Cytology Embryology

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M. Gorky Donetsk National medical university

CYTOLOGY

AND

GENERAL

EMBRYOLOGY

Donetsk

2011

Edited by

academician of the Highest School of Ukraine,

Doctor of Medical Sciences,

Professor Barinov E.F.

Barinov E.F., Sulayeva O.N., Tereschuk B.P.,Khlamanova L.I., Chereshneva E.V., Gatina K.I.,

Prylutskaya I.A.


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METHODS OF HISTOLOGICAL INVESTIGATION

AIM:To get acquainted with a microscopic technique, main stages of preparing histological specimens

and their investigation

Rules of working with a light microscope

1. Open your drawing-pad and put your microscope on the left half of the drawing-pad in front of you.

2. Place the low magnification objective opposite the microscope table opening at the distance 1.5cm.

3. Check up the position of the small objective. Centre the small objective - turn it to the click.

4. Looking through the eyepiece, illuminate the visual field with mirror using the concave side of the mirror.

5. Put the specimen on the microscope table with coverslip upwards. Place the section opposite the

objective lens.

6. Look from the side, move the microscope tubus down, leave a minimal distance between the objective

and the specimen (up to 1cm).

7. Look through the ocular lens and slowly raise the tubus with macroscrew until the image is clear.

8. Observe the whole specimen under a low magnification.9. Choose the part of the specimen that you need to study under a high magnification. Centre it precisely

in the visual field.

10. Raise the tubus a little, turn the objective turret and install the higher objective lens.

11. Look from the side, very carefully move the microscope tubus down until 1mm distance is left

between the lens and the specimen surface. Do not perform this operation looking through the eyepiece!

12. Look through the eyepiece and turn the microscrew until the image is clear.

13. When you have finished your work, raise the tubus by turning the macroscrew and turn the objective

turret, returning to the small objective.

14. Remove the specimen from the microscope table and put it on the desk.

Main rules of specimen investigation in light microscopyTo learn the general structure of a specimen it is necessary to study it under a low magnification. Profound

investigation of the specimen is possible while studying it under a high magnification when you can study the

shape, proportions and disposition of the cells.

Illustration includes studying a specimen and its drawing in detail.

Notice that colour and scale of reproduction of histological structures is of great importance. It should be

performed with coloured pencils in a drawing-pad with corresponding indications.

Main steps of preparing of tissue specimens for a light microscopy

1. Selection and fixation of the material for investigation (formalin, ethyl alcohol, special mixtures)

2. Dehydration (substitutions) in ethyl alcohol

3. Clearing (xylene)

4. Infiltration (ethyl alcohol, xylol)

5. Embedding (in paraffin or celloidin)

6. Sectioning with microtome (6-8mkm thick)

7. Mounting (glass slide)

8. Removal of paraffin

9. Rehydration

10. Staining and covering.

Histological dyes

1. Nuclear or alcaline: hematoxylin, carmine, saphranin.

2. Cytoplasmic or acidic: eosin, acid fushcin, picrin acid, orange.

3. Special dyes: orsein, sudan, osmic acid.4. Heavy metal impregnation: silver, gold.


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LESSON 1

THEME: GENERAL ORGANIZATION OF CELL. PLASMA MEMBRANE.

BACKGROUND:Eukaryotic cell is an elementary living system which consists of three basic elements:

plasmolemme, cytoplasm and nucleus. Cells provide cell division, passing genetic information, body growth,adaptation processes, physiological and reparative regeneration. Plasmolemme plays an important role in thecell performing protective function, providing metabolism, intracellular homeostasis, shape of the cell, its abilityto move, in that way regulating life processes, creating different systems within multicellular organism by

providing morphological and informational bound in the intercellular cooperation and association of cells withextracellular matrix. Alterations in structure and functions of plasmolemme cause development of different

pathological processes (insulin-independent diabetes mellitus and many others)

AIM OF STUDY:Be able to differentiate cells and their derivatives in histological specimens andelectron photographs. In the electron photos be able to differentiate plasmolemme, intercellular junctions.

TO ACHIEVE THIS AIM ONE HAS TO(practical procedures):1. Differentiate cells, their components in histological sections and electron photographs.2. Differentiate histological elements in histological sections and electronogramms.3. Identify the structure and features of biological membrane.4. Interpret the structure and functions of plasmolemme.5. Differentiate intercellular junctions in the electron photographs, understand their structure and functions.

TO ACHIEVE THE AIM IT IS NECESSARY TO CENTRE AROUND THE FOLLOWING POINTS:

1. Cell as a basic form of multi-cell organism existence. Cell theory.2. Histological elements as structural parts of human body. Types of histological elements.3. Main principles of eukaryotic cell structure, its main parts.

4. Biological membrane: its structure, chemical characteristics and functions.5. Plasma membrane, its structure, chemical and functional characteristics of its layers.6. Functions of plasmolemme and their morphological representation.7. Intercellular junctions: types, structure and functions.

REFERENCES:1. Junqueira L. C., Carneiro J., Kelley R. O. Basic histology. A Lange medical book.Tenth edition: Appleton& Lange, 2008.2. Burkitt H. G., Young B., Heath J. W. Wheaters Functional Histology. A text and colour atlas. 5th edition:Churchill Livingstone, 2005.3. Ross M. H., Romrell L. J., Kaye G. Histology. A text and atlas. 5d ed. Baltimore: Williams & Wilkins, 2007.

WHAT MUST YOU KNOW? (INSTRUCTION FOR YOUR SELF-LEARNING)Tissues and organs of human body consist of different histological elements.The histological elements

are stated below:Cellular:

1. Cells.2. Syncytium (a multinucleated protoplasmic mass formed by the secondary union of originally separate cells);3. Symplast (a multinucleated cell that has formed by fusion of separate cells);4. Postcellular structures - (erythrocytes, thrombocytes).

Non-cellular:5. Extracellular matrix.

The smallest living entity of an organism is the cell. In contrast to single-celled organisms that are

independent entities, the cells of higher organisms form functional units. In accordance with their function, thecells are differentiated by size, shape, and the degree of definition of certain characteristics. Size and shape are


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often closely linked to a cells specific properties.For all cells of the body there are a certain basic structure. The cell consists of the:1. cytoplasm containing the cell organelles,2. nucleus,3. surrounding cell membrane (plasmolemma).

Plasmolemma (plasma membrane) consists of:1. Glycocalix

The layer of oligosaccharides is called glycocalix. A layer of carbohydrate chains covers the outer plasmamembrane. They are part of the glycolipids and glycoproteins of the cell membrane, forming the surface coat ofthe cell. The chemical structure of the glycocalyx is laid down genetically and it is specific for each cell. Theglycocalix serves as a cell-specific antigen. By this structure cells can recognize each other as self and non-self.

2. Membrane(also surrounds the cell organelles and the nucleus).The elementary membrane consists of a double lipid layer, in which the fat-soluble components face

each other while the water-soluble parts form the inner and outer boundaries (a three-layered structure). The

structure of the plasma membrane cannot be resolved by light microscopy. In high-resolution electron micrographs,the plasma membrane appears as a tripartite structure: two dark lines separated by a light region.An electron-microscopic section demonstrates a three-layered structure (fig. 1.1): this includes a double

layer of lipids in which two layers of lipid molecules are arranged so that their lipid-soluble parts (fatty acids)oppose each other (light middle line) while the water-soluble ends form the outer and inner boundaries of thecell membrane (dark outer and inner lines).

The outer and inner dark lines seen in electronmicrographs of plasma membranes are the result of thedeposition of osmium onto the hydrophilic portions of the lipidmolecules.

The membrane contains lipids:1.Phospholipids (phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine).

The lipid molecules form a lipid bilayer with anamphipathic character (it is both hydrophobic and hydrophilic).The fatty acid chains of the lipid molecules (tails) face eachother, making the inner portion of the membrane hydrophobic(i.e., having no affinity for water). The surfaces of themembrane are formed by the polar head groups of the lipidmolecules, thereby making the surfaces hydrophilic (i.e.,having an affinity for water).

2. Cholesterol makes the membrane less fluid and more mechanically stable.

3. Glycolipids (sphingolipids): galactocerebroside and ganglioside are substances, which are responsiblefor special function of nerve tissue (fig. 1.2).

The double lipid layer is infiltrated with proteins. In most membranes, protein molecules constituteapproximately half of the total membrane mass.

Most of the proteins are embedded within the lipid bilayer or pass through the lipid bilayer completely.These proteins are called integral membrane proteins. Integral membrane proteins can move within the planeof the membrane; this movement can be compared to the movement of icebergs floating in the ocean. Theother types of protein, calledperipheral membrane proteins, are not embedded within the lipid bilayer. They

are associated with the plasma membrane by strong ionic interactions, mainly with integral proteins on both theextracellular and infra-cellular surfaces of the membrane.

Fig. 1.1. Electron micrograph of cell membrane (arrow).


