). Now it is known that the cytoplasm contains organelles. These organelles lie suspended in the cytoplasmic matrix or cytosol. With the advent of electron microscopy it became possible to observe the cell membrane itself. The cell membrane received the name plasmalemma (derived from the Greek lemma meaning bark) for its resemblance to the bark of trees.

Cytoplasm The cytoplasm contains organelles (they are constant structures of the cells) and inclusions (they are inconstant structures). Organelles are described as membranous (membrane-limited) and nonmembranous. A.The membranous organelles include: (1) plasma (cell) membrane; (2) rough-surfaced endoplasmic reticulum (rER); (3) smooth-surfaced endoplasmic reticulum (sER); (4) Golgi apparatus; (5) mitochondria; (6) lyzosomes; (7) peroxysomes; B. The nonmembranous organelles include: (1) ribosomes (both those attached to the rER membrane and those free in the cytoplasm) (2) centrioles (and their derivatives); (3) filaments (of various varieties); (4) microtubules. Microtubules and filaments form the cytoskeleton elements.

Cell membrane The cell membrane is not visible under the light microscope, because the total thickness of the cell membrane is about 8 to 10 nm. With the electron microscope it appears as if it is formed of three layers: the outer dark layer, the inner dark layer, and the light layer (between the outer and the inner layers). That is why it was called the trilaminar membrane. Sometimes (with low resolution of electron microscope) we can observe only a dark line. The cell membrane is formed of phospholipids, proteins, and carbohydrates. The poshospholipid molecule is composed of two parts: hydrophobic and hydrophilic lipids. The outer dark layer and the

inner dark layer of the cell membrane are due to the presence of phospholipids. Large integral proteins are situated between phospholipids. They extend from one side of the cell membrane (the outer dark layer) to the other (the inner dark layer). Large integral proteins perform important functions in cell metabolism, regulation, and integration. They can serve as receptors of enzymes, a pump, or any combination of these functions. Peripheral proteins form a noncontinuous layer in the lipid layers (outer and inner). Cholesterol molecules are present in the cytoplasmic aspect of the cell membrane. Some carbohydrates are attached to large integral proteins and phospholipids. Carbohydrates attached to phospholipids are termed glycolipids; carbohydrates attached to proteins are termed glycoproteins. Glycolipids and glycoproteins form a cell coat or glycocalyx. This outer layer may be very thick or thin, depending on the cell function. Functions of the glycocalyx:

1. It takes part: (a) in cell recognition; (b) in the formation of intercellular adhesion (aids in holding adjacent cells together); (c) in adsorption of molecules to the cell surface.
2. It serves as receptor sites for hormones. Functions of the cell membrane: (1) Passive diffusion of small molecules. The cell membrane can allow dissolved gases, essential ions, and oxygen to pass into the cell. It also allows carbon dioxide and other metabolic wastes to leave the cell; (2) Active transport. In order to pass through the cell membrane, large molecules such as sugar, amino acids, and fatty acids should be combined with some catalysts. This combination requires energy. That is why this transport was termed active. (3) Selective transport. The presence of the receptors on the outer surface of the cell membrane allows it to select and determine which materials can enter the cell and which substances are to leave the cell; (4) Transport of different material by endocytosis is the process of vesicular transport when it involves substances entering the cell. Two forms of endocytosis are recognized:  phagocytosis (the Greek for cell eating ). For example, the ingestion of bacteria or other cells.  pinocytosis (the Greek for cell drinking ). For example, the ingestion of bulk fluid.

Phagocytosis: When a solid particle comes in contact with the cell membrane, the membrane gradually surrounds the particle from all it sides, forming a bag. This bag is pinched off from the cell membrane and moves inside the cytoplasm. It is called a phagosome.

Pinocytosis: The plasma membrane invaginates to form small pits or caveolae. Gradually these pits will separate from the cell membrane. They have been referred to as pinocytosis vesicles.

(5) Vesicular transport by exocytosis. Exocytosis is the name given to the process of vesicular transport when it involves substance leaving the cell. For example: the liver produces a very low density lipoprotein (VLDL), which is released to the blood through exocytosic vesicles. VLDL is a component of blood serum that controls the dispersal of serum lipids, a factor important in the development of atherosclerosis. (6) Sodium–potassium pump function. The cell membrane is continually pumping sodium ions to the outside of the cell. As a result the concentration of sodium is higher in the tissue fluid than in the cytoplasm. The concentration of potassium ions is higher in the cytoplasm than in the tissue fluid. The sodium–potassium pump is responsible for the cell membrane polarization. It keeps the cell membrane with a positive charge on the outside and with a negative charge on the inside.

Cell membrane modifications The cell membrane is fluid and a dynamic system, not a static structure. It participates in the functional and metabolic activities of the cell. It forms special structures (microvilli and cilia) and junctions. (1) Microvilli. These microvilli are finger-like projections from the apical pole of the surface of certain cells. They increase the surface of the apical poles of cells. Microvilli are found in cells active in the transport of material, such as absorptive cells in the intestinal epithelium, or secretory cells. Microvilli have very similar sizes and shapes regardless of the cell type. Microvilli contain actin microfilaments, which are anchored to the plasma membrane at the tip and sides of the microvillus and extend to the apical pole of the cell.

(2) Cilia. They are situated on the apical pole of the epithelial cells lining the trachea and bronchi. They are special motile structures associated with the extracellular transport of material such as mucus, debris or cells. Each cilium is composed of nine double microtubules and a pair of central microtubules. (3) Cellular junctions. There are some types of cellular junctions. A. The tight or occluding type. This type of junction is created by localized sealing of the outer leaflets of adjacent plasma membranes. These junctions prevent the passage of water between the adjacent cells. These junctions are located between the epithelial cells. B. Junctions of adherent type. In this type of junction there is no direct contact between the cell membrane. They are separated by a wide intercellular space (20 nm). This space is filled with adhesive material. These junctions provide lateral adhesion between epithelial cells: (a) zonula adherens, (b) macula adherens (desmosome), (c) hemidesmosomes (only on the basal part of the epithelial cells), and (d) fascia adherens (located only between the cardiac muscle cells). C. The gap junction appears as two parallel, closely apposed plasma membranes separated by a gap of 2nm. Many special channels (or passageways) between two cells are packed at the site of the gap junction. Gap junctions allow small molecules and ions to pass directly between the cells without entering the extracellular space. These junctions are located between (a) epithelial cells, (b) smooth muscle cells, and (c) cardiac muscle cells.

Cytoskeleton The cytoskeleton is associated with the cell membrane more than the other organelles. The cytoskeleton consists of two classes of structural elements: microtubules and filaments. A. Microtubules. They are present in all kinds of the cells. They are cylindrical structures. They are formed of protein known as tubulin. Most of the microtubules are anchored in a region near the cell centriole, from which spindle fibers radiate. Microtubules are found in mitotic spindle fibers, centrioles, axoneme of cilia, basal bodies of cilia, elongating cell processes (such as axons), and cytoplasm, generally. Functions of microtubules: (1) cell migration (elongation and movement); (2) intercellular transport; (3) movement of chromosomes during mitosis; (4) maintenance of cell shape; (5) beating of cilia and flagella.

B. Filaments. They are divided into two groups: microfilaments and intermediate filaments. (a) microfilaments (actin and myosin filaments) In muscle cells, actin and myosin microfilaments are organized into specific myofibrils. In nonmuscle cells actin may constitute up to 10% of the total protein. Actin microfilaments are often grouped as bundles close to the cell membrane. It should be noted that actin filaments are visible with EM, whereas myosin is not. Yet myosin is also present, as shown by immunocytochemistry. Functions of microfilaments:

1. Anchorage and movement of the membrane proteins;
2. Movement of the plasmalemma (as in endocytosis and exocytosis);
3. Formation of the structural core of microvilli on absorptive cells;
4. Extension of cell processes;
5. Locomotion of cells. (b) intermediate filaments perform a mechanical function, i.e., the supporting or stiffening of cells and coordinating the activity of intracellular organelles. There are five major classes of intermediate filaments. This division depends on the special protein present in the tissues.
6. Keratin filaments occur in epithelial cells as tonofilaments (bundles of tonofilaments form tonofibrils).
7. The protein vimentin forms intermediate filaments that enmesh the nucleus. They appear to control the location of the nucleus. Vimentin filaments are present in the cells of mesenchymal origin (in connective tissue cells, some smooth muscle cells, and endothelial cells).
8. Desmin filaments are particulary abundant in smooth muscle cells.
9. Neurofilaments are present in neuronal cells.
10. Glial fibrillary acidic protein forms glial filaments. These filaments are present in neuroglial cells (special supporting cells that are in intimate physical contact with neurons).

Lecture 2 Membranous and Nonmembranous Organelles (V.L. Goryachkina)

Mitochondria The main function of mitochondria is known to provide energy for cells. They are found in large numbers in metabolically active cells. For example, liver cells, skeletal muscle fibers and cardiac muscle cells contain apporoximately 2000 mitochondria, whereas inactive cells contain very few mitochondria (lymphocytes). Note, that red blood cells and terminal keratinocytes of the epidermis do not contain mitochondria. Mitochondria can be elliptical, ovoid, spherical, discoidal, rod-shaped, etc. It is very difficult to observe them with light microscope (LM), because they measure 0.2 to 2 nm in width and 2 to 7 nm in length. It is necessary to use special staining to identify them with LM. Under the light microscope mitochondria are seen to be rods or threads, which accounted for their name (derived from the Greek mitos meaning “thread”). Each mitochondrion is covered with two membrane layers. The outer membrane is a unit membrane, about 5 to 7 nm thick. The outer membrane is thought to play a role in controlling the movement of substances into and out of the mitochondria and the uptake of substances and release of ATP. The inner membrane is a unit membrane too, about 5-7 nm thick. The inner membrane forms folds, which are termed cristae. Each crista projects into the inner chamber (mitochondrial matrix). Toward the matrix, the inner membrane encloses elementary particles. The enzymes for oxidative phosphorylation are located in these particles. The synthesis of ATP from ADP and phosphates takes place here. The matrix is filled with a fluid. Mitochondrial DNA and ribosomes are situated within the matrix. The enzymes for the Krebs cycle, lipid and protein synthesis, the enzymes for oxidation of fatty acids are located within the matrix too. In addition to enzymes, some granules are present in the matrix. These granules are the storage sites of Ca++^ and Mg++^ ions. Functions of mitochondria. Mitochondria are energy producers of cells. Energy is produced by the Krebs cycle and oxidative phosphorylation. Mitochondria also oxidize and synthesize fatty acids, concentrate ferritin, and accumulate cations in granules.

