Histology & Its Study
Histology is the study of cells, tissues and organs as seen with a microscope. The microscopes commonly used in class-rooms and in laboratories are light microscopes. Magnified images of objects are seen through these microscopes by the use of glass lenses. The maximum magnification possible with a light microscope is about 1500 times.
Early histological observations were, of necessity, empirical. With the development, in recent years, of refined methods for preparation and study of tissues, and because of accompanying developments in our knowledge of the chemical composition of cells, and of constant chemical transformations within them, we now have a much better comprehension of the physiological and biochemical significance of microscopic structures. Some of the techniques that have contributed to the development of this knowledge are briefly summarized below.
Traditional Histological Methods
The earliest histological observations were made on unfixed tissue (usually teased to make a flat preparation). The first significant advance was the discovery of chemicals for fixation and for staining of tissues. The next major development was the invention of instruments (called microtomes) for cutting thin sections of tissue. These sections could be mounted on glass slides and stained.
The process of fixation preserves a tissue by denaturing its proteins. It also makes the handling of tissue, and the preparation and staining of sections, more efficient. Numerous fixatives are known, the most commonly used being formaldehyde. (Formaldehyde is a gas. This gas dissolved in water is formalin).
Before a tissue can be sectioned it has to be given a firm consistency. One way of doing this is to freeze the tissue and cut sections while it is still frozen (such sections being called frozen sections). Techniques for the production of frozen sections have undergone great refinement and at present they are prepared using a microtome enclosed in a refrigerated chamber. Such an instrument is called a cryostat. Preparation of frozen sections is the fastest method of examining a tissue. The technique allows the examination of pieces of tissue removed by a surgeon, while the patient is still on the operating table, making it possible for the surgeon to plan his operation keeping in mind the nature of disease.
Apart from freezing a tissue, it can be made suitable for sectioning by embedding it in a suitable medium, the most common being paraffin wax. Such paraffin sections can be thinner than frozen sections, and reveal more details of structure. However, some materials (e.g., fat) are lost during the process of embedding tissues in paraffin wax.
The commonest staining procedure used in histology is haematoxylin-eosin staining. In sections stained with this procedure nuclei are stained blue, and most other components are seen in varying shades of pink. Numerous other staining methods are available for demonstrating specific tissue elements.
In the last few decades many new discoveries in the field of histology have become possible because of the development of the electron microscope (usually abbreviated to EM). This microscope uses an electron beam instead of light; and electromagnetic fields in place of lenses. With the EM magnifications in excess of 100,000 times can be achieved. The structure of a cell or tissue as seen with the EM is referred to as ultrastructure.
For electronmicroscopic studies small pieces of tissue are fixed very rapidly after removal from the animal body. Special fixatives are required (the most common being glutaraldehyde). Very thin sections are required, and for this purpose tissues have to be embedded in media that are harder than wax. Epoxy resins (e.g., araldite) are used. The microtomes used for cutting sections are much more sophisticated versions of traditional microtomes and are called ultramicrotomes. Thin sections prepared in this way are also very useful in light microscopy. They reveal much more detail than can be seen in conventional paraffin sections.
Before sections are examined under an electronmicroscope they are often treated with solutions containing uranium or lead, to increase contrast of the image. Osmium tetroxide acts both as fixative and staining agent and has been extensively used for preparing tissues for electronmicroscopy.
In conventional EM studies (or transmission electronmicroscopy) images are formed by electrons passing through the section. Wide use is also made of scanning electronmicroscopy in which the images are produced by electrons reflected off the surface of a tissue. The surface appearances of tissue can be seen, and three dimensional images can also be obtained. Specially useful details of some tissues (e.g., membranes) can be obtained by freezing a tissue and then fracturing it to view the fractured surface.
In many cases the chemical nature of cellular and intercellular constituents can be determined by the use of staining techniques. Lipids and carbohydrates (glycogen) present in cells are easily demonstrated. The presence of many enzymes can be determined by placing sections in solutions containing the substrate of the enzyme, and by observing the product formed by action of enzyme on substrate. The product is sometimes visible, or can be made visible using appropriate staining agents.
For enzyme studies, the use of frozen sections is essential. Good frozen sections can be obtained by using cryostats (mentioned above).
Specific molecules within cells can be identified in tissue sections stained with antibodies specific to the molecules. The technique enables chemical substances to be localized in cells with great precision. Such studies have greatly enhanced our knowledge of chemical transformations taking place within cells.