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Six broad categories of membrane proteins have been defined in terms of their function (fig. 1.3): pumps,channels, receptors, linkers, enzymes, and structural proteins. The categories are not mutually exclusive;e.g., a structural membrane protein may simultaneously serve as a receptor, an enzyme, a pump, or any combinationof these functions.

Pumps serve to transport certain ions, such as Na+, actively across membranes. Pumps also transportmetabolic precursors of macromolecules, such as amino acids and sugars, across membranes, either by themselvesor linked to the Na+pump (fig. 1.4).

Channels allow the passage of small ions and molecules across the plasma membrane in either direction,i.e., passive diffusion. Gap junctions formed by aligned channels in the membranes of adjacent cells permit

passage of ions and small molecules from the cytoplasm of one cell to the cytoplasm of the adjacent cells. Receptor proteins allow recognition and localized binding of ligands (molecules that bind to the

extracellular surface of the plasma membrane) in processes such as hormonal stimulation, coated-vesicle

endocytosis, and antibody.Linker proteins anchor the intracellular cytoskeleton to the extracellular matrix. Examples of linker

Fig. 1.3. Plasma membraneproteins functions.

Fig. 1.2. Plasma membrane structure.

1 membrane; 2 glycocalyx; 3 submembrane layer; 4 hydrophilic zone of phospholipid molecules; 5 hydrophobic

zone of phospholipid molecules; 6 integral and 6b - semiintegral proteins; 7 peripheral proteins; 8 carbohydratechains of glycocalyx; 9 microtubules; 10 microfilaments.

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proteins include the family of integrins that linkcytoplasmic actin filaments to an extracellularmatrix protein (fibronectin).

Enzymes have a variety of roles. Adenosinetriphosphatases (ATPases) have specific roles in ion

pumping: ATP synthase is the major protein of theinner mitochondrial membrane, and digestiveenzymes such as disaccharidases and dipeptidasesare integral membrane proteins.

Structural proteins are visualized by thefreeze fracture method, especially where theyform junctions with neighboring cells. Often,certain proteins and lipids are concentrated inlocalized regions of the plasma membrane to carryout specific functions. Examples of such regions can

be recognized in polarized cells, such as epithelial

cells.

Modified fluid-mosaic modelmeans that integral membrane proteins may move to a different regionof the plasma membrane. The lateral diffusion of proteins is often limited by physical connections betweenmembrane proteins and intracellular or extracellular structures.

3. Submembrane layer(cortical layer of cytoplasm) - consists of microtubules and microfilamenteswhich include contractile proteins - actin and myosin. This layer maintains the plasma membrane shape andmovement.

Properties of plasmolemma: The plasma membrane is flexible, semi-permeable, and experiencesactive transport and potential differences across it.

Functions of plasmolemma are as follows:1. Barrier, or protection of cell.2. Selective transportof substances.3. Antigenic determinants (cell passport)4. Receptors of PM determines the regulation of cell by systemic and local factors (hormons,

neurotransmitters, growth factors).5. Firm attachmentto other cells or a basal lamina; membrane specializationsfor this are: intercellular

junctions and receptors.6. Movement of the cellitself (migration) by pseudopodial, filipodial, or lamellipodial extensions and

the release of any firm attachments, or by flagellate activity, e.g., by sperm.

Transport of materialsin and out of the cell served by (fig. 1.5):

(a) diffusion(selective) through the membrane;Molecules move freely in aqueous solutions or in gases, and differences in concentration equilibrate by

diffusion. In this process, molecules diffuse toward the lower concentration until the concentrations even out.Small molecules, such as the respiratory gases O2 and CO2 as well as water pass through the cell membraneunimpeded (free diffusion). Pores through the membrane (channel proteins, membrane pores) or mobile transport

proteins (carriers) facilitate the passage (facilitated diffusion) of nutrients (e. g., glucose and amino acids inthe cells of the intestinal mucosa) and ions.

(b) active transportthrough the membrane;

Fig. 1.4. Na-K ATPase.


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Active transport is the transport of substances through the cell membrane by means of an energy-consuming transport system(transport ATPase). Such a transport process can move a substance through themembrane against a concentration gradient These active transport processes are served by specialized proteinsin the cell membrane that can move several ions simultaneously.

(c) endocytosis, and its more scaled-up forms - pinocytosis and phagocytosis (fig. 1.6);

Large molecules, such as proteins, enter (endocytosis) through the cell membrane by so-called vesiculartransport. During this process, substances are attached in part to the outside of the cell by membrane-bound

receptors, enclosed by a part of the plasma membrane, and moved into the interior of the cell as a membranewrapped vesicle (receptor-mediated endocytosis). Depending on the size of the absorbed particle, this process

Fig. 1.5. Transport of materials in and out of the cell.

Fig. 1.6.Endocytosis: A - E photomicrograph, B - scheme.

1 - plasmolemma; 2 - plasmolemma invagination (pit); 3 - endocytotic vesicle; 4 - ligand, called a receptor related

with; 5 - clathrin; 6 - endosome.

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may also be called pinocytosis or phagocytosis.Pinocytosis is the ingestion of fluid and small protein molecules via small vesicles, usually smaller

than 150 nm in diameter. Pinocytosis is performed by virtually every cell in the organism, and it is constitutive;i.e., it involves a continuous dynamic formation of small vesicles at the cell surface.

Phagocytosisis the ingestion of large particles such as cell debris, bacteria, and other foreign materials.

In this process, large vesicles (larger than approximately 250 nm in diameter) called phagosomes are produced.Receptor-mediated endocytosisallows entry of specific molecules into the cell. In this mechanism,

receptors for specific molecules, called cargo receptors, accumulate in well-defined regions of the cell membrane.These regions eventually become coated pits. Cargo receptors recognize and bind to specific molecules thatcome in contact with the plasma membrane. Clathrin molecules then assemble into a basket-like cage that helpschange the shape of the plasma membrane at that site into a vesicle-like invagination for transport into the cells.

(d) exocytosis

Large molecules, such as proteins, exit (exocytosis) through the cell membrane by so-called vesiculartransport. In exocytosis, products synthesized in the cell are enclosed in membranous vesicles and, by coalescenceof these vesicles with the inside of the plasma membrane, reach the extracellular space

In the constitutive pathway, substances designated for export are continuously delivered in transportvesicles to the plasma membrane. Proteins that leave the cell by this process are secreted immediately aftertheir synthesis and exit from the Golgi apparatus.

In the regulated secretory pathway, specialized cell, such as endocrine and exocrine cells and neurons,concentrate secretory proteins and transiently store them in secretory vesicles within the cytoplasm. In thiscase, a regulatory event (hormonal or neural stimulus) must be activated for secretion to occur.

Communication and transduction. Each cell collaborates with different (adjacent and distant) cells ofhuman body cells for development, growth, homeostasis, regeneration, and its own particular task. The importanceof the cell membrane in receiving and sending the necessary signals is stressed by the number of examples given:

(a) The binding of hormones and transmitters to receptors on the membrane.

(b) The binding of the immune cells membrane receptor to an antigen.(c) Chemical stimuli are transduced into nerve impulses in chemoreceptors; mechanical stimuli inmechanoreceptors.

(d) Chemotactic agents act on phagocytic cells to attract them to their targets.

Specializations of the plasma membraneinclude:1) microvilli are responsible for increase of transport area (surface),2) cilia with basal bodies, which can move and determine the movement of material outside of the cell.3) stereocilia - is a specialised structure of some sensor cells4) pinocytotic vesicles reflect the activity of endocytosis;5) infoldings often associated with mitochondria to active transport of ions through PM,6) intercellular junctions,

Intercellular junctions(fig. 1.7)perform mechanical or chemical relations between cells.Mechanical junction (fig. 1.9) is due to adhesion, desmosomes, hemidesmosomes, interdigitations.The chemical relations between cells are mediated by gap junctions (nexuses) (fig. 1.8).Chemical isolation is due to tight junctions (fig. 1.10).

Tight junction (zonula occludens) represents an area of fusion of the outer leaflets of the plasmamembranes of two adjacent cells.

Morphology: Tight junctions seal adjacent epithelial cells in a narrow band 0,1-0,5 mkm wide justbeneath their apical surface.

Molecules: This cell junction contains the adhesion molecule, E-cadherin. Tight junction an intercellular

junction at which adjacent plasma membranes are joined tightly together by interlinked rows of integralmembrane proteins - occludins, limiting or eliminating the intercellular passage of molecules.


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Fig.1.7. Intercellular junctions.Scheme.

1 - tight junction; 2 - desmosome;

3 - gap junction; 4 - adherent junction;

5 - plasmolemma.

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5 5

Fig. 1.9. Mechanical junctions between cells and cells with a basal membrane: A - E photomicrograph ofdesmosome 36 000; B - scheme of desmosome structure; C - scheme of hemidesmosome structure.

1 - plasmolemma; 2 - intermediate filaments of cell cytoskeleton, 3 - cytoplasmic plate; 4 - electron-dense layer in

the desmosome, 5 - cleft between two plasmolemms, 6 - desmoplakins and desmogleins; 7 - hemidesmosome, 8 - basal

plate, 9 - anchoring filaments, 10 - reticular fibers.

