William A Beresford MA, D Phil ©
Professor of Anatomy
Anatomy Department, West Virginia University, Morgantown, USA

Chapter 2 CYTOLOGY l

l Body components

These are cells, extracellular substances, and body fluids. Fluids can have their suspended solid constituents viewed microscopically as smear preparations (see Blood; Chapter l7.A), but are otherwise of limited histological interest. Extracellular substances are important for the cells whose environment they form: they reflect and help control cellular activities, aside from their critical structural mechanical properties. Individual materials can be seen and localised by histochemistry.

2 Cells: chemical constitution and fixation

  1. Composition: much water; proteins, nucleic acids, lipids, carbohydrates, amino acids, minerals, hormones, vitamins, etc.
  2. Fixation stabilizes mainly proteins, and protein conjugates. These substances are used as building materials for the firmer structures of the cell. Lipids, minerals, glucose, and smaller molecules are usually lost from the section during processing. What is left is a skeleton of structures - membranes, granules, filaments - made up of proteins, polypeptides, polysaccharides and some other macromolecular materials. The special steps of histo- and cytochemistry (Chapter 30.C) preserve and reveal some smaller molecules.

3 Cells: living properties and specialization
1 Properties of cells: (a) general - communication, respiration and energy storage and release, synthesis, excretion, growth, differentiation, reproduction; (b) specialized - irritability to stimuli (excitability), motility, contractility, conductivity, absorption, phagocytosis, secretion.
2 During development from the fertilized oocyte, a great variety of cells is formed in the mammal, each kind specializing in a certain function, e.g., secretion, but many activities, such as energy production, are common to all cells. The cells of the four primary tissues - epithelial, connective, nervous and muscular - are divided along lines of specialized function, e.g., muscle for contractility and excitability.

4 Cell morphology
1 Cells performing a given function have a characteristic size, form and fine structure adapted to that task. However it may help at this stage to think in terms of a composite cell having all the features the various cells of the body display.
2 The cell is defined as a distinct entity by having a thin skin or plasmalemma/cell membrane separating off from the outside a soft, viscous, almost fluid cytoplasm, in which are suspended a number of firmer, recognizable structures - organelles and inclusions - and one or more nuclei. The nucleus, likewise, is a mass of material enclosed in nuclear membranes.

5 Cell components
1 Cytoplasm: 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.

2 Cell or plasma membrane/plasmalemma

3 Nuclear membrane
(a) Is doubled with a 20 to 25 nm perinuclear space between the two membranes. Each is of unit membrane with a trilaminar nature similar to that of the plasma membrane.
(b) Many of the apparent interruptions or pores/fenestrations through the doubled membrane are covered by a very thin 'diaphragm', actually granular and fibrillar in nature.
(c) Transport between nucleus and cytoplasm takes place at assemblies of proteins at the pore complexes.
(d) The outer nuclear membrane sometimes leads on out to a membrane system existing in the cytoplasm.

4 Organelles
Structures 1-8, below, are organelles - actively participating cytoplasmic bodies of characteristic structure and behaviour.
Points to note: (a) their morphology in light microscopy; (b) morphology in EM; (c) multiple or single; (d) any special location in the cell; and (e) functions.

1. Endoplasmic reticulum
(a) Is the name given to the cytoplasmic membrane system of many parallel membranes and tubules in communication with one another. This system of closed channels (cisternae) leads towards the Golgi complex.
(b) Two varieties of endoplasmic reticulum (ER) are seen.
....(i) Granular/rough/GER has fine granules, ribosomes of ribonucleo-
... protein (RNP), l5 nm in diameter, in clusters studding the outer surface
... of the parallel membranes.
...(ii) Agranular/smooth lacks the ribosomes and is more tubular. This
... kind is associated with cholesterol metabolism among other things, whereas
... the granular variety is related to protein synthesis, e.g., enzyme
... formation.

2. Ribosome particles: may lie free in the cytoplasm in small clusters (polyribosomes/polysomes) unrelated to membranes. This is noted particularly in growing cells.

3. Golgi body/complex/apparatus
(a) This usually takes one area near to the nucleus and often in a specific place, e.g., supra-nuclear in cuboidal epithelial cells.
(b) It consists of a complex of stacked smooth cisternae-enclosing lamellae, tubules, and vesicles of various sizes.
(c) It is more disorderly in appearance than the smooth endoplasmic reticulum and has more closed sacs or vesicles, often with dark staining material within them.
(d) 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, the juxta-nuclear vacuole reveals the site of the Golgi structure as a pale negative image.
(e) Its tasks are the concentration and preparation for storage of proteins, and completing the synthesis of complex sugars.

4. Microtubules: with a diameter of 20-25 nm; seen lying in the cytoplasm only with EM or fluorescent tagging; they give form to the cell, and are responsible for much intracellular transport and some movements.