Many molecules (e.g., aminoacids) injected into an animal become incorporated into the tissues of the animal. Sometimes it is possible to replace a normal aminoacid with a radioactive substitute. For example if a radioactive isotope of thymidine is injected, it becomes incorporated in proteins in place of normal thymidine. The sites of presence of the radioactive material can be determined by covering tissue sections with a photographic emulsion. Radiations emerging from radioactive material act on the emulsion.
After a suitable interval the emulsion is ‘developed’. Grains of silver can be seen under the microscope at sites where the radioisotope was present.
Units of measurement used in histology
The study of histology frequently involves the measurement of microscopic distances. The units used for this purpose are as follows.
1 micrometer or micron (μm)= 1/1000 of a millimetre (mm).
1 nanometre (nm)= 1/1000 of a micrometer.
Cells, Tissues And Organs
The human body, like that of most other animals and plants, is made up of units called cells. Cells can differ greatly in their structure. However, most of them have certain features in common. These are described in this chapter.
Aggregations of cells of a common type (or of common types) constitute tissues. Apart from the cells many tissues have varying intercellular substances that may separate the cells from one another. Organs (e.g., the heart, stomach or liver) are made up of combinations of various kinds of tissue.
A cell is bounded by a cell membrane (or plasma membrane) within which is enclosed a complex material called protoplasm. The protoplasm consists of a central, more dense, part called the nucleus; and an outer less dense part called the cytoplasm. The nucleus is separated from the cytoplasm by a nuclear membrane. The cytoplasm has a fluid base (matrix) which is referred to as the cytosol or hyaloplasm. The cytosol contains a number of organelles which have distinctive structure and functions. Many of them are in the form of membranes that enclose spaces. These spaces are collectively referred to as the vacuoplasm.
From what has been said above it is evident that membranes play an important part in the constitution of the cell. The various membranes within the cell have a common basic structure which we will consider before going on to study cell structure in detail.
Basic Membrane Structure
When suitable preparations are examined by EM the average cell membrane is seen to be about 7.5 nm thick. It consists of two densely stained layers separated by a lighter zone, thus creating a trilaminar appearance (Fig. 1.1A).
Cell membranes are made up predominantly of lipids. Proteins and carbohydrates are also present.
Lipids in cell membranes
It is now known that the trilaminar structure of membranes is produced by the arrangement of lipid molecules (predominantly phospholipids) that constitute the basic framework of the membrane (Fig. 1.1B).
Each phospholipid molecule consists of an enlarged head in which the phosphate portion is located; and of two thin tails (Fig. 1.2). The head end is also called the polar end while the tail end is the non-polar end. The head end is soluble in water and is said to be hydrophilic. The tail end is insoluble and is said to be hydrophobic.
When such molecules are suspended in an aqueous medium they arrange themselves so that the hydrophilic ends are in contact with the medium; but the hydrophobic ends are not. They do so by forming a bi-layer.
The dark staining parts of the membrane (seen by EM) are formed by the heads of the molecules, while the light staining intermediate zone is occupied by the tails, thus giving the membrane its trilaminar appearance.
Because of the manner of its formation, the membrane is to be regarded as a fluid structure that can readily reform when its continuity is disturbed. For the same reasons proteins present within the membrane (see below) can move freely within the membrane.
Some details regarding the lipid content of cell membranes are as follows.
- As stated above phospholipids are the main constituents of cell membranes. They are of various types including phosphatidylcholine, sphingomyelin, phosphatidylserine, and phosphatidyl-ethanolamine.
- Cholesterol provides stability to the membrane.
- Glycolipids are present only over the outer surface of cell membranes. One glycolipid is galactocerebroside which is an important constituent of myelin. Another category of glycolipids seen are ganglionosides.
Fig. 1.2: Diagram showing the structure of a phospholipid molecule (phosphatidyl choline) seen in a cell membrane
Proteins in cell membranes
In addition to molecules of lipids the cell membrane contains several proteins. It was initially thought that the proteins formed a layer on each side of the phospholipid molecules (forming a protein-phospholipid sandwich). However, it is now known that this is not so. The proteins are present in the form of irregularly rounded masses. Most of them are embedded within the thickness of the membrane and partly project on one of its surfaces (either outer or inner). However, some proteins occupy the entire thickness of the membrane and may project out of both its surfaces (Fig. 1.3). These are called transmembrane proteins.
The proteins of the membrane are of great significance as follows.
- They may form an essential part of the structure of the membrane i.e., they may be structural proteins.
- Some proteins play a vital role in transport across the membrane and act as pumps. Ions get attached to the protein on one surface and move with the protein to the other surface.
- Some proteins are so shaped that they form passive channels through which substances can diffuse through the membrane. However, these channels can be closed by a change in the shape of the protein.