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Fig. 1.8. Gap junction (nexus junction):A - E photomicrograph 36000; Scheme.

1 gap junction; 2 connexins.

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Fig. 1.10. Scheme of tight junctionstructure .

1 - plasmolemms of neighboring cells,2 - cleft between neighboring

plasmolemms; 3 - ZO-2 protein; 4 - ZO-

1 protein, 5 - actin; 6 - ZO-3 protein,

7 - transmembrane domain of occludin;

8 - extracellular domain of occludin;

9 - cytoplasmic domain of occludin;

10 - claudin.

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Tight junctions perform two vital functions: They prevent the passage of molecules and ions through thespace between cells. So materials must actually enter the cells (by diffusion or active transport) in order to passthrough the tissue. This pathway provides control over what substances are allowed through.They block themovement of integral membrane proteins between the apical and basolateral surfaces of the cell.

Adherent junction isa type of intercellular junction that links cell membranes and cytoskeletal elementswithin and between cells, connecting adjacent cells mechanically. Adherens junctions provide strong mechanicalattachments between adjacent cells. They hold cardiac muscle cells, epithelial cells tightly together as the heartexpands and contracts. They seem to be responsible for contact inhibition.

Morphology: The intermediate junction (zonula adherens) is characterized by the presence of anintercellular space 10-20 nm wide, separating areas of cytoplasmic density occurring in each of the participatingcells.

Molecules:Adherens junctions are built from: cadherins transmembrane proteins whose extracellularsegments bind to each other and whose intracellular segments bind to catenins. Catenins are connected to actinfilaments.

Desmosome (macula adherens).Morphology:The structure of a desmosome, or macule adherens, is fairly uniform in most tissues

examined to date: within each cell, at the region of localized contact of two cells, there is a dense plaqueadjacent to the cell membrane, made up of converging cytoplasmic actin microfilaments (tonofibrils). The twocell membranes do not appear modified. Within the intercellular substance 20-30 nm wide, there is a densecentral lamina. Very slender filaments run between the central lamina and the adjacent cell membranes.

Molecules:C-cadherin are an essential component of desmosomes. Desmosomes are biochemicallycomplex structures containing many different filamentous proteins, some of which are desmosome specific.Specific adhesion proteins (adherins) have been identified in cytoplasmic plaques. Other protein components ofdesmosomes are desmoplakins and desmogleins. The desmosomes also contain intermediate filaments of variousmolecular weights.

Hemidesmosomes (half-desmosomes) are observed at the attachment points of epithelial basal cells tothe basement lamina. The half-desmosome is morphologically somewhat similar to the desmosome: there is athickening of a limited area of the cytoplasm of a basal cell adjacent to the cell membrane, upon which convergecytoplasmic fibrils. However, the apposed basement membrane shows merely a slight thickening, which containsslender filaments. An intermediate thickening, or membrane, is usually present within the fibrils of thehemidesmosome. Hemidesmosomes serve as connectors between the extracellular matrix and the intermediatefilaments in the cytoplasm of the cell.

The Gap Junctions (Nexus Junctions) have multiple functions: they provide cell-to-cell communicationsof essential metabolites and ions and may serve as electrical synapses.

Morphology:The Gap Junctionsis a narrowed portion of the intercellular space containing channelslinking adjacent cells and through which pass ions, most sugars, amino acids, nucleotides, vitamins, hormones,and cyclic AMP. In electrically excitable tissues, these gap junctions transmit electrical impulses via ioniccurrents. The junction is composed of seven layers, three of which are electrontranslucent and are sandwichedin between electron-dense layers. The central electron-lucent zone (or gap) is composed of small hexagonalsubunits, forming the channels of communication between adjacent cells.

Molecules:The gap junction channels are composed of a diverse family of proteins, named connexins.


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LESSON 2

THEME: CELL. CYTOPLASM. HYALOPLASM, ORGANELLES.

BACKGROUND: The vast majority of life processes is going on within the cytoplasm: catabolism and

digestion of organic substances and creation of energy, synthesis of proteins, lipids and carbohydrates specificfor the cell, their accumulation and secretion. These processes provide specific cell functions, as well as

functioning of the whole multi-cellular organism.

AIM OF STUDY: To be able to differentiate cytoplasm and its key structures hyaloplasm, organelles,

inclusions within the eukaryotic cell on light and electron microscopy. Differentiate organelles within the normal

cell for better understanding of their functions and possible changes under condition of pathology.

TO ACHIEVE THIS AIM ONE HAS TO(practical procedures):

1. Differentiate the basic structures of cytoplasm: hyaloplasm, organelles, inclusions.

2. Distinguish hyalopasm in histological specimen and electronograms.

3. Distinguish organelles in histological specimen and electronogramme, interpret their structure and functions.4. Analyze cell function on basis of the structure of cytoplasm and organelles interaction.


1. Main components of the cell cytoplasm hyaloplasm, organelles, inclusions.

2. Hyaloplasm definition, basic elements, features, importance.

3. Organelles definition, classification.

4. Membrane organelles ( rough and smooth endoplasmic reticulum, Golgi apparatus, lysosomes, peroxisomes,

mitochondria), their structure and functions.

5. Non-membrane organelles (ribosomes, centrioles, microtubules, microfilaments), their structure, functions.

6. Organelles with specific functions (microvilli, cilia, tonofibrills, miofibrills, neurofibrills).

REFERENCES:

1. Junqueira L. C., Carneiro J., Kelley R. O. Basic histology. A Lange medical book.Tenth edition: Appleton

& Lange, 2008.

2. Burkitt H. G., Young B., Heath J. W. Wheaters Functional Histology. A text and colour atlas. 5th edition:

Churchill Livingstone, 2005.

3. Ross M. H., Romrell L. J., Kaye G. Histology. A text and atlas. 5d ed. Baltimore: Williams & Wilkins, 2007.

WHAT MUST YOU KNOW? (INSTRUCTION FOR YOUR SELF-LEARNING)

CYTOPLASM

The cytoplasm is the component of the cell, located between the nucleus and the cell membrane. Depending

on the type and origin of the cell, the cytoplasm may present a variegated light microscopic appearance. Its

shape, size, and staining properties vary greatly and will be described in detail for the various tissues and organs.

In living cells, there is an intense movement of particles within the cytoplasm.

Cytoplasm consists of three components - hyaloplasm (cytomatrix), organelles and inclusions.

Hyaloplasm(cytomatrix) is the so-called soluble phase of the cell, consisting mostly of water, dissolved

solutes, and larger molecules in suspension tending to link repetitively with covalent bonds giving the cytoplasm

a dense, viscous colloidal sol or gel consistency. It contains molecules of different size and shape (e.g., electrolytes,

metabolites, RNA, and synthesized proteins). In most cells, it is the largest single compartment.

Organellesare theobligatory components of cytoplasmwhichdetermine the specification of cell.

There are several types of organelles according to their significance, morphological features and functions.

Classification of organelles:

1. According to the significance it has been divided all organelles on (into) two types:

- organelles of general significance- specific organelles which present only in certain types of cells. For example, in muscle fibres we can


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see myofilaments or myofibrils. Nervous cell has neurofibrils and chromatophilic substance. Sperm cells include

tail.

2. According to the morphplogical features organelles have been designed as

- membranous - which are bounded by a membrane. This type includes: endoplasmic reticulum (rough

and smooth), Golgi complex, mitochondria, lysosome, peroxisome.

- unmembranous which are not bounded by a membrane. They are as follows: ribosomes, centrioles,microtubules, filaments.

3. According to function it has been shown several functional apparatus as follows:

- apparatus of protein synthesis and lipid and carbohydrates metabolism: endoplasmic reticulum, Golgi

complex, ribosomes.

- apparatus of intracellular digestion: lysosomes.

- apparatus of cell movement and maintain of cell shape cytoskeleton,plasma membrane.

- energy-requiring and energy-generating apparatus: mitochondria

and many others specialised apparatus in certain types of cells.

Characteristics of membranous organelles:

1. Endoplasmic reticulum.Morphological features: It is a cytoplasmic closed system of unit membranes forming tubular canals and

flattened sacs or cisternae that subdivide the cytoplasm into a series of compartments (fig. 2.1). This system

leads towards the Golgi complex.

There are two varieties of endoplasmic reticulum:

1) Granular/roughhas the membranes of the endoplasmic reticulum that are covered with numerous

attached granules composed of ribonucleic acid (RNA) and proteins (RNP granules or ribosomes, l5 nm in

diameter. RER forms a closed system not open (closed) to cell exterior but continuous with outer surface of

nuclear envelope. In general, rough endoplasmic reticulum is abundant in cells with marked synthesis of proteins

for exportfor instance, in the pancreas or the salivary glands. In light microscopy, the RNA-rich cytoplasmic

areas stain bluish with hematoxylin and the cell is said to be basophilic (its cytoplasm stained by hematoxylin

and has blue colour). This feature is commonly observed in metabolically active cells.