Rough-surfaced endoplasmic reticulum - granular reticulum (rER) Under the electron microscope rER appears as a series of interconnected, membrane-limited flattened sacs called cisternae with ribosomes studding to the exterior surface of the membrane. Under the light microscope rER is basophilic in staining due to the presence of the ribosomes. Some rER is present in every kind of the cell. However, the quantity of the rER cisternae depends on the cell function. It should be noted that any cell containing a well- developed rER will produce proteins for export. For example: fibroblasts of connective tissue wich secrete proteins and other substances that constitute the extracellular matrix (this matrix contains many proteins: collagen, elastin, etc.), and plasma cells wich make up and secrete immunoglobulins (antibodies). Pancreatic acinar cells are involved in the synthesis of proteins for export. Functions of rER: The rER takes part in the synthesis of (1) enzymes contained within the lysosomes; (2) integral proteins of the cell membrane; (3) proteins for export from the cell. Smooth-surfaced endoplasmic reticulum (sER) The sER consists of short anastomosing tubules that are not associated with ribosomes. The sER tubules are often continuous with rER. The amount of sER in the cells varies, depending on their type, and is prominent in only certain cell types. Functions: (1) The synthesis of lipids, cholesterol, lipoproteins, and steroid hormones. For example, liver cells containing a well-developed sER are known to produce the lipoproteins found in blood. (2) The synthesis of glycogen. Liver cells contain large amounts of sER (enzymes for glycogen formation are associated with sER). (3) Since the sER takes part in cholesterol production, it is indirectly involved in membrane formation. (4) Drug detoxification. The liver is known to be concerned with the detoxification of certain drugs. That is why hepatocytes contain many sER tubules. (5) Calcium storage. (6) Transport of different material within the cell.

Ribosomes They are formed in the nucleus. (1) Ribosomes consist of two subunits: (a) the small subunit contains one rRNA molecule (ribosomal RNA) and 24 different proteins; (b) the large subunit contains two molecules of rRNA and 40 proteins. (2) Messenger RNA (mRNA) passes through the cleft between subunits. Ribosomes may occur free in the cytoplasm or be attached to the membrane of endoplasmic reticulum. I. Free ribosomes. They are scattered freely in the cytoplasm and are responsible for its basophilia. Growing cells, cancer cells, dividing cells contain many free ribosomes. It should be noted that free ribosomes are divided into two types: monoribosomes and polyribosomes or polysomes. These polysomes were termed clusters, because they contain many ribosomes attached to mRNA. These polysomes together with mRNA form a spiral structure. II. Attached ribosomes. They are attached to the outer surface of the membrane of endoplasmic reticulum. Functions:  Free ribosomes synthesize proteins, which remain in the cell, e.g., (a) keratinocytes, (b) some precursors of erythrocytes, (c) muscle fibers contain a lot of free ribosomes. In addition, most mitochondrial enzymes are synthesized by free polysomes and transferred into that organelle.  Attached ribosomes synthesize proteins for export.

Golgi apparatus The Golgi complex consists of (1) flattened sacs (saccules), (2) a stack of sacs consisting of two faces:  the forming face associated with small transfer vesicles;  the maturing face; the formation of lysosomes and glycoproteins and the accumulation of secretory granules occur here. (3) transfer vesicles, which convey proteins to sacs (to the forming face). Functions of the Golgi apparatus:  formation of lysosomes  formation of glycoproteins  accumulation of secretory granules  excretion of secretory granules. Lysosomes In 1955 Cristian de Duve was the first to postulate from the biochemical data the existence of unusual organelles containing hydrolytic enzymes. De Duve and his coworkers proposed the name lysosome (the Greek lysis meaning dissolution) Lysosomes are present in almost all kinds of cells. Their number varies greatly from one cell to another, depending on its type and function. It was established that lysosomes contain nearly 100 different hydrolytic enzymes: proteases, acid phosphotase, etc. Morphofunctionally, lysosomes can be subdivided into primary lysosomes and secondary lysosomes. The lysosomes that bud off the maturing face of a stack of Golgi apparatus are termed primary lysosomes. They contain only enzymes; they are not involved in the digestive activity. Primary lysosomes are lined by a unit membrane; they measure from 25 nm to 0.05 mcm in diameter and contain a homogeneous, fine granular material. Primary lysosomes may interact with the material brought into the cell from the outside or with broken down organelles. The fusion of primary lysosomes with phagosomes results in the formation of secondary lysosomes. Thus, secondary lysosomes contain both the digestive enzymes and the material to be digested. Secondary lysosomes measure from 0.5 to 1.5 mcm in diameter and are enclosed by a unit membrane. When secondary lysosomes cannot digest the materials, they turn into debris-filled vacuoles that are called residual bodies. Residual bodies or tertiary lysosomes may remain in the cell throughout its life. For example, in nerve cells it is possible to see such bodies; in nerve cells, they have been called "the age pigment" or lipofuscin granules. Thus, lysosomes play a significant role in breaking down different materials in the cells. The absence of certain lysosomal enzymes can lead to the pathological accumulation of undigested substrate in residual bodies, resulting in some disorders referred to as lysosomal storage diseases. More than 20 storage diseases due to the deficient activity of lysosomal enzymes have been identified. For example, the

absence of lysosomal galactosidase in nerve cells produces concentric lamellated structures in residual bodies that accumulate in the nerve cells and interfere with normal function. In glycogen-storage disease, type II α-glycosidase is absent in lysosomes. As a result, hepatocytes become enlarged by stored glycogen-filled vesicles, which cannot be metabolized. A contrasting group of lysosomal diseases is due to the intracellular break up of lysosomes. For example, in gout or uratic arthritis, urate crystals form in the synovial cavities and other connective tissue spaces as a result of the genetically induced high level of uric acid in the body fluids. Leukocytes engulf these crystals. As they become incorporated into secondary lysosomes, these crystals disrupt the lysosomes, and as a result the latter lose the hydrolytic enzymes. The leukocytes are destroyed, and the enzymes, released into the tissue, induce inflammation characteristic of uratic arthritis. Thus, lysosomes are involved in intracellular digestion. Lysosomes not only maintain the health of normal cells but are also of great importance in the defense of the body against certain bacterial invaders. Peroxisomes (microbodies) They are present in all cells. Peroxisomes are lined by a unit membrane; they measure from 0.2 to 0.8 mcm in diameter. Peroxisomes contain nearly 20 enzymes: catalase, peroxidase, etc. Accounting for their name, peroxisomes generate hydrogen peroxide as an oxidation product. But hydrogen peroxide is quite toxic to cells, and peroxisomes, by their catalase content, break it down to oxygen and water. Peroxisomes are capable of generating energy. This process takes place during the oxidation of various substrates by molecular oxygen. This energy is dissipated, generating heat. Peroxisomes are involved in the metabolism of purine bases (parts of nucleic acids). It is noteworthy that most mammals oxidize uric acid to allantoin or allantoic acid. Their peroxisomes contain urate oxidase, organized as paracrystalline arrays of hollow tubules. But the peroxisomes of birds, monkeys, and humans lack this enzyme and metabolize purines only to uric acid, the end product of purine metabolism. High levels of uric acid cause gout, a painful disease with joint inflammation and chalky deposits, due to the disturbance of purine metabolism. Peroxisomes possess alcohol dehydrogenase and thereby degrade alcohol, thus reducing the degree of intoxication and alcohol-induced liver and nerve damage.

Another function of peroxisomes is the conversion of noncarbohydrates (amino acids and

fatty acids) to carbohydrates. This phenomen is called gluconeogenesis.

Centrioles These organelles are paired rod-shaped bodies. They lie close to the nucleus in the center of a cell. They were termed centrioles (derived from the Greek kentro, meaning “to a center” or “central location”). This part of the cell was called the cell center, centrosome or centrosphere. Each rod-shaped centriole consists of nine triplets of microtubules oriented parallel to a long axis of the organelle. The centrioles in resting cells are arranged at right angles to each other but are not connected. Centrioles are surrounded by dense material (centriolar satellites) connected with each triplet. Centrioles and centriolar satellites constitute a general microtubule-organizing center in both interphase and mitosis. The centriolar satellites appear to be the actual points of polar attachment of the microtubules of the mitotic spindle. The functions of centrioles are as follows. (1) They play an important role in cell division. Before division centrioles duplicate themselves. Each pair of centrioles migrates to both sides of the dividing cell. Then centrioles induce the formation of microtubules of the spindle apparatus. In additon, centrioles are responsible for their depolymerization shortening during anaphase. (2) Ciliogenesis: centrioles migrate toward the cell surface and induce the formation of the cilia and axonemes of the spermatozoon tails. Inclusions The cytoplasmic inclusions are “nonliving”components of the cell. They include glycogen, neutral fat and other lipid droplets, pigment granules, and secretory granules. Glycogen exists as deposits in cytoplasmic matrix. Glycogen may be seen with the light microscope (LM) only after special fixation and staining procedures. Glycogen appears in electron micrographs as granules of 25 to 30 nm in diameter and clusters of such granules. The liver and striated muscle fibers contain large amounts of glycogen. Lipid inclusions (fat droplets) are stored in adipose or fat cells. Fat sometimes accumulates in liver cells and other types of cells. The most important pigment is hemoglobin, an iron-containing pigment in red blood cells. Melanin is usually a brown-to-black pigment found chiefly in the skin and its appendages and in the eye. It is also present in the substantia nigra.

Lecture 3 Nucleus and Cell Cycle (V.L. Goryachkina)

Nucleus and cell cycle The nucleus is a membrane-limited compartment that contains genetic information. The nucleus of nondividing cells, also called the interphase nucleus, consists of the following components: (1) chromatin organized as euchromatin and heterochromatin, (2) the nucleolus (or nucleoli), (3) membranous nuclear envelope, (4) nuclear skeleton, and (5) nucleoplasma. Chromatin It stores genetic information. Chromatin is a complex of DNA and proteins. Chromatin proteins include basic proteins called histone and other nonhistone proteins. These proteins connect with the folding of DNA strands and take part in the regulation of DNA activity. Histones connect the first steps of the folding. The length of DNA strand is about two meters. But the volume of the nucleus is measured in only microns. Therefore, DNA must be packed in the nucleus. After the first folding the DNA length decreases sixfold. Nonhistones take part in the following steps of the DNA folding. The chromatin visible with LM as dense granules or clumps is called condensed chromatin. The chromatin invisible with LM is called extended chromatin. The DNA of extended chromatin is active in providing information to direct protein synthesis. Extended chromatin is termed euchromatin (good chromatin), because it works; condensed chromatin was termed heterochromatin or other kind of chromatin (derived from the Greek heteros meaning “other”). Heterochromatin is disposed in three locations: (1) marginal heterochromatin is found on the periphery of the nucleus, (2) central heterochromatin is scattered as irregular clumps, and (3) perinuclear chromatin. Heterochromatin predominates in metabolically inactive cells (lymphocytes). Euchromatin predominates in active cells (nerve cells). Chromatin represents the DNA-protein complex of chromosomes, which are visible in dividing cells. Chromosomes are permanent components of the nucleus composed of chromatin fibers visible with EM. Chromatin fibers consist of nucleosomes interconnected by internucleosomal DNA (iDNA). Nucleosomes are the smallest units of chromatin. It is composed of a central globular histone octamer around which a DNA double helix winds in two full turns. This DNA in direct contact with histones was termed nucleosomal DNA (nDNA). Eight nucleosomes form nucleomeres. The following levels of the arrangement are chromonemma, chromatid, and chromosome. Nobody knows the arrangement of these structures. Nucleolus The nucleolus is a portion of chromosome. The nucleolus mainly consists of RNA and ribonucleoproteins; a small amount of DNA in the form of perinucleolar and intranucleolar chromatin; RNA polymerase and other enzymes, which are responsible for the synthesis of rRNA. Basophilia is due to the presence of RNA and DNA. The nucleolus is formed by (1) filamentous material (pars fibrosa) and (2) granular material (pars granulosa). The pars fibrosa consists of dense filaments about 5 nm thick, predominantly situated in the interior of the nucleolus. The pars granulosa consists of small particles 10 to 15 nm in diameter. Both the pars granulosa and the pars fibrosa contain RNA. The network formed by the pars granulosa and the pars fibrosa is called nucleonemma. (3) Perinucleolar chromatin represents the patches of heterochromatin attached to the periphery of the nucleolus. (4) Intranucleolar chromatin, single or twisted DNA filaments that penetrate the pars fibrosa. (5) The nucleolus matrix contains protein. The function of the nucleolus is to synthesize rRNA. Nuclear envelope Under the LM, the nuclear envelope appears as a single line. Under the EM, two membranes can be observed: outer and inner. The space between these membranes was termed the perinuclear cisternal space. The outer membrane is often rough, because it has many ribosomes on its surface. The outer membrane is continuous with the rER membranes. The inner membrane lies adjacent to the fibrous nuclear lamina. This lamina contains special filaments and provides internal support for chromatin, chromatin-associated proteins, the inner nuclear membrane, and nuclear pores. It should be noted that during cell division the nuclear envelope breaks into vesicles, which reconstitute the nuclear envelope at the end of the anaphase and in the telophase. Nuclear pores Nuclear pores are seen as round or octagonal interruptions of the nuclear envelope. Every nuclear pore contains 8 granules, which form the so-called annulus. From the pores, the filamentous material