5. Filaments: fine threads visible in EM, but may be aggregated into thicker fibrils visible in LM; in muscle cells they are a very important contractile element; in all cells they furnish a flexible skeleton articulated by the cell itself.

6. Mitochondria
(a) The many to be found in almost all cells may or may not have a special location within the cell.
(b) 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.
(c) They are hollow bodies enclosed in two unit membranes; the inner membrane projects inwards as plates or tubules called cristae, studded with small 9 nm wide elementary particles - rounded bodies on stalks. In the matrix of the intercristal space inside the mitochondrion, granules may be found.
(d) Mitochondria are very rich in the enzymes associated with the storage and release of energy, and with respiration, and fatty acid metabolism, etc. They may also store calcium.

7. Lysosomes
(a) Are round, single-membrane-limited, darkly staining bodies without cristae and containing another class of enzyme, hydrolytic or digestive.
(b) The material that they digest may be:
... expended organelles of the cell itself;
... extracellular matter engulfed by phagocytosis and membrane-enclosed in a phagosome;
... endocytotic vesicles.
Material resistant to digestion may persist as a residual body.

8. Centrosome/diplosome/division body/centriolar complex
(a) A body which lies alongside the nucleus at the cell centre or cytocentrum and is just visible as dots with LM. EM reveals that it consists of two centrioles lying at right-angles to one another. Each is an open-ended cylindrical body with a wall composed of nine bundles, each of three microtubules. These tubules help to initiate and control the aster, also microtubular, during cell division.
(b) A similar cylindrical structure is seen at the base of each cilium and is called a basal body/kinetosome. One way basal bodies and cilia develop is by a multiplication of centrioles.

5 Cell nucleus
(a) Nuclear membranes have been discussed in 5.3.
(b) Chromatin granules/karyosomes seen in the sap during the interphase period are chromosomes composed of deoxyribose-nucleic acid molecules (DNA) not fully uncoiled. The sex chromatin of female cells is an extreme example of not uncoiling, where one of the female's two X chromosomes continues in a condensed heterochromatin, rather than the dispersed euchromatin, form, although a few genes on that chromosome escape inactivation.
(c) The nucleolus seen as a dense body in most cells' nuclei with LM. is a condensation of protein and RNA and DNA, comprising granules supported by microfilaments. The dense branching strand is the nucleolonema.
(d) There is great variety in the size of the nucleus, its shape, the densities of staining of chromatin and nucleolus, and in the position of the sex chromatin, if present.
(e) In general, a large nucleus, with much pale euchromatin and a prominent nucleolus, is very active in the control of protein synthesis by transcribing mRNA from chromatin DNA. The nucleolus is mostly pre-ribosomal RNA. Nucleoli are prominent in nerve and Sertoli cells.

6 Cell inclusions
(a) Non-living, non-participating, poorly structured cell elements, very rarely seen in an intra-nuclear position; usually cytoplasmic.
(b) Examples:

7 Dynamic nature of the cell
The cell is not a static entity in life. Its chemical constitution and morphology are in continuous flux. Its complement of organelles is altering, with wearing-out and replacement, i.e., the cell is having to synthesize its own material. The cell itself represents a system of activities isolated to partial extent from an extracellular environment. Within the cell things are constantly being altered, moved around and joined up within the membranes. The membranes define temporary compartments separated from the cytoplasm, where particular activities can be confined and controlled by enzymes attached to the extensive membrane surfaces. Dynamic aspects of the cell's existence are partly deduced from a study of its morphology in specimens fixed in various states, partly from microscopical observations of living cells, and from histophysiological experiments outlined in Chapter 30.

8 Cell staining
l Cellular material contains a lot of water and has little natural colour. It was discovered that various dyes intended for textiles would selectively stain different structures in the cell. These stains may be discussed for how their chemical nature relates to the particular things in the cell with which they react and stain: for more details see Chapter 30.B.3.
2 Routine staining of human material usually employs haematoxylin, which can be made to react preferentially with nucleic acids and acidic groups of proteins. The nucleic acids are concentrated in the nucleus and the rough endoplasmic reticulum. The colour is usually some shade of blue or brown depending on the particular method of haematoxylin staining used.
3 A few cells have a lot of granular ER in their cytoplasm, which gives a strong staining reaction, and the cell is said to be basophilic/ basophil - liking basic stains and haematoxylin. This term depends on how the cytoplasm reacts: it is taken for granted that the nucleus will react positively with basic stains.
4 Most cells do not have such a concentration of GER that their cytoplasm stains significantly with haematoxylin. Consequently, another cytoplasmic stain is needed. Such a general stain is eosin, acidic in nature, which stains the cytoplasm of most cells red or pink. Some structures stain bright red. These are said to be strongly eosinophilic or acidophilic/acidophil - liking an acidic stain.
5 Three points should be mentioned.