Function: Granular endoplasmic reticulum takes place in secreting protein synthesis. It is a site of mRNA,

tRNA and amino acids interaction. This organelle functions in the synthesis of proteins destined to be transported

out of the cell, of proteins that comprise lysosomes, and of the proteins in the plasma membrane. Proteins

synthesized on the rough ER are transported to the Golgi complex in transport vesicles. In the Golgi, the proteins

destined for export out of the cell, or those that end up in the plasma membrane or in lysosomes, are sorted andsent to their appropriate destination.

Fig. 2.1. Structure of the endoplasmic

retuculum. Scheme: A - granular;

B - agranular.

1 - membranes; 2 - the internal cavity ofcisterns, 3 - ribosomes.

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2)Agranular/smoothis more tubular and consists of small, interconnecting channels or tubules within

the cell and lacks ribosomes on its cytosolic surface. The cytoplasm of cells which have a lot of Smooth ER is

stained by eosin and has red or pink colour. This is said to be eosinophilic or acidophilic.Smooth ER is

especially abundant in steroid hormone secreting cells.

Function:The smooth ER is the site for the synthesis and metabolism of fatty acids and phospholipids,

steroid hormones, and cholesterol, for the detoxification of drugs in the liver, adds lipid portion of lipoproteins(liver) and for the storage of calcium in muscle.

2. Golgi body/complex/apparatus.

Location: This usually takes one area near to the nucleus and often in a specific place, e.g., supra-nuclear

in cuboidal epithelial cells.

Light microscopic images of cells active in protein synthesis and secretion can exhibit an unstained area

of cytoplasm that corresponds to the Golgi in electron micrographs. Plasma cells secrete antibody molecules

(immunoglobulins) into the extracellular matrix. The region of their cytoplasm adjacent to the nucleus appears

clear and unstained, whereas the remainder of the

cytoplasm is basophilic as a result of the presence

of stacks of rough endoplasmic reticulum. Theunstained area of the plasma cells marks the

location of the Golgi complex.

Morphological features: It consists of a

complex of stacked smooth flattened cisternae

with dilated ends, tubules, and vesicles of various

sizes (fig. 2.2).

Cisternae segregated into convex (cis),

medial (middle), and concave (trans)

compartments. The Golgi is polarized so that the

cis region faces the endoplasmic reticulum

(forming face) and the trans region faces thesecretory vesicles in cells manufacturing proteins

for export. Small transitional vesicles are near

convex face. Large condensing vacuoles located

near concave face. Cis cisternae stain with

osmium, medial saccules stain for nicotinamide

adenine dinucleotide phosphatase (NADPase),

trans saccules react with thiamine

pyrophosphatase (TPPase)

Identification: In LM after special silver

staining, the Golgi apparatus may be seen as a

tangled network. With routine haematoxylin

staining in certain cells, e.g., active osteoblasts or

plasmocytes, the juxta-nuclear vacuole reveals

the site of the Golgi structure as a pale negative

image.

Functions: The Golgi plays important roles

in sorting plasma membrane and intracellular

membrane traffic, in the modification of

carbohydrates on proteins (e.g., acylation and

sulfation), and in the packaging of proteins that

will be secreted by the cell.

3. Mitochondria.Location: Because mitochondria generate ATP, they are more numerous in cells that use large amounts

B

Fig. 2.2. Ultramicroscopic structure of Golgi

complex : A - scheme; B - E photomicrograph

10000.

1 - flat cisterns, 2 - vacuoles; 3 - transport vesicles.

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of energy, such as striated muscle cells and cells engaged in fluid and electrolyte transport. Mitochondria also

localize at sites in the cell where energy is needed, as in the middle piece of the sperm, the intermyofibrillar

spaces in striated muscle cells.

Morphological features: Mitochondria are small, usually elongated structures, usually less than 0.5 m in

width and less than 7 m in length. Each mitochondrion is composed of two membranes, located one within the

other (fig. .2.3).

Outer and inner membranes separated by ~20 nm space. The outer shell of the mitochondrion is a

continuous, closed-unit membrane. Running parallel to the outer membrane is a morphologically similar inner

membrane that forms numerous crests or invaginations (cristae mitochondriales), subdividing the interior of the

organelle into a series of communicating compartments. Cristae may be tubular but are usually shelf-like. A

homogeneous material or mitochondrial matrix fills the interior of the organelle. Matrix contains dense matrix

granules, mitochondrial DNA, tRNA, rRNA and mRNA.Identification: In LM they can be stained with special methods and appear as coloured rods or granules.

In EM their shape tends to be tubular or spheroid.

Functions: The key role of the mitochondria within the cell is that of carriers of energy-producing complex

enzyme systems. Several oxidative systems have been identified within the mitochondria: Krebs cycle enzymes,

fatty acid cycle enzymes, and the enzymes of the respiratory chain, including the cytochromes. Most importantly,

the formation of energy-producing adenosine triphosphate (ATP) from phosphorus and adenosine diphosphate

(ADP) takes place within the mitochondria. The ATP is exported into the cytoplasm where it serves as an

essential source of energy for the cell. They may also store calcium. Tubular cristae prevalent in steroid

synthesizing cells and include specific enzymes for steroidogenesis.

The mitochondria possess their own DNA that is independent of nuclear DNA and is responsible for

independent protein synthesis and for the mitochondrial division cycle. This supports the concept that the

mitochondria are quasi-independent organelles, living in symbiosis with the host cell, which they supply with

energy. Two genetic systems exist within a cell, one vested in the mitochondria and the other in the nucleus. The

two systems are interdependent.

4. Lysosomes.

Morphological features:

In electron microscopic preparations, the lysosomes may be identified as spherical or oval structures of

heterogeneous density and variable diameter (fig. 2.4). Lysosomes contain a collection of hydrolyticor digestive

enzymes, acid phosphatase being the first one identified, that serve to digest the phagocytized material, and are

surrounded by a unique membrane that resists hydrolysis by their own enzymes. There are more then 50

hydrolytic enzymes. The lysosomal membrane possesses highly glycosylated specific membrane proteins thatprotect the membrane from digestion by lysosomal enzymes. These lysosome-specific proteins are synthesized

3

1 2

4

5

Fig. 2.3. Scheme of ultramicroscopic structure of

mitochondria with plate cristae.

1 - the outer mitochondrial membrane;

2 - the inner mitochondrial membrane;3 - intermembrane space, 4 - matrix;

5 - cristae.


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in the rER, transported to the Golgi apparatus. In

addition, lysosomes contain proton (H+) pumps

that transport H+ions into the lysosomal lumen,

maintaining a low pH (-4.7).

There are primary and secondary types of

lysosomes. Primary lysosomes are the newlyformed lysosomes, which arise as complete and

functional organelles budding from the Golgi

apparatus. Primary lysosomes are round, TEM-

lucent vesicles that have not participated in a

digestive event. Secondary lysosomes formed from

fusion of primary lysosome withphagosome(see

below).

Identification: In the greater majority of

cells, lysosomes cannot visualized by light

microscopy. Exceptions to this statement are the

granules of two types of white blood cells,neutrophils and eosinophils, which can be seen by

light microscopy.

Functions:

The lysosomes, or cell disposal units, are the organelles participating in the removal of phagocytized

foreign material. Occasionally, the lysosomes also digest obsolete fragments

of cytoplasm and organelles, such as mitochondria, for which the cell has no further use.

Depending on the nature of the digested material, different pathways deliver material for digestion within

the lysosomes. In the digestion process, most of the digested material comes from endocytotic processes;

however, the cell also uses lysosomes to digest its own obsolete parts, nonfunctional organelles, and unnecessary

molecules. Three pathways for digestion exist:

Extracellular large particles such as bacteria, cell debris, and other foreign materials are engulfed inthe process of phagocytosis. A phagosome, formed as the material is internalized within the cytoplasm,

subsequently fuses with a lysosome to create a phagolysosome (secondary lysosome).

Extracellular small particles such as extracellular proteins, plasma membrane proteins, and ligand-

receptor complexes are internalized by endocytosis and receptor-mediated endocytosis. These particles follow

the endocytotic pathway through early and late endosomal compartments and are finally delivered to lysosomes

for degradation.

lntracellular particles such as entire organelles, cytoplasmic proteins, and other cellular components

are isolated from the cytoplasmic matrix by endoplasmic reticulum membranes, transported to lysosomes, and

degraded in the process called autophagy (removal of cytoplasmic components, particularly membrane-bounded

organelles, by digesting them within lysosomes).

In addition, some cells (e.g., osteoclasts involved in bone resorption and neutrophils involved in acute

inflammation) may release lysosomal enzymes directly into the extracellular space to digest components of the

extracellular matrix.

More than 20 diseases are due to lysosomes altering and storage of a heterogeneous material in the

cytoplasm.

5. Peroxisome.

Morphological features: Peroxisomes (peroxide + soma) are membrane-bound spheres of moderate

electron density with 0,5-1,2 mkm in diametre. The matrix of peroxisome contains some specific enzymes, such

as catalase, peroxidase D- fnd L-aminooxydase. In some species, but not humans, a crystalline nucleoid is

present that is composed of urate oxidase (fig. 2.5).