extends into both the cytoplasm and the nucleus. The pores are closed by the diaphragm with a central granule. The pore and annulus together are called the nuclear pore complex. Nuclear pores may occupy 3 to 35% of the nuclear surface. Functions of nuclear pores: (1) They regulate the passage of proteins between the nucleus and cytoplasm. For example, some nuclear proteins (histones, lamines) are produced in the cytoplasm. Thus, nuclear pores serve for the passage of these substances into the nucleus. (2) They also regulate the passage of mRNA and ribosomes into and out of the nucleus. Nuclear skeleton The nuclear skeleton (matrix) is a sponge structural framework (consisting of proteins). It is formed by 2- to 3-nm thick microfibers associated with microfibrils 20 to 30 nm in diameter. Both fibrils are connected with the nucleolus, the inner membrane, the lamina fibrosis, and the nuclear pore. The nuclear cytoskeleton is believed to play a role in the arrangement of the chromatin of meiotic and mitotic chromosomes. Nucleoplasm The nucleoplasm consists of nucleoproteins and a number of enzymes, which participate in the DNA and RNA synthesis. Functions of the nucleus:

1. Regulation of cell differentiation, maturation, and function.
2. Replication and transmission of genetic message to new cells in spite of cell divisions.
3. The synthesis of messenger, transfer, and ribosomal RNA and their transport into the cytoplasm. The cell cycle An essential feature of the cells is their ability to divide and reproduce. In addition, after cell death, an adult needs to be compensated for by the production of new cells. Cells reproduce due to duplication of their contents and division. The phases involved in cell replication can be regarded as a cell cycle. The cell cycle is the sum of morphological and biochemical events occuring in the life of a cell between its formation and the end of its division into two daughter cells. The cell cycle is divided into two main phases: mitosis and interphase. Interphase is divided into 3 phases. The interval after mitosis is called the first “gap” or G 1 phase. During this phase, the cell growth, protein (enzymes) and RNA synthesis, as well as the formation of receptors on the cell surface take place. During the "S" phase, DNA reduplication (DNA synthesis) and hystone synthesis occur. The interval between the S phase and mitosis is called the second “gap” or G 2. G 2 is a short phase of preparation for mitosis. The synthesis of proteins (tubulins) for the spindle apparatus occurs during this phase. In most tissues, the majority of the cells have to differentiate after mitosis. The beginning of differentiation is associated with the cell leaving the cycle. They are said to be in the G 0 (“outside”) phase. As a rule, G 0 cells leave the cycle from the G 1 phase. Occasionally, some cells leave the cycle in the G 2 phase. The cells of the adult body can be classified into three populations: static, stable, and renewing according to their mitotic activity. (1) Static cell population consists of cells that will not divide (nerve cells, cardiac muscle cells). (2) Stable cell population consists of cells that divide episodically when they are stimulated by injury to become more mitotically active (liver cells). (3) Renewing cell population: blood cells, epidermis, epithelial cells of the lining the alimentary canal. After division, one daughter cell will differentiate; the other will remain in G 0 phase or rest phase. The latter cell was called “stem cell” or “reserve cell”. After division of a stem cell, the formation of two types of the cell occurs: a new stem cell in the G 0 phase (to maintain the stem cell population) and a daughter cell (which differentiates along one cell line). Stem cells may be in the G 0 phase and only come to reenter the cycle if there is a demand (e.g., following cell death). It should be noted that every cell has its own program of activity and death. Approximately 20 years ago a special gene was discovered in DNA. It was called a lethal gene or the gene of death. When this gene begins to work, the DNA is the first to be destroyed, then the nucleus is broken into fragments. This process was called apoptosis. Apoptosis is a normal process of programmed cell death. This mechanism of death is induced in cells when they are damaged, useless to the body, or dangerous. Apoptosis is the key mechanism of the developing organism. For example, our fingers are separated, because the embryonic cells that joined them died during development by apoptosis. Apoptosis takes place not only during the embryonal development, but in adult life. For example, this process can be observed in mammary glands. During pregnancy and lactation they are large, but in the absence of sucking the secretion of milk ceases, and the mammary glands begin to regress due to apoptosis.

Lecture 4 Introduction to Tissues and Tissue Development Initial Stages of Embryonic Development (T.V. Boronikhina)

Tissues are aggregates of cells and extracellular material organized to perform distinctive functions. There are four basic tissue groups: epithelial tissues; a group of internal medium tissues including blood, lymph, and all types of connective tissues; muscular tissues; and nervous tissue. All cells of the mature organism and all tissue types are derived from a single cell called a zygote. Two processes occur in this development: cell proliferation (division) and cell differentiation. Differentiation is the development of specialized cell types from stem cells, during which changes in the cell structure and biochemistry occur for the cell to perform distinctive functions. The process of cell determination precedes cell differentiation. Determination is the process of a cell’s particular fate choice, when the cell differentiation pathway is chosen. The differential genome activity underlies determination. Each cell contains a complete genome established in the zygote during fertilization, the DNAs of all cells are identical. As cells develop, the genetic material does not change but some genes are expressed (activated) and others repressed (inactivated). Only a small percentage of the genome is expressed in each cell, so that a portion of RNA synthesized by this cell type is specific. Because determination occurs step-wise, the cells can exist in different states of differentiation and possess different potentialities. Potentiality is the capability of a cell for differentiation that has not yet been realized. According to their potentialities, cells may be distributed in some populations. Stem cells are pluripotential; they usually give rise to several cell types. Their immature offspring is oligopotential; their potentialities become less. Mature cells are unipotential; they have chosen the only way of differentiation. The zygote is a totipotential cell, because it serves as the progenitor for all the kinds of body cells (single produces whole). Embryonic development Cell differentiation and tissue development begin in embryogenesis. The embryogenesis includes the following stages: fertilization, cleavage, gastrulation, differentiation of germ layers and formation of the axial organs, and the last period (the longest and most complex) of histogenesis and organogenesis. Fertilization Fertilization is the sequence of events, by which a sperm fuses with an ovum to form a unicellular organism called a zygote. The mature sperm is a microscopic, free-swimming, and actively motile cell consisting of a head and a tail or flagellum. The head, forming most of the bulk of the sperm, includes the nucleus whose chromatin is greatly condensed. The anterior two thirds of the nucleus is covered by the acrosome, a membrane-limited organelle containing the enzymes that facilitate sperm penetration of the corona radiata and zona pellucida during fertilization. The sperm tail consists of three segments: the middle piece, the principal piece, and the end piece. The junction between the head and the tail is called the neck. The tail provides the sperm motility, which assists in its transport to the site of fertilization. The middle piece of the tail contains the mitochondria generating the energy for sperm motility. With respect to the sex chromosome constitution, there are two kinds of normal sperm: 22 + X and 22 + Y. The mature ovum is the secondary oocyte arrested at the metaphase of the second meiotic division (female meiosis may be completed only after fertilization). The secondary oocyte released at ovulation is surrounded by the zona pellucida and a layer of follicular cells called the corona radiata. Compared with ordinary cells and, notably, the sperm the ovum is truly large. It has an abundance of cytoplasm containing organelles, yolk granules, RNA, morphogenetic factors, and cortical granules. The ovum nucleus is haploid (22 + X), euchromatic, and metabolically active. The ovum is immotile and transported down the oviduct passively by the movement of cilia. The usual site of human fertilization is the uterine tube ampulla. The fertilization process requires from 12 to 24 hours. Capacitation of sperms Before a mature motile sperm can penetrate the oocyte surroundings, it must undergo capacitation. This process consists of enzymatic changes that result in the removal of a thick glycoprotein coat formed during sperm incubation in the epididymis from the plasma membrane over the acrosome. This coat is known to prevent the early acrosome reaction. No morphological changes are known to occur during the capacitation process. The capacitating ability inheres in some substances of the female genital tract secretion. It takes sperms about 7 hours to be capacitated.