6 Haematoxylin and eosin, then, are used as a routine procedure to stain the tissues - cells, and some extracellular materials, such as hyaline cartilage matrix (by haematoxylin), and collagen (by eosin) - and make things in general easier to see. When the interest is in specific cell features, special methods are used: for example, methods for glycogen granules, fibrils, etc. After performing the special staining, a counterstain is often used to reveal the nuclei or some other aspects of the general background to the special feature.

9 Cytological description of an individual cell
In light microscopy involves: (l) relative and absolute size; (2) shape; (3) number of nuclei; (4) shape and size of nucleus/nuclei; (5) intensity of nuclear staining; (6) amount of cytoplasm; (7) staining affinity of cytoplasm, e.g., basophilic, acidophilic (eosin), argentophilic (silver stains), or chromophobe (liking no stain); (8) granular cytoplasm; (9) nature of any inclusions, for instance, melanin pigment, fat, carbon, bacteria, zymogen granules, glycogen, mucus; (l0) specializations of the cell membrane, e.g., cilia, a brush/striated border (many microvilli); (ll) distinctive organelles in cytoplasm and their position, e.g., prominent Golgi complex, many fibrils, numerous orderly mitochondria giving another striated effect, Nissl substance (GER) in nerve cells; (l2) whether the cell is in some phase of mitosis or meiosis; (l3) the cell's surroundings; (l4) manifest properties of the living cell, e.g., motility, phagocytosis, contractility.

10 Aspiration cytology
The above cellular detail discriminates pathological, e.g., malignant, change in cells. These can be obtained, single and clumped, from any tissue or organ of the body, where a needle can be introduced to suck out cells to make a smear for fixing and staining - fine-needle aspiration cytology (FNA).

11 Cell division
l Review from biology what happens to (a) the centrioles and spindle, (b) the nuclear chromatin and nucleolus, (c) the nuclear membrane, (d) the cell membrane, and (e) the cytoplasm and its organelles and inclusions,
2 during the various phases: pro-, meta-, ana-, telo-phase and interphase.
3 During interphase the chromatids duplicate themselves by an exact replication, when DNA has to be synthesized.
4 Pursue biochemists and molecular biologists for their accounts of the molecular controls on cell division, progress through the cell cycle Powerpoint, and the continue working-proliferate-die decisions, and the medical relevance.

12 Apoptosis
1 The orderly or programmed death of cells is needed to balance cell proliferation in mature renewing tissues, such as blood and epithelia. Also, one strategy of development is to overproduce cells, then select, e.g., for the survival of correctly connected neurons, or of lymphocytes reactive to non-self antigens. Thirdly, if an organ cannot work properly, e.g., a gland has a blocked main duct, or its hormonal drive stops, many cells die by apoptosis.
2 For apoptosis, endonucleases are activated which break up the chromatin, chopping up the DNA, transcription slows and stops, organelles clump, and the GER dilates. Caspases digest relevant cellular materials and structures. The cell and internal membranes bleb out. Finally the shrunken cell is phagocytosed by macrophages.[Caspases - cysteinyl aspartate-specific proteinases]
3 With LM, only the increasing nuclear density and cell shrinkage are noticeable, unless special cytochemical methods to detect apoptotic events (2 above) are used.
4 Apoptosis is intentionally unobtrusive, to remove single cells without provoking inflammation or upsetting tissue function.
5 Quite often, the inclination of cells is to die, and they need signals not to undergo apoptosis.

13 Stem cells
For a stable population, the corollary to cell death is cell renewal. This requires:
..(i) the proliferation of cells;
..(ii) an enduring population of stem cells;
..(iii) controls (+ & -) that promote division of stem cells to maintain their numbers - self-replication;
..(iv) controls that cause differentiation of certain of the stem cells to become the determined/committed precursors of the mature cells of the tissue;
..(v) factors to promote division of the precursors/progenitors and their further differentiation. The controlling factors include cytokines (Chapter 8.F).

More is known about the ensuing progenitor cells than about the stem cells. Although not essential to the concept of stem cells, at step (iv) above, stem cells usually give rise to more than one lineage of differentiated cells, in order to furnish the needed diversity of cell types in blood and most epithelia. One mechanism for this is the asymmetric cell division, wherein the daughter cells of a mitosis differ.

The next chapter (Cytology II) reviews in more detail the structures of the cell, emphasizing their functions.

William A Beresford, Anatomy Department, School of Medicine, West Virginia University, Morgantown, WV 26506-9128, USA - - e-mail: -- wberesfo@wvu.edu -- wberesfo@hotmail.com -- beresfo@wvnvm.wvnet.edu -- fax: 304-293-8159