Functions:

1) The key enzyme of peroxisome is catalasa, an enzyme that decomposes hydrogen peroxide to waterand oxygen (2 H

20

2>2 H

2O + 0

2). Thus, peroxisomes protect the cell from the effects of hydrogen peroxide,

Fig. 2.4. Ultramicroscopic structure of

lysosomes. 19 000.

1 - primary lysosome, 2 - secondary lysosome

(fagolysosome).

1

2


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which could cause irreversible damage to many important cellular constituents.

2) Anoter inportant function of peroxisome is detoxification (the elimination of a variety of toxic

compounds).

3) Moreover peroxisomes contain enzymes involved in lipid metabolism and take place in b-oxidation of

fatty acids

4) Peroxisomes also contain certain enzymes that take place in the formation of the bile acids in heparcells.

Characteristics of unmembranous organelles:

1. The Ribosome.

Location: In the cytoplasm, the ribosomes may be either floating free or they may be attached to theouter surface of the endoplasmic reticulum. Morphological features: The ribosomes are submicroscopic particles

and are composed of RNA and proteins in approximately equal proportions. They are ubiquitous and have been

identified in practically all cells of animal and plant origin. Each ribosome is composed of two, approximately

round subunits of unequal size (fig 2.6).

Ribosomes may be joined together by strands

of messenger RNA (mRNA) to form aggregates or

polyribosomes that thus resemble a string of beads.

The string may be either open or closed. Ribosomes

are attached to the membranes of the endoplasmic

reticulum by the larger subunit.

Functions: It appears likely that the two types

of ribosomes exercise different functions: the free

ribosomes are primarily engaged in the production of

proteins for the cells own use, whereas attached

ribosomes are responsible for protein production for

export. A marked concentration of ribosomes (and

hence proteins) confers upon the cytoplasm a

basophilic staining.

2. The Cytoskeleton.

The cytoskeleton is unique to eukaryotic cells. It is a dynamic three-dimensional structure that fills thecytoplasm. This structure acts as both muscle and skeleton, for movement and stability.

Fig. 2.5. Ultramicroscopic structure ofperoxisome. 33 000.

1 - peroxisome; 2 - crystalloid core;

3 - glycogen inclusions.

2

1

3

2

3

4

5

1

Fig . 2.6. The structure of the ribosome. Scheme.

1 - small subunit 2 - large subunit 3 - mRNA 4 - tRNA,

5 - a signal peptide.


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The cytoskeleton acts as a track on which cells can move organelles, chromosomes and other things.

Some examples are:

- Vesicle movement between organelles and the cell surface, frequently studied in the squid axon.

- Cytoplasmic streaming

- Movement of pigment vesicles for protective coloration

- Discharge of vesicle content for water regulation in protozoa- Cell divisioncytokinesis

- Movement of chromosomes during mitosis and meiosis

Cells have protein motors that bind two molecules, and using ATP as energy, cause one molecule to shift

in relationship to the other. Two types of these protein motors are myosin and actin, and dynein or kinesin and

microtubules (fig. 2.7).

These families of proteins all have a motor end, but may have several kinds of different molecular

structures on the binding end. When these proteins bind, they can cause many different molecules, organelles,

etc. to move. To the right is an example of the different binding ends found in the kinesin family of motors.

When linked to other microtubules, protein motors can cause motion if the ends are fixed or extend the

lengths of the fiber bundles if the ends are free.

Broken motors: In healthy individuals, the protein dystrophin is part of the linkage between the cellular

cytoskeleton and the adhesive proteins on the outside of the cell. In Duchenne Muscular Dystrophy, however,

the gene that codes for dystrophin is defective, resulting in muscle degeneration and finally death. This disease

is X-linked recessive and occurs in 1 out of every 3,500 males.

Cytoskeleton

The skeleton of the cells and, hence, the structures maintaining their physical shape, facilitating their

motion, and providing structural support to all cell functions, is provided by a family of fibrillar proteins. The

cytoskeleton is fundamentally composed of three types of fibrillar proteins, initially classified by their diameter

in electron microscopic photographs: the actin filaments (microfilaments, tonofilaments), intermediate filaments,

and microtubules.

Intermediate Filaments

Intermediate filaments are about 10 nm diameter and provide tensile strength for the cell, they are larger

than actin microfilaments and smaller than microtubules (fig. 2.8). Several subspecies of intermediate filaments

proteins have been identified, differing from each other by relative molecular mass and anatomic distribution.

Perhaps the best known of the intermediate filaments are the keratins, which have been extensively studied in

the epidermis of the skin. There are several subfamilies of keratin filaments (proteins) forming pairs, each

composed of one basic and one acidic protein. Each type of squamous epithelium (skin, cornea, other epithelia)may be represented by a special pair of proteins of high relative molecular mass. With the change of epithelial

Fig. 2.7. Proteins which associated with microtubules. Scheme.

(+ end) (- end)

endocytotic vesicle

lysosome

dyneins

kinesins

receptor

mitochondria (energyofATP)


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type from a single layer to multilayer epithelium, different keratin genes, producing proteins of increasing molecular

mass are activated. This mechanism may be important in understanding the change known as squamous

metaplasia.

Examples of the cytoskeleton: in epithelial cells (skin), cells of the intestine, all three types of fibers are

present. Microfilaments project into the villi, giving shape to the cell surface. Microtubules grow out of the

centrosome to the cell periphery. Intermediate filamentsconnect adjacent cells through desmosomes.

Microfilaments

The ubiquitous actin filaments, measuring 5 to 7 nm in

diameter, are observed in all cells of all vertebrate species. In

electron microscopy, they can be recognized as bundles of

longitudinal cytoplasmic filaments (fig. 2.9) crisscrossing the

cytoplasm and often converging on specific targets such as

desmosomes. The actin filaments are found within virtually

all structural cell components and interact with many other

proteins that regulate their length. The fundamental structureof these elongated fibrillar proteins is helical, with two different

ends: this latter feature allows the filaments to attach to two

different molecules and function as an intermediary polarized

link. The actin filaments are easily polymerized (i.e., they

form structures composed of several actin units). This is

probably the mechanism that allows actin filaments to form

tight meshworks in conjunction with other proteins. Among

the latter, it is important to mention the links of actin filaments

to a contractile protein, myosin, accounting for motion and

contractility of cells. Other linkages occur with transmembrane

proteins, such as spectrin, ensuring the communicationsbetween the cell membrane and cell interior. Thus, actin

microfilaments perform several essential functions within cells

as linkage filaments coordinating the activity of divergent cell

components. Microfilaments can also carry out cellular movements including gliding, contraction, and cytokinesis.

Microtubules

Microtubules (fig. 2.9) are an integral component of cilia, flagella, and centrioles.

3

1

2 1

3

Fig. 2.8 The structures of the apical cytoskeleton

in epithelial cells. 47 000.

1 - actin filaments, 2 - terminal network,

3 - intermediate filaments.

Fig. 2.9. Microtubules and microfilaments: A - scheme; B - Electron micrograph,

39 000.1 - microtubules, 2 - microfilaments.

2

1

B

2

1


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Microtubules, measuring between 22 and 25 nm in diameter, have long been recognized and identified by

light microscopy as the constituents of the mitotic spindle. Microtubules are hollow, tube-like structures, which

appear to be universally present in all cells, and are synthesized from precursor molecules of tubulin. They are

composed of subunits of the protein tubulin-these subunits are termed alpha and beta. Microtubules are polarized,

they have one minus and one plus end; hence, they can be attached to two different molecules and form a

bridge between them.Microtubules act as a scaffold to determine cell shape, and provide a set of tracks for cell organelles

and vesicles to move on. The principal role for microtubules and associated proteins is their participation in

cellular events requiring motion. Cilia and flagella are a good example of this function in which microtubules

perform a sliding movement in association with a protein, dynein, and an energy-producing system, adenosine

triphosphate (ATP).

The mitotic spindle is synthesized by the cells undergoing mitosis from molecules of tubulin. During cell

division, the centrioles serve as an organizing center for the mitotic spindle. From the centrioles, located at the

opposite poles of the cell, the microtubules attach to the condensed double chromosomes arranged at the

metaphase plate and participate in the migration of the single chromosomes into the two daughter cells. Once

the mitosis is completed, the spindle microtubules are depolarized and redistributed in the cytoplasm.

3. The Centriole.

The centrioles are cytoplasmic organelles that play a key role during cell division. Each interphase animal

cell contains a pair of centrioles, short tubular structures, usually located in the vicinity of the concave face of

the Golgi complex. As the cell is about to enter mitosis, another pair of centrioles appears, and each pair travels

to the opposite poles of the cell and becomes the anchoring point of the mitotic spindle. The origin of the second

pair of centrioles has not been fully clarified; apparently it is synthesized de novo from precursor molecules in

the cytoplasm. This event is induced and directed in an unknown fashion by the original pair of centrioles. Each

pair of centrioles is surrounded by a clear zone, the centrosome, which, in turn, is surrounded by a slightly

denser area or the astrosphere. Within each pair, the centrioles are placed at right angles to each other. Thus, in

a fortuitous electron micrograph (fig. 2.10), one centriole will appear in a longitudinal section and the other in

cross section. In the cross section, each centriole appears as a cylindrical structure with a clear center and ninetriplets or groups of three microtubules.