Acrosome reaction The acrosome reaction may occur after sperm capacitation. This reaction consists of structural changes. The outer membrane of the acrosome fuses at many places with the overlying cell membrane of the sperm head, and the fused membranes then rupture, producing multiple perforations through which the enzymes leave the acrosome. The enzymes released from the acrosome facilitate the passage of the sperm through the oocyte envelopes. Hyaluronidase enables the sperm to penetrate the corona radiata. Acrosin appears to cause lysis of the zona pellucida, thereby forming a pathway for the sperm. Penetration The sperm head is attached to the surface of the secondary oocyte. The plasma membranes of the oocyte and sperm fuse and then break down at the point of contact. The sperm nucleus and centriole enter the ovum cytoplasm, leaving the sperm’s plasma membrane and its tail outside, attached to the oocyte plasma membrane, where they rapidly degenerate. Cortical reaction or zona reaction As soon as the first sperm passes through the zona pellucida, a zona reaction occurs. The zona reaction is produced by cortical granules containing lysosomal enzymes. After penetration, the cortical granules open and release the enzymes that modify the physicochemical characteristics of the zona pellucida, converting it to impermeable to other sperms and preventing polyspermy. Although several sperms may penetrate the zona pellucida, usually only one sperm enters the ovum and fertilizes it. There is some experimental evidence that aged oocytes do not release cortical granules. As a result, the zona reaction does not take place, and multiple penetrations of sperms occur. Polyspermy results in the development of a nonviable embryo. Events after penetration The secondary oocyte completes the second meiotic division, forming a mature ovum and the second polar body. The ovum nucleus is known as the female pronucleus. The sperm nucleus enlarges to form the male pronucleus. The human zygote looks like a synkaryon. Then, the male and female pronuclei approach each other in the centre of the zygote, where they come in contact and lose their nuclear membranes. The maternal and paternal chromosomes intermingle at the metaphase of the first mitotic division. Fertilization is completed. Biological significance of fertilization The restoration of the diploid number takes place: the fusion of two haploid germ cells produces a zygote, a diploid cell with 46 chromosomes, the usual human number. Because half of the chromosomes come from the mother and half from the father, the zygote contains a new combination of chromosomes and genetic material that is different from those of the parents. Moreover, the embryo sex is determined at fertilization by the kind of the sperm that fertilizes the ovum. Fertilization by an X-bearing sperm produces an XX zygote, which normally develops into a female embryo. Fertilization by a Y-bearing sperm produces an XY zygote, which normally develops into a male embryo. Fertilization initiates a series of rapid mitotic cell divisions called the zygote cleavage. The cleavage of a secondary oocyte may occur without fertilization. This process is called parthenogenesis. There is evidence that the human oocyte may start to undergo parthenogenetic cleavage, but this does not result in organized development. Cleavage Cleavage is a process of successive rapid mitotic divisions without the growth of the daughter cells called blastomeres. As G1 phase in these mitotic cycles is not present, the cell cytoplasm volume does not increase; blastomeres become progressively smaller until they acquire the size of most of the somatic body cells. Thus, the normal nucleus to the cytoplasm volume ratio is restored. That is why this stage of development is termed segmentation or cleavage, but not division. Blastomeres do not leave the cell cycle; they never begin to differentiate. They do not use their genome; they synthesize proteins on maternal RNA. Through some first mitotic divisions, blastomeres retain the zygote property, namely, totipotentiality. Occasionally, two or more blastomeres are separated, and each develops into an embryo. Thus, identical twins appear. Initially, the embryo is under the control of maternal informational macromolecules that have accumulated in the ovum cytoplasm during oogenesis. Later, development depends on the activation of the embryonic genome, which encodes various growth factors and other macromolecules required for normal progression to the blastocyst stage.

Human cleavage occurs in the oviduct for 3 days and then in the uterus for 3 to 4 days. Cleavage begins with the first mitotic division of the zygote, then, the morula – a solid ball of 12 to 16 blastomeres that looks like a mulberry – is formed. The morula enters the uterus as it is forming. Cleavage ends with the blastula formation. Human cleavage is holoblastic (total); it means that the entire cytoplasm is cleft. Human cleavage is unequal; it means that blastomeres are different in size, i.e., small and large cells. Human cleavage is asynchronous; it means that blastomeres divide at different time and may be even or odd in number. Blastocyst formation Some fluid passes into the morula forming a cavity; blastomeres are also able to produce fluid. As fluid increases, it separates the morula cells into two parts: (1) an outer cell layer called the trophoblast consisting of small light blastomeres, from which the chorion and the placenta develop; and (2) an inner cell mass called the embryoblast consisting of large dark blastomeres, from which the embryo proper and some provisional organs arise. The human blastula looks like a cyst and is called the blastocyst: the trophoblast forms its wall; the embryoblast is attached to the inner side of the trophoblast and projects into the fluid-filled blastocyst cavity. The blastocyst is still surrounded by the zona pellucida and lies free in the uterine secretion before implantation. Gastrulation Gastrulation is a process of highly integrated cell movements whereby the germ layers, namely, the ectoderm, endoderm, and mesoderm, are formed. The gastrulation events are as follows: extensive cell rearrangement and segregation, mitotic cell divisions, and cell differentiation. These events result in the formation of the germ layers. In mammalian species, gastrulation is a two-stage process, and all morphological changes take place only in the embryoblast. During the first stage of gastrulation (on the 7th day of development), the embryoblast is transformed into a bilaminar embryonic disk by the mechanism called delamination. The embryonic disk is composed of a superior layer called the epiblast consisting of high columnar cells and an inferior layer called the hypoblast consisting of cuboidal cells adjacent to the blastocyst cavity. The epiblast gives rise to all the three germ layers of the embryo (the ectoderm, mesoderm, and endoderm). The hypoblast is probably displaced to extraembryonic regions. During the second stage of gastrulation (on the 15th day of development), morphological changes take place only in the epiblast; the hypoblast does not take part in embryo formation. The second stage of gastrulation is characterized by the formation of the primitive streak. This structure forms as follows: epiblastic cells from the cranial embryo end proliferate and migrate to the caudal embryo end along the disk margins and converge at the caudal end of the disk; these cell currents then turn toward the midline and elongate back, to the cranial end. Concurrently, a narrow primitive groove develops on the top of the primitive streak. Since the primitive streak appears, it is possible to identify the embryo’s craniocaudal axis, its dorsal and ventral surfaces, and its right and left sides. The primitive streak is the origin of the embryonic mesoderm and the embryonic endoderm. The epiblastic cells move along the primitive streak and enter the primitive groove. They lose their attachment to the rest epiblastic cells and migrate inward between the epiblast and the hypoblast. The early-migrating cells are those that replace the hypoblastic cells to become the endoderm. The later-migrating cells begin to spread laterally, ventrally, and cranially to form a layer called the mesoderm. As soon as the primitive streak gives rise to the mesoderm and the endoderm, the cells that remain in the epiblast are referred to as the embryonic ectoderm. Thus, the gastrulation is completed; as a result, the embryo looks like the trilaminar disk. Differentiation of the germ layers and the formation of the axial organs The complex of the axial organs includes the following structures: the notochord, the neural tube, and the mesodermal somites. At this point, the last stage of embryogenesis begins. The interaction between the germ layers is important for initiating histogenesis and organogenesis, i.e., the creation of specific tissues and organs. Firstly, the notochord is formed, concurrently with the second stage of gastrulation. The cranial end of the primitive streak thickens to form the primitive knot (Hensen’s nodule) with a central indentation known as the primitive pit. The primitive pit then extends through the epiblast to form the notochordal canal. Cell masses surrounding the primitive knot migrate through this canal, reach the hypoblast, and turn cranially to form the cellular rod or the column-like process called the notochord. It grows between the ectoderm and endoderm, from the primitive knot toward the cranial end of the disk. The wing-like mesoderm is on each side of the notochordal process.

The functions of the notochord are as follows: (1) it forms a midline axis of the embryo; (2) it does not give rise to the skeleton (the skeleton arises from the sclerotome), but it is the structure, around which the vertebral column forms; the notochord degenerates and disappears where it is surrounded by the vertebral bodies, but persists as the nucleus pulposus of the intervertebral disks; (3) it also induces the overlying ectoderm to form the neural plate, i.e., the embryonic induction of neurulation. Neurulation or the neural tube formation includes the formation of the neural plate, the neural groove with two folds, and the fold closure forming the neural tube. As the notochord develops, the embryonic ectoderm over it thickens to form the neuroectoderm or the neural plate. The developing notochord and the mesoderm on each side of it induces this process. On about the 18th day, the neural plate invaginates along the central axis to form the neural groove with two neural folds on each side. By the end of the third week, the neural folds begin to converge and fuse, converting the neural groove to the neural tube. The neural tube is then separated from the ectoderm that is referred to as the surface (covering) ectoderm and differentiates into the skin epidermis. The neural tube is the primordium of the central nervous system consisting of the brain and the spinal cord. As the neural folds fuse, some ectodermal cells lying along and over each fold are not incorporated in the neural tube. They appear as a cell mass between the neural tube and the covering ectoderm, constituting the neural crest. The latter gives rise to the spinal ganglia and the ganglia of the autonomic nervous system, as well as Schwann cells, the meningeal covering of the brain and the spinal cord (the pia mater and the arachnoid), the skin pigment cells, and the adrenal gland medulla. The mesoderm on each side of the notochord and the neural tube thickens to form the longitudinal columns of the paraxial mesoderm. Each paraxial mesoderm is continuous laterally with the intermediate mesoderm, which gradually thins laterally into the lateral mesoderm. The paraxial mesoderm begins to divide into paired cuboidal bodies called somites. This series of mesodermal tissue blocks is located on each side of the developing neural tube. Somites are then subdivided into three regions: myotome that gives rise to skeletal muscles; dermotome that develops into the skin dermis, and sclerotome, from which bone and cartilaginous tissues arise. The intermediate mesoderm (somite cords) differentiates into nephrogonadotome that develops into the kidneys and gonads. Within the lateral mesoderm the space called the coelom appears, dividing the lateral mesoderm into two layers: (1) the parietal layer, somatopleure, and (2) the visceral one, splanchnopleure. The coelom is then divided into the following body cavities: the pericardial, pleural, and peritoneal ones. The cells of the parietal and visceral layers give rise to the mesothelium lining these cavities. The splanchnopleure takes part in the development of the heart (the myocardium and the epicardium are derived from it) and the adrenal gland cortex. Some cells from the mesoderm migrate and are disposed among the axial organs, they form a loose origin called the mesenchyme. The mesenchyme gives rise to the blood, all types of connective tissue, smooth muscle cells, blood vessels, microglial cells, and endocardium. The embryonic endoderm develops into the epithelium of the gastrointestinal tract, the liver, the pancreas, the gallbladder, and the epithelial parts of the lungs.

Lecture 5 Epithelial Tissue (S.L. Kuznetsov)

The epithelia are a diverse group of tissues, which, with rare exceptions, cover or line all body surfaces, cavities, and tubes. Epithelia thus function as interfaces between different biological compartments. As such, epithelia mediate a wide range of activities such as selective diffusion, absorption and/or secretion, physical protection, and containment; all these major functions may be exhibited at a single epithelial surface. For example, the epithelial lining of the small intestine is primarily involved in absorption of the products of digestion, but the epithelium also protects itself from intestinal contents by the secretion of a surface coating of mucus. All epithelia are supported by a basement membrane. Basement membranes separate epithelia from underlying supporting tissues and are never penetrated by blood vessels; epithelia are thus dependent on the diffusion of oxygen and metabolites from adjacent supporting tissues. The classification of epithelia is based on: (1) the number of cell layers: a single layer of epithelial cells is termed simple epithelium, whereas epithelia composed of several layers are termed stratified epithelia, and (2) the shape of the component cells: this is based on the appearance in sections taken at right angles to the epithelial surface. In stratified epithelia, the shape of the outermost layer of cells determines the descriptive classification. Cellular outlines are often difficult to distinguish, but the shape of epithelial cells is usually reflected in the shape of their nuclei.

Epithelia may be derived from ectoderm, mesoderm or endoderm, although in the past it was thought that true epithelia were only of ectodermal or endodermal origin. Two types of epithelia derived from mesoderm, i.e., the lining of blood and lymphatic vessels and the linings of the serous body cavities, were not considered to be epithelia and were termed endothelium and mesothelium, respectively. By both morphological and functional criteria, such distinction is of little practical value; nevertheless, the terms endothelium and mesothelium are still used to describe these types of epithelium. Simple epithelia Simple squamous epithelium is composed of flattened, irregularly shaped cells forming a continuous surface. The term “squamous” derives from the comparison of the cells to the scales of a fish. Like all epithelia, this delicate lining is supported by an underlying basement membrane. The basement membrane is rarely thick enough to be seen with routine light microscopy. Simple squamous epithelium is found to line the surfaces involved in passive transport (diffusion) of gases (as in the lungs) or fluids (as in the walls of blood capillaries). Simple squamous epithelium also forms the delicate lining of the pleural, pericardial, and peritoneal cavities where it permits the passage of tissue fluid into and out of these cavities. Reflecting the minimal metabolic activity of these cells, the nuclear chromatin is condensed and the cytoplasm contains few organelles. Simple cuboidal epithelium represents an intermediate form between simple squamous and simple columnar epithelium. The distinction between tall cuboidal and low columnar epithelium is often very slight. In section perpendicular to the basement membrane, the epithelial cells appear square, leading to its traditional description as cuboidal epithelium; on surface view, however, the cells are actually polygonal in shape. The nucleus is usually round and located in the centre of the cell. Simple cuboidal epithelium usually lines small ducts and tubules, which may perform excretory, secretory, or absorbtive functions; examples are the small collecting ducts of the kidney, salivary glands, and the pancreas. Simple columnar epithelium is similar to simple cuboidal epithelium, except that the cells are taller and appear columnar in sections at right angles to the basement membrane. The height of the cells may vary from low to tall columnar, depending on the degree of functional activity. The nuclei are elongated and may be located at the base, in the centre or, occasionally, in the apical part of the cytoplasm. This is known as polarity. Simple columnar epithelium is most often found on highly absorptive surfaces such as in the small intestine, although it may constitute the lining of highly secretory surfaces such as that of the stomach. Simple columnar ciliated epithelium as a type of simple columnar epithelium is traditionally described separately because of the presence of surface specializations called cilia on the apical surface of the majority of the cells. Among the ciliated cells there are some nonciliated cells, which usually perform a secretory function. Cilia are much larger than microvilli and are badly visible with the light microscope. Each cillium consists of a finger-like projection of the plasma membrane, its cytoplasm containing a motile specialization of the cytoskeleton. Each cell may have up to 300 cilia that, along with those of other cells, beat in a wavelike manner, generating a current, which propels fluid or minute particles over the epithelial surface. Simple columnar ciliated epithelium is not common in humans, except in the female reproductive tract. Another variant of simple columnar epithelium is described in which the majority of the cells are also usually ciliated. The term pseudostratified is derived from the appearance of this epithelium in section, which conveys the erroneous impression that there is more than one layer of cells. In fact, this is a true simple epithelium, since all the cells rest on the basement membrane. The nuclei of these cells, however, are disposed at different levels, thus creating the illusion of cellular stratification. Not all the ciliated cells extend to the luminal surface; such cells are capable of cell division, providing replacements for lost or damaged cells. Pseudostratified columnar ciliated epithelium may be distinguished from true stratified epithelia by two characteristics. Firstly, the individual cells of the pseudostratified epithelium exhibit polarity. Secondly, cilia are never present on stratified epithelia. Pseudostratified epithelium lines the larger airways of the respiratory system in mammals and is therefore often referred to as respiratory epithelium. Stratified epithelia Stratified epithelia consist of two or more layers of cells. They mainly perform a protective function, and the degree and nature of the stratification is related to the kinds of physical stresses to which the surface is exposed. The classification of stratified epithelia is based on the shape and structure of the surface cells, since the cells of the basal layer are usually cuboidal in shape.

Stratified squamous epithelium consists of a variable number of cell layers, which exhibit transition from a cuboidal basal layer to a flattened surface layer. The basal cells divide continuously. During the process, the cells undergo first maturation, then degeneration. The surface cells show the process of degeneration; this is particularly evident in the nuclei, which become progressively condensed (pyknotic) and flattened before ultimately disintegrating. Stratified squamous epithelium is well adapted to withstand abrasion, since the loss of surface cells does not compromise the underlying tissue. It is poorly adapted to withstand dessication. This type of epithelium lines the oral cavity, pharynx, esophagus, anal canal, uterine cervix and vagina, i.e., the sites, which are subject to mechanical abrasion but which are kept moist by glandular secretion. The specialized form of stratified squamous epithelium constitutes the epithelial surface of the skin (epidermis) and is adapted to withstand the constant abrasion and desiccation to which the body surface is exposed. During maturation, the epithelial cells undergo a process called keratinization, resulting in the formation of a tough, non-cellular surface layer, consisting of the protein named keratin. Keratinization may be induced in normally non-keratinizing stratified squamous epithelium such as that of the oral cavity when it is exposed to excessive abrasion or desiccation. Transitional epithelium is a form of stratified epithelium that lines the urinary tract in mammals where it is highly specialized to accommodate a great degree of stretch and to withstand the toxicity of urine. This epithelial type is so named, because it has some features, which are intermediate (transitional) between stratified cuboidal and stratified squamous epithelia. In the contracted state, transitional epithelium appears to be about four to five cell layers thick. The basal cells are roughly cuboidal, the intermediate cells are polygonal, and the surface cells are large and rounded and may contain two nuclei. In the stretched state, transitional epithelium often appears only two or three cells thick, and the intermediate and surface layers are extremely flattened. The intercellular, luminal, and basal surfaces of epithelial cells exhibit a variety of specializations. Intercellular surfaces of epithelial cells are linked by several different types of membrane and cytoskeletal specializations. These cell junctions permit the epithelia to form a continuous cohesive layer, in which all of the cells “communicate” and cooperate to meet the particular functional requirements of the epithelium. Occluding junctions, also known as tight junctions, are located immediately beneath the luminal surface of simple columnar epithelium (e.g., intestinal lining), where they seal the intercellular spaces so that the luminal contents cannot penetrate between the lining cells. Each tight junction forms a continuous circumferential band or zonule around the cell and is thus also known as a zonula occludens. Adhering junctions tightly bind the constituent cells of the epithelium together and act as anchorage sites for the cytoskeleton of each cell so that the cytoskeletons of all cells are effectively linked into a single functional unit. Adhering junctions are of two morphological types. Deep to the tight junctions of columnar epithelial cells, an adhering junction forms a continuous band (the zonula adherens) around the cell, providing structural reinforcement to the occluding junction. Secondly, adhering junctions in the form of small circular patches or spots called desmosomes (the macula adherens) are circumferentially arranged around columnar cells deep to the continuous adhering junction. The combination of the zonula occludens, the zonula adherens, and circumferentially arranged desmosomes is known as the ajunctional complex. Desmosomes (spot adhering junctions) are also widely scattered elsewhere in epithelial intercellular interfaces, binding the whole epithelial mass into a structurally coherent whole. Adhering junctions and communicating junctions are not exclusive to epithelia and are also present in cardiac and visceral muscle where they appear to serve similar functions. The luminal surfaces of epithelial cells may incorporate three main types of specialization: cilia, microvilli and stereocilia. Cilia are relatively long, motile structures, which are easily resolved by light microscopy. In contrast, microvilli are short, often extremely numerous projections of the plasma membrane, which cannot be individually resolved with the light microscope. Stereocilia are merely extremely long microvilli usually found only singly or in small numbers in odd sites such as the male reproductive tract. Basal surfaces. The interface between all epithelia and underlying supporting tissues is marked by a non-cellular structure known as the basement membrane, which provides structural support for epithelia and constitutes a selective barrier to the passage of materials between the epithelium and supporting tissue. The basal plasma membranes of some simple epithelia, which are very active in ion transport (e.g., the cells of the kidney tubules), exhibit deep basal folds. These greatly enhance surface area and provide an arrangement by which the energy-providing mitochondria can be situated in intimate association with

the plasma membrane. Hemidesmosomes, a variant of the desmosome, are present on the inner aspect of the basal plasma membrane adjacent to the basement membrane and provide a means of anchorage of the cytoskeleton to the basement membrane and underlying supporting tissue. Glandular epithelia The epithelium, which is primarily involved in secretion, is often arranged into structures called glands. Glands are the invaginations of epithelial surfaces, which are formed during embryonic development by proliferation of epithelium into the underlying tissue. The glands, which maintain their continuity with the epithelial surface, discharging their secretion onto the free surface via a duct, are called exocrine glands. In some cases, the glands have no ducts. The secretory products of such glands, known as endocrine glands, pass into the bloodstream. Their secretions are known as hormones. Nevertheless some endocrine glands develop by migration of epithelial cells without the formation of a duct. Exocrine glands may be broadly divided into simple and compound glands. Simple glands are defined as those with a single, unbranched duct. The secretory portions of simple glands have two main forms, tubular or acinar (spherical), which may be coiled and/or branched. Compound glands have a branched duct system, and their secretory portions have morphological forms similar to those of simple glands. There are three modes of discharge of secretory products from the cells. Merocrine – the secretion may occur by exocytosis from the cell apex into a lumen, so neither the cell membrane nor the cytoplasm become part of the secretion. This is the usual mode of secretion of all gland cells. Apocrine – a small portion of the apical part of cytoplasm is released along with the secretory product. This is an unusual mode of secretion and applies to lipid secretory products in the breasts and some sweat glands. Holocrine secretion involves the discharge of whole secretory cells with subsequent disintegration of the cells to release the secretory product. Holocrine secretion occurs principally in sebaceous glands. In general, all glands have a continuous basal rate of secretion, which is modulated by nervous and hormonal influences. The secretory portions of some exocrine glands are embraced by contractile cells, which lie between the secretory cells and the basement membrane. The contractile mechanism of these cells is thought to be similar to that of muscle cells. These cells are called myoepithelial cells. The simplest exocrine glands are goblet cells. Goblet cells are modified columnar epithelial cells, which synthesize and secrete mucus. They are scattered among the cells of many simple epithelial linings (of the respiratory and gastrointestinal tracts, etc.). The distended apical cytoplasm contains a dense aggregation of mucigen granules which, when released by exocytosis, combine with water to form the viscous secretion called mucus. Mucigen is composed of a mixture of neutral and acidic proteoglycans (mucopolysaccharides) and therefore can readily be demonstrated by the PAS method, which stains mucigen pink. The “stem” of the goblet cell is occupied by a condensed, basal nucleus and crammed with other organelles involved in mucigen synthesis. Simple tubular glands have a single, straight tubular lumen into which the secretory products are discharged. In this example, the entire duct is lined by secretory cells; the secretory cells are goblet cells. In other sites mucus is secreted by columnar cells, which do not have the classic goblet shape but function in a similar way. Simple tubular glands may be found in the large intestine. Sweat glands are almost the only example of simple coiled tubular glands. Each consists of a single tube, which is tightly coiled in three dimensions. Portions of the gland are thus seen in various planes of section. Sweat glands have a terminal secretory portion lined by simple cuboidal epithelium, which gives way to a nonsecretory (excretory) duct lined by stratified cuboidal epithelium. Simple branched tubular glands are found mainly in the stomach. The mucus-secreting glands of the pyloric part of the stomach are shown in this example. Each gland consists of several tubular secretory portions, which converge into a single, unbranched duct of a wider diameter; it is also lined by mucus- secreting cells. Unlike the cells of the large intestine, these mucous cells are not goblet-shaped. Simple acinar glands occur in the form of pockets in epithelial surfaces and are lined by secretory cells. In this example of the mucus-secreting glands of the penile urethra, the secretory cells are pale- stained compared to the non-secretory cells lining the urethra. Note that the term acinus can be used to describe any rounded exocrine secretory unit. A simple branched acinar gland consists of several secretory acini, which empty into a single excretory duct. Sebaceous glands provide a good example of this type of the gland. Their mode of secretion is holocrine, i.e., the secretory product, sebum, accumulates within the secretory cells and is discharged by degeneration of the cells.