Another derivatives of microtubules complex are ciliae and flagella.

Each cilium or flagellum contains 11 microtubules, of which two are single and located within the center,

and nine are double (doublets) and located at the periphery. Dynein arms attached to the microtubules serve

as the molecular motors (fig. 2.11). Defective dynein arms cause male infertility and also lead to respiratory

tract and sinus problems.

Fig. 2.10. Ultramicroscopic

structure of the cell center.

Electronic micrography, 30 000.

1 - centrioles in cross section;

2 - longitudinal section of centrioles;

3 - triplets of microtubules.

3

2

1


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Fig. 2.11. Cilia and flagella structure.


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LESSON 3

THEME: CELL INCLUSIONS.

BACKGROUND: Inclusions are one of the component of cytoplasm that reflect some specific features

(metabolism, age, functional activity, the stage of secretory cycle and others) of different cells in normal andpathological conditions. Several diseases have been shown to be caused by defective metabolic steps, resulting

in appearance or increase of certain inclusions. This fact is often used to diagnostics in biopsies of tissue taken

from patients with diseases that store iron (eg, hemochromatosis, hemosiderosis), glycogen (glycogenosis),

glycosaminoglycans (mucopolysaccharidosis), and sphingolipids (sphingolipidosis) in tissues.

AIM OF STUDY:To determine cell inclusions, their type and functional significance. To link the quantity

and quality of different inclusions with organelles activity.


1. Determine the cell inclusions in different cells stained with different dyes.

2. Differentiate storage inclusions in different cells.

3. Identify the stages of secretory cycle by the secretory inclusions.4. Explain the structural and functional links between inclusion and different organelles.


1. What is inclusion as the component of the cell cytoplasm.

2. Morphofunctional types of inclusions.

3. Methods of inclusions identification.

4. Diagnostic significance of different inclusions quantity.

REFERENCES:


& Lange, 2008.2. Burkitt H. G., Young B., Heath J. W. Wheaters Functional Histology. A text and colour atlas. 5th edition:




Cell inclusions - These are usually transitory and non-obligatory components of the cytoplasm. (Non-

living, non-participating, poorly structured cell elements, often seen in cytoplasm).

Cell inclusions may be different in shape, size and thin structure:

Examples:

Fat dropletsappearing as vacuoles in ordinary light microscopy preparation, but usually preserved by

electron microscopy procedures as dark rounded bodies.

Lamellar bodiescontain lipids to be secreted.

Glycogen granulesvisible as dark l5-30 nm wide granules in EM, e.g., in cardiac muscle and liver cells.

Secretion granules, e.g. zymogen, in pancreatic cells.

Pigments, e.g., melanin (skin cells), lipofuscin (old neurons), haemosiderin (natural), carbon (exogenous

- from outside the body).

Crystalls, e.g., in testicular interstitial cells.

Bacteriaand viral inclusion bodies (pathological).

INCLUSIONS CLASSIFICATION

Thus, cell inclusions form a wide variety in morphology, functions and chemical composition, group, that

according to functional significance has been divided on four types:1. Storage inclusions (storage of calories/energy)


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2. Secretory granules

3. Excretory inclusions

4. Pigments.

1. Storage of calories/energy.This type of inclusions usually composed of accumulated metabolites, such as:

a) lipids

b) carbohydrates

c) yolk inclusions are typical only for egg cell.

Characteristics of lipids inclusions:

Morphological features: lipidsinclusions usually form droplets.

Routine processing methods for microscopy generally extract lipid from tissues and therefore lipid droplets

within cells appear as unstained vacuoles.

The selective method for lipid demonstration (identification) is histochemical staining of frozen sections

with osmium (Fig. 3.1 - lipids are visible as black droplets) or sudan (lipids have orange colour)

Under the electron microscope lipids droplets are of variable size and electron density and are not

bounded by a membrane. Usually these droplets are associated with organelles that take place in lipid metabolism:

smooth endoplasmic reticulum and mitochondria.

Location: You can see lipid droplets in:

a) adipose tissue orb) liver cell, where lipid droplets are used as fatty acids and glycerine storage.

c) adrenal cortex cells, where lipid droplets are used as storage of cholesterol, that are needed for steroid

hormones synthesis. In this case lipid droplets are associated with specific mitochondria - with tubular crists

and smooth endoplasmic reticulum.

Characteristics of glycogen inclusions:

Morphological features: glycogen inclusions usually form granules.

Routine processing methods for microscopy generally extract glycogen from tissues and these inclusions

are not visible.

The selective method for glycogen demonstration (identification) is histochemical staining with PAS

glycogen granules become magenta (Fig. 3.2).

1 2 3

4

5

Fig. 3.1. The inclusion of lipids in liver cells (impregnation osmic acid and safranin):

1 - liver cells; 2 - a drop of fat; 3 - cytoplasm; 4 - nucleus; 5 - nucleolus.


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Electron microscopy: After impregnation with lead salts, this substance appears as collections of coarse,

irregular electrondense particles. They often are associated with smooth endoplasmic reticulum cisterns.

Location: Glycogen appears as granules in different cells, mostly in hepatocytes, muscles.

2. Secretive inclusions.

Secretive inclusions are typical for cells with secretive abilities: for example, cells of endocrine and

exocrine glands (pancreas, parathyroid gland and others), blood granulocytes, mast cells and others.

In brief, secretive inclusions are secretive product of certain cells. This type of inclusion is resulted for

Golgi apparatusactivity and reflects accumulation in the cell secretive products. They may be different inchemical composition, but always include proteins:

- proteins,

- lipoproteins,

- glycoproteins.

Morphological features: Secretive inclusions form membrane-bound granules with rounded shape, which

well are visible under the electron microscope. The size and thin structure of granules in cells are different and

specific for certain cell types. It can be used for morphological diagnostic (determination) of these cells. In

addition, the dynamics of secretive granules amount permits to detect the secretive activity and the stage of

secretive cycle. It increases during accumulation of secretive products and decreases when secret emerge

outside the cell (in extracellular medium).

Under the light microscope secretive products may appear in different forms, according to their chemical

composition and quantity. They can react both with acid and base dyes, or can be identified by histochemical

methods.

3. Pigment inclusions

Deposits of coloured substances - pigments - are often found in different cells. They may be synthesized

by the cell (eg, in the skin melanocytes) or come from outside the body (eg, carotene).

This group of inclusions includes a wide variety of chemical substances which have different colours.

They are:

- melanin - have black or dark brown colour. This type of inclusions form dense intracellular membrane-

limited granules in cells of the epidermis of the skin and in the pigment layer of the retina

- Lipofuscin - have yellowish-brown colour (Fig. 3.3).- Haemoglobin and myoglobin (iron-contained pigments that can link oxygen)

Fig. 3.2. The inclusion of glycogen in liver cells (staining by Best):

1 - liver cells, 2 - a piece of glycogen, 3 - cytoplasm; 4 - nucleus;, 5 - nucleolus.

1 2 3

4

5


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Diagnostic significance: One of the most common pigments is lipofuscin present mainly in permanent

cells (eg, neurons, cardiac muscle) that increases in quantity with age. Its chemical constitution is complex. It is

believed that granules of lipofuscin derive from secondary lysosomes and represent deposits of undigestible

substances.

Pathological forms of inclusion. The development of some disease is due to altering of methabolic

processes in several cells. Lysosomes play an important role in the metabolism of several substances in the

human body and consequently many diseases have been ascribed to deficiencies of lysosomal enzymes. In

metachromatic leukodystrophy, there is an intracellular accumulation of sulfated cerebrosides caused by lack oflysosomal sulfatase. In most of these diseases, a specific lysosomal enzyme is absent or inactive, and the

digestion of certain substances (glycogen, cerebrosides, gangliosides, sphingomyelin, glycosaminoglycans, etc)

does not occur. As a result, these substances accumulate in different cell types, interfering with their normal cell

function. This diversity of affected cell types explains the variety of clinical symptoms observed in these diseases.

1

2

3

4

Fig. 3.3. Pigment inclusions - lipofustsyn in nerve cells:

1 - nerve cell; 2 - inclusion of lipofuscin; 3 -nucleus; 4 - nucleolus.


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LESSON 4

THEME: THE CELL NUCLEUS.

BACKGROUND:Nucleus is the main part of living cell. It performs many functions and controls

realisation of different processes, such as cell division, differentiation, adaptation, regeneration and others.AIM OF STUDY: To find and analyze the cell nucleus under light and electron microscopy, interpret

functional state of cells and determine pathological features.