Brunner’s glands of the duodenum are the example of compound branched tubular glands. Their duct system is branched, thus defining the glands as compound glands. The secretory portions have a tubular form, which is branched and coiled. Compound acinar glands are those in which the secretory units are acinar in form and drain into a branched duct system. This type of glands consists of numerous acini, each of which drains into a minute duct. These minute ducts, which are just discernible in the centre of some acini, drain into a system of branched excretory ducts of increasing diameter and are lined by simple cuboidal epithelium. The pancreas is a good example of this type of glands. Compound tubulo-acinar glands have three types of secretory units, namely, branched tubular, branched acinar, and branched tubular with acinar end-pieces called demilunes. The submandibular salivary gland is the classic example. It contains two types of secretory cells, mucus-secreting cells and serous cells; the former stain poorly, but the latter, which have a protein-rich secretion (digestive enzymes), stain strongly due to their large content of rough endoplasmic reticulum. Generally, the mucous cells form tubular components, whereas the serous cells form acinar components and demilunes. Endocrine glands are ductless glands. The secretory products diffuse directly into the bloodstream. The secretory products are known as hormones and control the activity of cells and tissues usually far removed from the site of secretion. Most endocrine glands consist of clumps or cords of secretory cells surrounded by a rich network of small blood vessels. Each clump of endocrine cells is surrounded by a basement membrane, reflecting its epithelial origin. Endocrine cells release hormones into the intercellular spaces from which they diffuse rapidly into surrounding blood vessels.

Lecture 6 Blood and Lymph (T.V. Boronikhina)

Blood, lymph, and all types of connective tissue constitute the group of internal medium tissues. The tissues of this group are characterized by the following features: they originate from mesenchyme, display a variety of cells, contain a well-developed extracellular matrix, and maintain body homeostasis. Blood is a liquid tissue, which circulates throughout the body in the closed system of vessels. Its volume in an average adult is approximately 5 liters. The predominant function of blood is transport. Blood carries gases, nutrients, waste products, hormones, antibodies, and electrolytes throughout the body. Blood is involved in defence reactions of the body, such as phagocytosis, immunity, inflammation, and blood clotting. Blood also helps maintain homeostasis in the body, regulates the osmotic and acid– base balance as well as body temperature. Blood is composed of formed elements suspended in the fluid intercellular material known as plasma. The relative volume of the formed elements and plasma is about 40 to 45% and 55 to 60%, respectively. This value is called a hematocrit. Plasma is the fluid extracellular amorphous ground substance of blood. Plasma contains water (90%), organic substances: proteins, hormones, glucose, cholesterol (9%), and inorganic salts (1%). Albumins, globulins, and fibrinogen are the important blood proteins. The plasma composition is studied in the course of biochemistry. Formed elements are the major area of interest in histology. They include erythrocytes (postcellular structures), leukocytes (true cells), and platelets (cell fragments). The number of formed elements in a certain blood volume is called the blood formula or hemogram. Each cubic millimetre of blood contains 4 to 5 million erythrocytes, 4,000 to 9,000 leukocytes, and 180,000 to 320,000 platelets. Each liter of blood contains 4 to 5×10^12 erythrocytes, 4 to 9×10^9 leukocytes, and 180 to 320×10^9 platelets. The morphology of the blood formed elements is studied in blood smears. The specimens are not embedded in paraffin and sectioned. A drop of blood is placed directly on a slide and spread thinly over the surface of the slide, with the edge of another slide to produce a monolayer of cells. This preparation is air-dried and stained according to Romanovsky–Giemsa method, with the use of two dyes: azure II and eosin. On the basis of their appearance after staining, white blood cells are divided into granulocytes and agranulocytes. Blood is a readily available specimen that can be withdrawn from a patient without any complications. Blood chemistry and blood cytology reveal much about the patient’s health. These examinations are not too difficult, and all patients undergo them at the beginning of treatment.

Erythrocytes Erythrocytes are the most prevalent cells in peripheral blood: each cubic millimetre of blood contains approximately 5×10^6 red cells. Erythrocytes are produced in the red bone marrow from stem cells; their life span is approximately 120 days in the circulation. Red cell destruction occurs in the spleen, and, partially, in the liver. Erythrocytes are biconcave discs, measure from 7 to 8 m in diameter, and stain from light salmon to pink in the blood smear. The biconcave-disc shape of erythrocytes considerably enlarges the aggregate surface area of all red cells. The immense capacity of blood to bind and transport gases is partially due to the tremendous surface area of red cells. Erythrocytes are extremely elastic and deform readily when passing through the smallest blood vessels. Erythrocytes are surrounded by a typical plasma membrane. On the external surface of plasmalemma, the determinants for the A, B, and O blood groups, as well as the Rh-factor, reside. The ultrastructure of erythrocytes is not rich: neither organelles nor nuclei are present in the mature erythrocyte, except for microfilaments of the proteins spectrin and actin. These proteins are associated with the internal aspect of the cell membrane and serve as cytoskeleton for erythrocytes, maintaining their morphology. Since erythrocytes do not possess mitochondria, their energy requirements are met by glycolysis. Nuclei and organelles are lost during erythropoiesis in the bone marrow to clear erythrocyte cytoplasm for hemoglobin. About one-third of the erythrocyte mass is hemoglobin (Hb), a protein composed of four globin chains and an iron-containing porphyrin called heme. When hemoglobin is not present in sufficient amount, either because the normal amount in each cell is decreased or because the amount in each cell is sufficient but the cells are reduced in number, the condition clinically shows up as anaemia. There are several types of normal hemoglobin in humans known as A 1 , A 2 (adult), and F (fetal) hemoglobin. The types of hemoglobin depend on the amino acid sequences of polypeptide chains. HbA 1 is the predominant type in adult blood. HbF is the principal form of hemoglobin in the fetus (in adult only 2%), and its persistence in a high percentage in the adult is indicative of certain forms of anaemia. Additionally, abnormal hemoglobin, such as HbS, may also be present in the human population. HbS is the result of a genetic alteration (point mutation) that causes sickling of the red blood cells. This abnormality in shape occurs in sickle cell anaemia. Peripheral blood contains immature red cells called reticulocytes, which contain aggregated reticular clusters of ribosomes. Reticulocytes comprise about 1% of all erythrocytes in the blood. Some hemolytic diseases and bleeding cause an abundance of reticulocytes. Erythrocytes transport oxygen from pulmonary alveoli to peripheral tissues and carbon dioxide from peripheral tissues to pulmonary alveoli. In the lungs, a region of high partial pressure of oxygen, hemoglobin preferentially picks up oxygen to form oxyhemoglobin. In the peripheral tissues, a region of high partial pressure of carbon dioxide, oxyhemoglobin releases its oxygen, exchanging it for carbon dioxide, to form carbhemoglobin. Carbon monoxide also binds to the hemoglobin molecule to form carboxyhemoglobin. This bond is much more tenacious than that of oxygen and may cause the death of an individual. The erythrocyte plasma membrane also takes part in the transport of amino acids and polypeptides. Platelets A cubic millimetre of blood contains from 180,000 to 320,000 platelets. Platelets are not cells: they are cell fragments derived from megakaryocytes in the bone marrow. Megakaryocytes are giant polyploid cells. When platelets are formed, small bits of cytoplasm are separated from the peripheral regions of the megakaryocyte. The life span of platelets is less than 2 weeks in circulation, and they are destroyed in the spleen and in the liver. Platelets are lenticular in shape and measure 2 to 4m in diameter. In blood smears, they appear as round or oval particles that may be clustered in small or large masses. Platelets are surrounded by the plasma membrane that has deep surface invaginations connected with a tubular system, which probably functions in sequestering calcium ions. Platelets have a central granulomere, which stains purple in blood smears, and a peripheral hyalomere, which stains faintly. The hyalomere is rich in microfilaments and microtubules. The small bundles of these cytoskeletal elements lie just beneath the plasmalemma and encircle the periphery of the platelet, maintaining its morphology. The granulomere is composed of - granules, dense bodies, occasional mitochondria, a few lysosomes, and clusters of glycogen particles. The -granules are from 300 to 500 nm in diameter, contain fibrinogen, platelet thromboplastin, and the

growth factor. The dense bodies are from 250 to 300 nm in diameter, contain pyrophosphate, ADP, ATP, serotonin, and calcium ions. Platelets are important for blood coagulation and clot formation. When the wall of a blood vessel is cut or broken, platelets adhere to the ruptured end of the vessel. The platelets will aggregate into a platelet clot at the site of vessel injury and will release, among other substances, serotonin and thromboplastin. Serotonin, a potent vasoconstrictor, causes the vascular smooth muscle cells to contract, thereby reducing the local blood flow at the site of injury. Thromboplastin initiates the series of reactions that leads to the formation of a fibrin clot. Thromboplastin transforms prothrombin into thrombin, and the latter, in turn, transforms fibrinogen into fibrin. These reactions require the presence of calcium ions. Platelets aggregate rapidly, adhering to each other and to fibrin. Masses of aggregated platelets and fibrin are the basis for clots. After the definitive clot has been formed, platelets bring about clot retraction, probably as a function of actin filaments in the hyalomere. Finally, after the clot has served its function, platelets are presumably responsible for clot dissolution, probably by releasing lysosomal enzymes into the clot. Thromboplastin is present in the plasma as well as in the platelets. Platelet-free blood coagulates, though much more slowly, and lymph, which has no platelets, coagulates too. Pathologically, the platelets may agglutinate and give rise to colourless intravascular clots or thrombi. Deficiency of circulating platelets is clinically known as thrombocytopenia and characterised by slow blood coagulation. Leukocytes or white blood cells A cubic millimetre of blood contains from 4,000 to 9,000 leukocytes. They are true cells, containing nuclei and organelles in their cytoplasm. Leukocytes are movable, they can move like an amoeba. White blood cells must be regarded as transitional cells in the blood; they leave the blood through the walls of capillaries and venules to enter the connective tissue, where they perform their specific functions. Leukocytes participate in protective reactions of the body, such as immunity and inflammation. Leukocytes are subdivided into two groups: granulocytes and agranulocytes depending on the presence or absence of specific granules in their cytoplasm. Granulocytes include neutrophils, eosinophils, and basophils. Agranulocytes include lymphocytes and monocytes. The percentage of various leukocytes is called the leukocytic formula: neutrophils constitute 60 to 70%; eosinophils, 2 to 5%; basophils, 0.5 to 1%; lymphocytes, 20 to 30%; and monocytes, 3 to 11%. Neutrophils Neutrophils are the most common leukocytes in normal human peripheral blood; they comprise about 60 to 70% of all leukocytes. Neutrophils are round cells 12 to15 m in diameter. The nucleus has three to five lobes connected to each other by thin threads of chromatin. The cytoplasm contains two types of membrane-bounded granules: primary (nonspecific, azurophilic) and secondary (specific) granules. The azurophilic granules, which appear early in granulopoiesis and occur in all granulocytes as well as in monocytes and lymphocytes, are lysosomes. Primary granules comprise about 20% of the granule population, stain with azure, are visible under the light microscope, and are diagnostic for neutrophils. These granules are lysosomes, which contain various hydrolytic enzymes that function in phagocytosis. They also contain the enzyme myeloperoxidase, producing bactericidal molecular oxygen from hydrogen peroxide (H 2 O 2 ). Specific granules (secondary) comprise about 80% of the granule population, are small (0.1 to 0. m in diameter), do not stain well, and are not visible under the light microscope. They contain bactericidal substances, namely, lysozyme, phagocytin, collagenase, and alkaline phosphatase. Specific granules also contain the protein lactoferrin, which binds ferric ions required for bacterial multiplication. The neutrophil cytoplasm includes a few mitochondria, a small Golgi apparatus, little endoplasmic reticulum, and occasional free ribosomes. Glycogen deposits are plentiful. The life span of neutrophils is less than 1 week. The neutrophil functions are the phagocytosis and destruction of bacteria; hence, neutrophils are microphagocytes. Neutrophils form the first line of defence during acute inflammation. Bacteria can be phagocytosed after opsonization. During opsonization, microorganisms are coated with immunoglobulin or complement proteins. Neutrophils recognize the immunoglobulin coating on the opsonized bacterium rather than a component of the bacterial cell wall itself. Thus, opsonization facilitates the phagocytosis of bacteria by neutrophils. Neutrophils migrate from the bloodstream between endothelial cells to enter the connective tissue. Neutrophils first adhere to bacteria and then engulf them in the membrane-bound phagosome. Phagosomes fuse with secondary granules, and bactericidal substances kill the bacteria. Then primary

granules release their hydrolytic enzymes into phagolysosome, digesting the microorganisms. Ultimately, neutrophils may kill themselves. The accumulation of dead neutrophils, macrophages, microorganisms, and tissue fluid constitutes pus. Immature neutrophils have horseshoe-shaped nuclei and are named band neutrophils. Under normal conditions, no more than 5% of band forms are seen in smears. But if there is a great need for neutrophils in the body, e.g., in cases of infection and inflammation, band neutrophils increase in number. This increase is called a shift in the leukocytic formula to the left. Basophils Basophils comprise from 0.5 to 1% of all leukocytes. Often, several hundred white blood cells must be examined in the blood smear before one basophil is found. Basophils are round cells from 8 to 10 m in diameter. The basophil has an S-shaped nucleus that is frequently masked by numerous large specific granules. Primary (azurophilic) granules are also present. Specific granules are large (0.5 to 1.3 m in diameter), membrane-bounded, spherical structures. They contain heparin, histamine, peroxidase, and perhaps SRS-A. These granules stain metachromatically with the mixture of dyes used to stain blood smears. Metachromasia means a change in colour. This is a property of structures to change the colours of dyes, usually basic dyes. In our case, specific basophilic granules change the blue colour of azure for cherry. This property is considered to belong to histamine. The basophilic plasmalemma contains IgE receptors. When these receptors bind the immunoglobulin complex to specific antigens, the basophils degranulate to produce allergic reactions and, in extremely severe cases, anaphylactic shock. Basophils regulate tissue homeostasis and contribute to inflammation by releasing histamine, heparin, SRS-A, and the eosinophil chemotactic factor. Eosinophils Eosinophils comprise from 2 to 4% of all leukocytes. Eosinophils are round cells, from 10 to 14 m in diameter. The eosinophilic nucleus has two or three lobes. The cytoplasm contains relatively few organelles and many glycogen deposits. The specific granules are plentiful, and a few azurophilic granules are also present. Specific granules are large, ellipsoidal, membrane-bounded, and stain reddish orange. The electron micrographs depict an elongated crystalline core in these granules. The contents of specific granules are lysosomal enzymes, peroxidase, and the major basic protein. Primary granules are few in number. They contain acid phosphatase, arylsulfatase, and other hydrolytic enzymes. Eosinophils inactivate histamine and the slow-reacting substance of anaphylaxis released by basophils at inflammation sites. Eosinophils are implicated in inactivating and killing parasitic agents. The major basic protein from crystalloid has a poorly understood antiparasitic function. Eosinophils are believed to phagocytose antibody–antigen complexes too. Allergic reactions and parasite invasions cause an increase in the number of eosinophils in the blood. Lymphocytes Lymphocytes comprise 20 to 30% of all leukocytes; they are the most common agranulocytes. They are round, small cells, from 8 to 10 m in diameter. Lymphocytes have a round deeply staining nucleus that occupies most of the cell volume and a narrow rim of the light blue cytoplasm at the periphery. Three groups of lymphocytes can be identified according to size: small, medium, and large. In the bloodstream most lymphocytes are small- (more than 90%) or medium-sized. The ultrastructure of lymphocytes is not rich: small lymphocytes possess a few mitochondria, a poorly developed Golgi apparatus, and a rough endoplasmic reticulum, but contain numerous free ribosomes. A few lysosomes (azurophilic granules) are also present. There are two types of lymphocytes: T and B lymphocytes. They are classified according to their specific surface determinants and their site of differentiation. T lymphocytes are thymus-dependent. They have membrane-bound receptors, unique cell surface proteins (not antibodies) that appear during cell maturation in the thymus. These surface molecules are required to facilitate the recognition or binding of T cells to foreign antigens. B lymphocytes are so named, because they were first recognized as a separate population in the bursa of Fabricius in birds; while human B lymphocytes develop in the red bone marrow. B lymphocytes have plentiful immunoglobulins on the external surface of the plasma membrane that function as antigen receptors. There is a third group of lymphocytes, the so-called null cells, possessing no surface determinants. The null lymphocytes may include cells that are circulating hemopoietic stem cells or natural killer cells. These cell types are indistinguishable in blood smears or tissue sections;

immunocytochemical staining for different types of receptors on their cell surface must be used to identify them. Lymphocytes are the key cells in the immune system. T lymphocytes are primarily responsible for cell-mediated immunity, whereas B lymphocytes provide humoral immunity. When lymphocytes first encounter a specific antigen, they are stimulated to undergo several mitotic divisions and then differentiation into effector cells, i.e., the cells with specific functions. B lymphocytes differentiate into plasma cells. Plasma cells abound in rough endoplasmic reticulum and are involved in antibody production. T lymphocytes differentiate into cytotoxic T lymphocytes (killer, helper, and suppressor). Some B and T cells do not undergo differentiation into effector cells but serve as long-lived memory cells, circulating lymphocytes that can respond more rapidly to their specific antigen. Monocytes Monocytes comprise from 3 to 11% of all leukocytes. Monocytes are the largest of all circulating cells (12 to15 m in diameter). The monocyte has an acentric, kidney-shaped nucleus. In contrast to lymphocyte chromatin, monocyte chromatin stains uniformly, revealing a delicate network. The cytoplasm of monocytes is blue with numerous azurophilic granules. They are primary lysosomes containing peroxidase, acid phosphatase, and arylsulfatase among other enzymes. The life span of monocytes is probably less than 3 days in the bloodstream. Monocytes are direct precursors of macrophages. Monocytes migrate into connective tissue and differentiate into macrophages. In the peripheral blood, they are in transit from the bone marrow to the body tissues, where they will differentiate into the various phagocytes of the mononuclear phagocytic system, i.e., the connective tissue macrophages (histiocytes), osteoclasts, alveolar macrophages, perisinusoidal macrophages in the liver (Kupffer cells), and macrophages of the lymph nodes, spleen, and bone marrow. During inflammation, the monocyte leaves the blood vessel at the site of inflammation, transforms into a tissue macrophage, and participates in the phagocytosis of bacteria, other cells, and tissue debris. The monocyte–macrophage also plays an important role in immune responses by concentrating antigen and presenting modified antigens to lymphocytes to facilitate antibody production by immunocompetent cells. The monocyte– macrophage can secrete many substances: interferon, pyrogen, lysozyme, etc. Lymph Lymph, like blood, consists of fluid plasma in which various elements are suspended. Red blood cells and platelets are entirely absent, and granulocytes are few in number, the chief cellular elements are lymphocytes. The lymph plasma is similar to that of blood, but it is of less fixed constitution. It carries carbonic acid but very little oxygen. During digestion, the lymphatics of the intestine become filled with a large amount of fat globules. The lymph assumes white colour and is known as chyle. Many of the fat globules are removed before the lymph reaches the bloodstream.

Lecture 7 ConnectiveTissues (V.L. Goryachkina)

Connective tissue is a tissue of mesenchymal origin. Connective tissue provides structural and metabolical support for other tissues and organs throughout the body. Connective tissue contains blood vessels and mediates the exchange of nutrients, metabolites and waste products between tissues and the circulatory system. Classification of connective tissue

1. Embryonic: mesenchyme.
2. Adult (A,B,C). A. Connective tissues proper: Loose connective tissue (it is widely distributed in the body), as in dermis (papillary layer), mesentery, omentum, and the lamina propria of tubular epithelial organs. Dense connective tissue Irregular: as in periosteum, dermis (reticular layer), and organ capsules; Regular: as in tendons, ligaments, and cornea. Special connective tissue Adipose tissue: as in hypodermis (subcutaneous layer), omentum, mesentery, around the kidneys, etc. Reticular tissue : spleen, lymph nodes, red bone marrow. B. Cartilage C. Bone

Connective tissue consists of cells and extracellular matrix. The cells of connective tissues may be divided into three types according their function:  Cells responsible for the synthesis and maintenance of the extracellular matrix, e.g., fibroblasts (connective tissue proper), chondroblasts (cartilage), and osteoblasts (bone tissue);  Cells responsible for the storage and metabolism of fat. These cells are known as adipocytes;  Cells with defense and immune functions (see below). The extracellular matrix consists of the ground substance and fibers (collagen fibers, elastic fibers, and reticular fibers). The extracellular matrix is important for spatial organization and mechanical stability. Ground substance The ground substance is mainly composed of proteoglycans and hyaluronic acid. Proteoglycans are large molecules. They are composed of core proteins to which glycosaminoglycans (GAGs) are covalently bound. GAGs are large polysaccharides, which maintain turgor and determine the diffusion of substances through extracellular matrix. They can be divided into four groups.

1. Hyaluronic acid. (a) it is the main component of connective tissue; (b) it is widely distributed in loose connective tissue; (c) it has been extracted in significant amounts from cartilage, blood vessels, and skin. 2. Chondroitin sulfate. (1) It predominates in cartilage, bone, and blood vessels; (2) It has also been identified in the skin and cornea. 3. Dermatan sulfate. It is found mostly in the skin, but it also has been demonstrated in blood vessels, heart valves, tendons, and connective tissue of the lung. 4. Keratan sulfate and heparan sulfate occur in cornea and cartilage; heparan sulfate is found in the lamina lucida of basement membrane. GAGs have the following properties: (a) the high negative charge and (b) strongly hydrophilic behavior, with the exception of hyaluronic acid, covalently bound to proteins to form proteoglycans. Besides GAGs, the ground substance contains glycoproteins. They serve as adhesive molecules between the cells and extracellular scaffold. There are some types of glycoproteins in connective tissues.