TO ACHIEVE THIS AIM ONE HAS TO: (practical procedures):

1. Find out the cell nucleus in fixed cells stained with different dyes.

2. Detect the main components of the cell nucleus in a histological specimen under the light microscope

and interpret their chemical composition.

3. Determine the main components of the cell nucleus under the electron microscopy.

4. Estimate the relation between functional state of cell nucleus and cytoplasm.

5. Explain the functional state of the cell on the basis of structural organisation of its components.

TO ACHIEVE THE AIM IT IS NECESSARY TO CENTRE AROUND THE FOLLOWING

THEORETICAL POINTS:

1. The nucleus: its general morphological characteristics and functions.

2. The nucleus components, their chemical composition and morphological features.

3. Chromatin: its chemical composition, levels of condensation, chromatin types and their functions.

4. Morphological, functional and chemical characteristics of nucleolus.

5. Nuclear matrix, the organisation and role of karyoskeleton.

6. Nucleus envelope: structural basis of transport processes between nucleus and cytoplasm, pore

apparatus.

REFERENCES:1. Junqueira L. C., Carneiro J., Kelley R. O. Basic histology. A Lange medical book.Tenth edition: Appleton

& Lange, 2008.





The nucleus is the main part of the living cell. Its functions include: storage, realisation and reproduction

of the genetic information throughout whole cell life.

Cell nuclei of different tissues vary greatly in size, shape, location in cell and intensity of staining (fig.

1.4). These features depend on cell function and level of activity with regard to protein synthesis.

In most cells under the light microscope the cell nucleus appears as a rounded, elongated or irregular

shape basophilic structure, usually located in the centre of the cell. In mammalian tissues its diameter varies

between 5 and 10 mkm. Even in a single tissue you can detect cells with nuclei different in size and morphologic

features (fig. 4.1).

Using light and/or electron microscopy you can identify structural components of the cell nucleus.

The nucleus comprises the nuclear envelope, chromatin, nucleolus, and nuclear matrix (fig. 4.2).

Each cell type has its specific nuclear morphology. In general, the degree of activity of any cell may be

estimated by the structural appearance of its nucleus. For this purpose you have to analyze all structural

components of cell nucleus and their chemical composition.


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CHROMATINis the main part of nucleus.

The main chemical component of chromatin isDNAwhich carries most of the genetic information -

the information coded for different protein structure. Besides, DNA comprises less than 20% of nucleus mass.

The DNA codes for numerous different proteins that are not synthesised simultaneously in a cell. Moreover, the

total length of DNA molecule is very long. That is why DNA needs packing with certain genes selective

activation.

The nucleoproteinsplay the key role in regulation of DNA arrangement and gene activity regulation .

There are two types DNA-binding proteins: histonesand nonhistones.1) histones are relatively low molecular weight proteins with a high content of positively charged amino

acids. Functional significance: histones are involved in the packing and regulation of gene activity.

2) Nonhistones include enzymes responsible for DNA and RNA synthesis. These DNA-associated

proteins may be involved in gene activity regulation.

All nuclear proteins are synthesised in the cytoplasm and imported into the nucleus.

In addition, chromatin can include RNA, that represents newly synthesised messenger RNA which has

not passed into the cytoplasm yet.

B C

Fig. 4.1. In this picture you can see variety of the nucleus sizes and intensity of staining (A), shape

(picture B) and location in the cells (picture C). - The cells of the nervous tissue, 480.B - The blood cells. Different leucocytes have nuclei varying in shape.C - The white adipocyte, in which small dark elongated nucleus is located at the periphery of the cell.

B

Fig. 4.2. How does the cell nucleus appear under the light and electron microscope? In this pictures you

can see structural components of the nucleus under the light (A) and electrone microscope (B).


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HOW DOES CHROMATIN PACKING GET? (how is chromatin packed)

There are several stages of chromatin packing:

Firstly, the basic structural unitof chromatin is the nucleusome. Nucleosome consists of a core ofeight histonesand a short segment of DNA double helix(fig. 4.3 A) coiled around histones (fig. 4.3 B). Thisorganization of chromatin has been referred to as beads-on-a-string and corresponds to morphologicalform of euchromatin.

The nucleosomes form aggregates (fig. 4.3 C). The next higher order of chromatin organization is the

30-nmfibers. In this structure, nucleosomes coiled an axis (with six nucleosome per turn) to form the chromatin

fiber that organizes loops (looks like solenoid). (fig. 4.3 D). These forms of condensing chromatin organization

correspond to heterochtomatin.

The final stageof chromatin parking is a chromosome, which may be visible only during mitosis and

meiosis.

ESTIMATION of CHROMATIN PACKING AND ACTIVITIES BY MORPHOLOGICAL

FEATURES

The degree of DNA coiling varies during different stages of cell activity. According to the intensity of

chromatin packing there are two (morphofunctional types of chromatin.

Structural and functional statesof the chromatin: Except the cell division process you can see twodifferent typesof the chromatin, which can be distinguished with both the light and the electron microscopes.

Fig. 3.4. Stages of chromatin packing.

B

C

D

E

30


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They are euchromatin (or activechromatin) and heterochromatin(or inactivechromatin).Under the light microscopy euchromatin corresponds to a lightly stained zones (areas) in the

nucleus. The genetic information, which are coded in these zones, is accessible for reading during such process

as transcription (the first stage of protein synthesis) and replication (during S-period of cell cycle). That is why

highly active cell has a big light nucleus. But thestructural organizationof euchromatinis visible only in the

electron microscope.Heterochromatin is visible in the light microscope and appears as basophilic clumps which in

electron micrograph corresponds to coarse granules. Heterochromatin tends to be clamped around the periphery

of the nucleus, but also forms irregular clamps throughout the nucleus. This type of chromatin dominates in

nuclei of relatively inactive cells.

Diagnostic possibilities:The proportions between eu- and heterochramatin reflects the level of functional

activity. Relatively inactive cells have small dark nuclei in which heterochromatin dominates. In highly active

cells there are big light nuclei in which there is a big amount of active dispersed chromatin.

CAN YOU (How to) DETERMINE WHETHER CELL is MALE OR FEMALE ORGANISM FROM?

The reply is: Yes! For this aim you have to know What the sex chromatin is.SEX CHROMATIN:Careful study of the chromatin of mammalian cell nuclei reveals a heterochromatin

mass that is frequently observed in female cells but not in male cells. This chromatin clump is the sex chromatin

and is one of the pair of X chromosomesthat is visible in female cells during interphase. It remains tightly

coiled and visible, while the other X chromosome is uncoiled and not visible. The male has one X chromosome

and one Y chromosome as sex determinants; the X chromosome is uncoiled, and therefore no sex chromatin is

visible.

Morphological features:In human epithelial cells, sex chromatin appears as a small granule attached to

the nuclear envelope. The cells lining the internal surface of the cheek are frequently used to study sex chromatin.

Blood smears are also often used, in which case the sex chromatin appears as a drumstick-like appendage to

the nuclei of the neutrophilic leukocytes (fig. 4.4).

Diagnostic significance:The study of sex chromatin has wide applicability to medicine, because it

permits determination of genetic sex in patients whose external sex organs do not permit diagnosis of gender, as

in hermaphroditism and pseudohermaphroditism. It is essential for the study of other anomalies involving the

sex chromosomes, for examples, Klinefelters syndrome, in which testicular abnormalities, azoospermia, and

other symptoms are associated with the presence of XXY chromosomes in the cell.

NUCLEOLUS

The function of the nucleolus are synthesis of ribosomal RNA (rRNA) and ribosome assembly.

Chemical composition of nucleolus includes: 1) special zones of DNA that is known as nucleolar

organiser, 2) high amount of ribosomal RNA and 3) proteins, which are organized in complexes with rRNAmolecules to form ribosome subunits.

Fig. 4.4. Sex chromatin

inthe neutrophilic leukocyte

nucleus (arrow).


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Morphologic features:Under the light microscope nucleolus appears as a spherical structure, up to 1

mm in diameter, and rich in rRNA and proteins. It is usually basophilic when stained with hematoxylin and eosin.

As seen with the electron microscope, the nucleolus consists of three distinct components. They are:

1) The amorphous part, that looks as one or several pale-staining regions composed of nucleolar organiser

DNA sequences of bases that code for rRNA (fig. 5.4). In the human genome, five pairs of chromosomes

(13, 14, 15, 21, 22) contain nucleolar organizers. That is why the maximal amount of nucleoli in mammalian cellsnucleus is five. In this zone the transcription is realised.

2) The part fibrosa, that closely associated with the nucleolar organizers and consists of densely packed

5-10-nm ribonucleoprotein fibers. This part is composed of primary transcripts of rRNA genes.

3) The third component of the nucleolus is the pars granulosa, consisting of 15-20-nm granules (fig. 4.5),

presented by ribosome subunits.

You have to know that many different ribosomal proteins are synthesised in the cytoplasm, imported in

the nucleus and then become associated with rRNAs in the nucleolus to form different ribosomal subunits.

After that ribosome subunits migrate into the cytoplasm, where subunits being assembled into the fully active

ribosomes.

Diagnostic possibilities: The size and amount of nucleolus reflects the intensity of protein synthesis.

Large nucleoli are encountered in embryonic cells during their proliferation, in cells that are actively synthesizing

proteins, and in rapidly growing malignant tumors. The nucleolus disperses during cell division but reappears in

the telophase stage of mitosis.

NUCLEAR MATRIX

The nuclear matrix is the component that fills the space between the chromatin and the nucleoli in the

nucleus.

It is composed mainly of proteins, metabolites, and ions.

There are two types of nuclear matrix proteins. They are enzymes and fibrillar proteins which in order to

maintain structural stability and specific shape of cell nucleus arrange a supporting frameworkthat is known

as karyoskeleton (nucleoskeleton). This framework is connected with nuclear envelope by fibrous lamina, thatalso is a part of the nuclear matrix. The nucleoskeleton probably contributes to the formation of a protein base

1

1

2

2

Fig. 4.5 Morphology of the nucleolus under the electron

microscopy.1 granular component; 2 fibrillar component.


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to which DNA loops are bound.

Thus nuclear matrix arranges the supports of nuclear morphology, controls DNA and nuclear RNA

space organisation and regulates general nucleus methabolism.

NUCLEAR ENVELOPE

All cells nuclei are bounded by a membrane system, called the nuclear envelope.Functional significance:The nuclear envelope serves as dynamic interface that mediates the continuous

exchange between nucleus and cytoplasm.

Morphological features:Under the light microscope you can observe a border of nucleus as a thin

basophilic linesurrounding the nucleus. This is a thin layer of heterochromatin that lines and binds to theinternal surface of the nuclear envelope (fig. 4.2). But a single nuclear envelope(alone) can be observed

only under the electron microscopy.

Electron microscopy shows that the nucleus is actually surrounded by two parallel unit membranes

separated by a narrow (40-70-nm) space called the perinuclear cisterna. Together, the paired membranes

and the intermembranous space make up the nuclear envelope (fig. 4.6).

The internal membraneof the nuclear envelope is closely associated with a fibrous lamina(appearingas electron dense layer) - the part of nucleoskeleton, which varies in thickness from 80 to 300 nm, depending on

the cell examined. The fibrous lamina is composed of three main polypeptides, called laniinswhich bound onmembrane proteins and are linked with underlying chromatin.

The outer membrane is studded with ribosomes and polyribosomes. This portion of the nuclear envelope

is sometimes continuous with the rough endoplasmic reticulum (fig. 4.6). When covered with polyribosomes,

the nuclear envelope functions as rough endoplasmic reticulum, synthesizing polypeptide chains and segregating

them in the perinuclear cistern between its two membranes.The perinuclear space is continuous with cisterns of the endoplasmic reticulum.

Fig. 4.6. Ultramicroscopic structure of the nuclear envelope. Electronic micrography. Mag. 80 000.

1 - nucleus, 2 - outer nuclear membrane, 3 - perinuclear space (cisterna), 4 - inner nuclear membrane,5 - heterochromatin, 6 - ribosomes on the external surface of the nuclear membrane;

7 - cisterns of rough endoplasmic reticulum. Arrows indicate nuclear pores.

1

2

3

7

6

3

4

5

4

5

2

6

7


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The nuclear envelope contains numerous nuclear pores,(a) sites where the inner and outer membranes

become continuous to form circular gaps (fig. 4.7 A, B), that provide pathways between the nucleus and the

cytoplasm. Nuclear pores have an average diameter of 70 nm and are composed of eight subunits. The pores

are not open but are bridged by an electrondense membrane that forms a singlelayered diaphragm of protein.

This structure is thinner than the membranes that constitute the nuclear envelope. While the permeability of the

nucleus to molecules is variable, all pores are permeable to some macromolecules (mRNA, cytoplasmic proteins).

THERE ARE SEVERAL MECHANISMS OF THE SUBSTANCES EXCHANGE BETWEEN

NUCLEUS AND CYTOPLASM.

1. Low molecular substances are transported across the nuclear envelope according to regularities of

membrane transport in both directions.

2. Macromolecules can be exchanged through nuclear envelope by:

a) perinuclear space;

b) bulk transport;

c) pore complex.

3. Universal mechanism of nuclear-cytoplasm exchange - during cell division associated with nuclear

envelope disappearance.

3 4 5 3 1 2

B 4 5 6 2

3

1

47 nm

84 nm

115 nm

Fig. 4.7. This scheme demonstranes the structure of nuclear pore complex. Find in this picture the membranes

of the nuclear envelope (2, 3) that are continuous in sites of the pore complex, perinuclear spase (1), peripheral (4)

and central (5) granules of the nuclear pore complex and the fibrilles of pore diaphragm (6).


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LESSON 5

THEME: CELL CYCLE AND REPLICATION. CELL DIFFERENTIATION. CELL DEATH.

BACKGROUND: Even in a full-developed organism, many cells keep dividing because of cell

regeneration. New cells production is necessary for keeping the tissue integrity or for the organs restorationafter its alteration (wounds healing, reparation of the altered tissue etc.). Uncontrolled cell division is the

reason of malignant tumors outcome. Decrease of mitotic properties of cells can be a reason of growth and

regeneration altering in some organs.

In some tissues division may take place throughout all life, in others cells divide only under certain

conditions. To explain processes which take place in an organism for supporting cell amount (populations), it is

necessary to determine dependence between the degree of cell specialisation and its mitotis activity.

AIM OF STUDY:To determine cell life period by morphological features. To know the possible ways

of regulation of mitotic cell activity and cell death. To prognose the development of pathological changes in

cells, tissues, organs and possibilities of the cell reparation.


1. Determine the period of cell life.

2. Differentiate cell cycle periods by morphological features.

3. Identify kinds of cell division, their phases and essence.

4. Determine the structure features and role of chromosomes during the interphase and mitosis.

5. Define a level of cell differentiation

6. Explain the functional state of the cell on the basis of structural organisation of the cells components.

7. Detect cell death and its type.

TO ACHIEVE THE AIM IT IS NECESSARY TO FOCUS ON THE FOLLOWING

THEORETICAL POINTS:1. The cell life periods, their characteristics.

2. Cell replication. Mitosis, its phases.

3. Cell cycle, its periods and their morphological reflections.

4. Cell differentiation (specialisation): its morphological features.

5. Morphological characteristics of different functional types of mature cells.

6. Cell death: its types, morphological signs and mechanisms.

REFERENCES:


& Lange, 2008.




WHAT MUST YOU KNOW?(INSTRUCTION FOR YOUR SELF-LEARNING):

During embryogenesis a complex multicellular organism is developed from a single, fertilized egg cell -

zygota. This process involves cellular division, growthand progressive morphofunctional specialisation.

During emryogenesis and early postnatal period human tissues containe immature (i.e. relatively

unspecialised) cells. During human organism development cells believed to be capable of differentiation into all

types of cells found in mature organism. However some immature cells remain in fully mature tissues and

provide a source of cells required for replacement or repairment of tissues.

Thus in the adult human organism there are many different cells. Some of them perform individually

specialised functions. These cells are known as highly differentiated (or specialised) ones. In addition, toproduce restoration of specialised cells in fully developed tissues there is a certain amount of immature cells.


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The turnover rate of the cells varies greatly from one tissue to anotherrapid in the epithelium of the alimentary

canal (tract) and the epidermis, slow in the pancreas and thyroid. The life time period is different for particular

cells. By the end of their life period cells become old (aged) and then die.

Thus whole cell life time can be divided into different periods or states (fig. 5.1) as follows:

1) When cells are immature. These cells have ability to replication and pass through cell cycle.

2) When cells undergo differentiation and become highly specialised to perform individual functions.3) Old cells which have lower functional activities.

4) Cells that undergo cell death.

1. Immature cells.

The morphological features and functional properties of immature cells.

The key morphological features of unspecialized cells are big nucleus and a small amount of cytoplasm,

that designated high nuclear-cytoplasm index(fig. 5.2).

Fig. 5.1. Life cycle of cell.

A

B

Fig. 5.2. Morphological characteristics of unspecialised cells under light (A) and electron (B) microscopes.

The proportion of nucleus and cytoplasm volume called nuclear-cytoplasm index is known as the main criterion ofcell maturity detection.


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Under the electron microscope you can see a little amount of membrane organelles, as well as a lot of

free ribosomes in cytoplasm.

Immature cells are able to replication .

Replicationis necessary for increase of cell amount. This process occures not only during embryogenesis

but also in a fully mature organs and tissues.Division of these cells is necessary for realisation of physiological and reparative regeneration (provide

the maintenance of cellular amount in different tissues in normal conditions and after injury/alteration).

The mechanism of cells replication is known as mitosis.

MITOSIS

Mitosis (or mitotic division) of a single cell results in the formation of two daughter cells. Each of the

daughter cells receives a chromosomal karyotype identical to that of the parent cell. For realisation of this

process it is necessary to duplicate genetic information in parent cell and to distribute it identically to the

daughter cells. The time interval during w

Cytology Embryology

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