1. Fibronectin (surface glycoprotein of fibroblasts). It has affinity to collagen.
2. Laminin is a major component of the basement membrane.
3. Entactin is also a component of the basement membrane and binds to laminin. Fibers. Collagen fibers are composed of fibrillar protein – collagen. The collagens are a large family of proteins, which can aggregate to produce either filaments, fibers, or meshwork, which then interacts with other proteins to provide support in the extracellular matrix. There are at least 20 types of collagen polypeptids (-chains). They form different fibers. For example, type I collagen forms large bundles of collagen fibers. Their distribution is the skin, dermis, tendons, bone, ligaments, cornea, and loose connective tissue. Type II collagen forms small bundles of collagen fibers. Their distribution: hyaline and elastic cartilage, vertebral disks, vitreous of eye. Type III collagen forms small bundles of collagen (reticular) fibers. They are situated in the blood, vessels, bone marrow, spleen, lymph nodes, lung, and fetal skin. Type IV collagen forms sheet-like layers (basement membranes, the lens capsule). The formation of collagen fibers The production of collagen fibers involves series of events, some of which occur inside the cell, while other events occur outside the cell. Intracellular events include the uptake of amino acids (proline, lysine, etc.) by endocytosis, resulting in the production of polypeptide chains in the rER. A number of modifications of the polypeptide chains occur within the cisternae of rER and the Golgi complex. The resultant molecules are called procollagen. Then packing of the procollagen into secretory vesicles occurs inside the cell. These secretory vesicles move to the plasma membrane and leave the cell by means of exocytosis. Extracellular events include the formation of tropocollagen and the aggregation of tropocollagen into collagen fibers. Collagen fibers have a 68-nm banding pattern. This banding pattern is a reflection of the arrangement of tropocollagen in forming the fibril. Tropocollagen measures about 300 nm long with a head and a tail. In forming a fibril these molecules become aligned head to tail in overlapping rows with a gap between the molecules within each row. Thus, collagen fibers consist of bundles of fine collagen fibrils.

Elastic fibers Elastic fibers provide tissues with the ability to respond to stretch and distension. They are thinner than collagen fibers. Elastic fibers are composed of elastin and microfibrils. The protein elastin is rich in proline and glycine but is poor in hydroxyproline. Microfibrils are fibrillar glycoprotein. Elastin is synthesized by the same pathway as collagen. Cells Resident cells (fixed cell population). Fibroblasts, fibrocyte, myofibroblasts Macrophages Adipose cells Mast cells Undifferentiated cells Pericytes (in the wall of capillares and venules) Pigment cells

Immigrant cells (wandering population) Lymphocytes Plasma cells Neutrophils Eosinophils Basophils Monocytes Fibroblasts The fibroblast is a spindle-shaped or stellate cell. Its nucleus has a regular elliptic contour and sparse and scattered chromatin. The ultrastructural organization of the fibroblast reflects its function. The fibroblast contains well-developed rER on which the precursor polypeptides of collagen, elastin, proteoglycans, and glycoproteins are synthesized. Besides rER, it contains many free ribosomes, a well- defined Golgi apparatus, mitochondria, fat droplets, and primary and secondary lysosomes. Additionally, the bundles of actin microfilaments are prominent; they are implicated in cellular motility. The function of the fibroblasts is to produce extracellular matrix. The fibrocyte is a more elongated cell than the fibroblast. When the fibroblasts become less active during adult life, their nuclei undergo condensation and their cytoplasms appear less basophilic, they are then reffered to as fibrocytes. The fibrocyte contains decreased amounts of rER and other organelles. Myofibroblasts. This cell looks like a fibroblast. The myofibroblast contains rER, Golgi complex, mitochondria, etc. In addition, it contains bundles of longitudinally disposed actin filaments and dense bodies similar to those observed in smooth muscle cells. The myofibroblast plays a role in the contraction of wounds and may synthesize collagen fibers. Macrophages Macrophages are long-lived actively phagocytic cells. They are widely distributed throughout the body. They are derived from the blood monocytes. Monocytes leave the bloodstream and migrate to the tissues to turn into macrophages, for example,  connective tissue (histiocytes),  liver (Kupffer cells),  lung (alveolar macrophages),  lymph nodes (free and fixed macrophages),  spleen (free and fixed macrophages),  bone marrow (macrophages),  serous cavities (pleural and peritoneal macrophages),  bone tissue (osteoclasts),  the central nervous system (microglial cells). Macrophages and their precursors are assigned to the “mononuclear phagocyte system”. The minimal criteria for inclusion in this system are: (1) the derivation from the red bone marrow; (2) characteristic cell structure; (3) high level of phagocytic activity. It should be noted that the Russian scientist Elie Mechnikoff was the first who to describe the phagocytic cells. More than 100 years ago (1882) he found that vertebrates possessed two types of cells

able to fight invading organisms. These cells are microphages (small eaters) now known as neutrophils and macrophages (big eaters). The latter include monocytes and phagocytosing cells. The structure of macrophages. The main features of macrophages are abundant lysosomes and phagolysosomes, and numerous folds or fingerlike processes. Moreover macrophage contains a relatively large Golgi region, mitochondria, sER, and rER, and well-developed cytoskeletal elements. Bundles of actin-rich microfilaments are involved in adhesion, endocytosis, and movement. Note that macrophages can secrete a variety of important molecules. Secretory products of macrophages

1. Enzymes: (a) lysosomal hydrolases, (b) neutral proteinases (these enzymes participate in tissue remodeling and damage), (c) lysozyme (carbohydrase that digests the cell walls of many microorganisms).
2. Coagulation factors (V, VII, IX, X).
3. Molecules that regulate cell activities and cell proliferation: (a) interferon, an antiviral factor, (b) interleukin 1 (IL1), mitogenic protein for T-cells, (c) the tumor necrosis factor (cachetin), a protein able to kill transformed cells, (d) the angiogenesis factor – increases endothelial multiplication. Secretion is increased by hypoxia.
4. Other macromolecules: (a) endogenous pyrogen, a fever mediator and (b) apolipoprotein E playing an important role in the transport of cholesterol and triglycerides to the liver. It should be noted that macrophages take part in the immune response. The participation of macrophages in the immune response involves the processing and presentation of antigens to lymphocytes. Macrophages “present” antigens on their surface. A macrophage-bound antigen serves as a trigger in the proliferation of lymphocytes. For this reason this process was termed antigen-dependent proliferation and differentiation of lymphocytes, resulting in the appearance of effector cells: T-killers, T- helpers, T-suppressors, and plasma cells. Plasma cells They are antibody-producing cells derived from B-lymphocytes. A plasma cell contains a highly developed rER, a well-developed Golgi apparatus, few lysosomes and mitochondria. Light microscopy shows radially arranged clumps of dense heterochromatin adjacent to the nuclear membrane. This arrangement of chromatin gives the cartwheel appearance to the nucleus. In addition, the well-developed Golgi region gives the light macula appearance near the nucleus. Mast cells In 1877 Paul Erlich described these cells and named them "mast" cells (the German for “feeding”). He observed the granules within these cells and presumed that they represented stored nutrients; hence, he named them "mast". As a rule, mast cells are located around blood vessels. They are numerous in the dermis and in the connective tissue of the mucous lining of the respiratory and digestive tract, connective tissue of the lungs, and the serosal lining of the peritoneal cavity. They take part in the metabolism of heparin, histamine, and other mediators producing allergic reactions.

Mediators produced by human mast cells and basophils Mediator Function Histamine Increases vascular permeability and contracts smooth muscle cells Heparin Anticoagulant Eosinophil chemotactic factors Attract eosinophils and neutrophils Slow-reacting substance of Anaphylaxis or leukotriene C

Contracts smooth muscle cells

Note the information about anaphylactic shock: More than 100 years ago it was found that the second injection of an antigen had harmful effect, and indeed could be fatal. In 1893, this effect was called anaphylaxis (the Greek ana – again; aphylaxis – defenseless). Anaphylaxis is easily demonstrated in guinea pigs. If a guinea pig is injected a particular antigen and then after 10 to 14 days is injected the same antigen, it goes into what is called anaphylactic shock. This is manifested by difficulty in breathing and a rapid pulse rate; moreover, the animal may die from an inability to breathe. The reason for respiratory failure is that the smooth muscle cells of small

bronchi become so contracted that their lumen is too narrow to permit an adequate volume of air to enter and, in particular, to leave the lung. Another effect observed in anaphylaxis is that venules and capillaries become dilated and leaky so that the plasma escapes from them. As a result, blebs of plasma may form in the loose connective tissue directly under the epithelium. This phenomenon was called urticaria. It was discovered in 1914 that histamine has a profound effect on smooth muscle cells, causing dilation of capillaries and venules. Leaking plasma can be observed by EM, after breaking the tight junction. The allergic reaction is known to be mediated by IgE. They are produced by plasma cells in response to certain antigens called allergens. Mast cells and basophils have a high affinity for IgE. IgE attaches to mast cells, but the antigen-combining sites of antibody molecules are exposed. If the antigens reenter the body again, they react with these sites. The combination of antigen with IgE triggers the release of mediators (due to the discharge of granules), producing allergic disease.

The compartive analysis of loose and dense connective tissue Loose connective tissue Dense connective tissue

1. The fibers are thin and relatively sparse. 1. Thick fibers are arranged in bundles. High proportion of collagen fibers.
2. The ground substance is abundant and occupies more volume than the fibers.
   2. There is relatively a small amount of ground substance. Fibers predominate over cells.
3. Many kinds of cells are widely distributed. Distribution: (a) papillary layer; (b) around vessels and inner organs; (c) tunica mucosa, tela submucosa, and serous membrane
   3. The cells population is sparse (usually represented by only one type of cells).

irregular regular The fibers are oriented in various directions (the reticular layer of the skin).

The bundles of fibers are oriented in parallel rows to each other (tendons, ligaments).

Functions of the connective tissue proper:

1. Mechanical support (fibers).
2. Exchange of metabolites between blood and tissue (ground substance).
3. Protection against infection (wandering cells).
4. Regeneration after injury (fibroblasts).

Adipose tissue The adipose tissue is divided into two types: white and brown. White adipose tissue is composed of large fat cells or adipocytes. Every cell contains one flattened nucleus and a large single lipid droplet. If hematoxylin and eosin are used for staining, a hole will be seen instead of a lipid droplet. If special staining (sudan) is used, the lipid droplet stains orange. The EM enables one to see some organelles near the nucleus. It is noteworthy that fat cells are organized into groups called lobules. The lobules of fat cells are separated by partitions of loose connective tissue; they have been called septa. This connective tissue conducts blood vessels and nerves into adipose tissue. The sites of white adipose tissue:

1. It is present in the following fatty areas: under the skin (hypodermis), especially in females.
2. It is more condensed in the mammary glands and gluteal region (females).
3. Around the kidney and blood vessels.
4. In the mesentery, omentum, and the abdominal wall. Note that the white adipose tissue can be divided into two functional kinds: I. Storage adipose tissue with fat readily available for energy production (adipose tissue of the hypodermis, mesenteries, omenta, retroperitoneum). II. Structural adipose tissue with the role of an elastic pad, mechanical support and protection (adipose tissue in the orbits of the eye, articulations, palms, soles, cheeks, etc.). Note that this adipose tissue remains practically unchanged during fasting.