- Other proteins act as receptors for specific hormones or neurotransmitters.
- Some proteins act as enzymes.
Carbohydrates of cell membranes
In addition to the phospholipids and proteins, carbohydrates are present at the surface of the membrane. They are attached either to the proteins (forming glycoproteins) or to the lipids (forming glycolipids) (Fig. 1.4). The carbohydrate layer is specially well developed on the external surface of the plasma membrane forming the cell boundary. This layer is referred to as the cell coat or glycocalyx.
Membranes in cells are highly permeable to water, and to oxygen, but charged ions (Na+, K+) do not pass through easily.
THE CELL MEMBRANE
The membrane separating the cytoplasm of the cell from surrounding structures is called the cell membrane or the plasma membrane. It has the basic structure described above. We have seen that the carbohydrate layer, or glycocalyx, is specially well formed on the external surface of this membrane.
The glycocalyx is made up of the carbohydrate portions or glycoproteins and glycolipids present in the cell membrane. Some functions attributed to the glycocalyx are as follows.
- Special adhesion molecules present in the layer enable the cell to adhere to specific types of cells, or to specific extracellular molecules.
- The layer contains antigens. These include major histocompatibility antigens (MHC). In erythrocytes the glycocalyx contains blood group antigens.
- Most molecules in the glycocalyx are negatively charged causing adjoining cells to repel one another. This force of repulsion maintains the 20 nm interval between cells. However, some molecules that are positively charged adhere to negatively charged molecules of adjoining cells, holding the cells together at these sites.
The cell membrane is of great importance in regulating the activities as follows.
- The membrane maintains the shape of the cell.
- It controls the passage of all substances into or out of the cell. Some substances (consisting of small molecules) pass through the passive channels already described: this does not involve deformation of the membrane. Larger molecules enter the cell by the process of endocytosis described below.
- The cell membrane forms a sensory surface. This function is most developed in nerve and muscle cells. The plasma membranes of such cells are normally polarized: the external surface bears a positive charge and the internal surface bears a negative charge, the potential difference being as much as 100 mv. When suitably stimulated there is a selective passage of sodium and potassium ions across the membrane reversing the charge. This is called depolarisation: it results in contraction in the case of muscle, or in generation of a nerve impulse in the case of neurons.
- The surface of the cell membrane bears receptors that may be specific for particular molecules (e.g., hormones or enzymes). Stimulation of such receptors (e.g., by the specific hormone) can produce profound effects on the activity of the cell. Receptors also play an important role in absorption of specific molecules into the cell as described below.Enzymes present within the membrane may be activated when they come in contact with specific molecules. Activation of the enzymes can influence metabolism within the cell as explained below.When a receptor on the cell surface is stimulated this often activates some substances within the cell that are referred to as second messengers. Important second messengers are as follows.
- Adenylate cyclase: This enzyme changes the concentration of cyclic adenosine monophosphate (cyclic AMP) within the cell. In turn this can lead to alterations in many functions of the cell including protein synthesis and synthesis of DNA.
- Enzymes controlling cyclic GMP have effects that are usually opposite to those controlling cyclic AMP.
- Phosphoinositol (a phospholipid) affects calcium regulatory processes within the cell.
- Membrane proteins help to maintain the structural integrity of the cell by giving attachment to cytoskeletal filaments (page 21). They also help to provide adhesion between cells and extracellular materials.
- Cell membranes may show a high degree of specialisation in some cells. For example, the membranes of rod and cone cells (present in the retina) bear proteins that are sensitive to light.
Role of cell membrane in transport of material into or out of the cell
We have seen, above, that some molecules can enter cells by passing through passive channels in the cell membrane. Large molecules enter the cell by the process of endocytosis (Fig. 1.5). In this process the molecule invaginates a part of the cell membrane, which first surrounds the molecule, and then separates (from the rest of the cell membrane) to form an endocytic vesicle. This vesicle can move through the cytosol to other parts of the cell.
Fig. 1.6: Three stages in exocytosis. The fusogenic proteins facilitate adhesion of the vesicle to the cell membrane
The term pinocytosis is applied to a process similar to endocytosis when the vesicles (then called pinocytotic vesicles) formed are used for absorption of fluids (or other small molecules) into the cell.
Some cells use the process of endocytosis to engulf foreign matter (e.g., bacteria). The process is then referred to as phagocytosis.
Molecules produced within the cytoplasm (e.g., secretions) may be enclosed in membranes to form vesicles that approach the cell membrane and fuse with its internal surface. The vesicle then ruptures releasing the molecule to the exterior. The vesicles in question are called exocytic vesicles, and the process is called exocytosis or reverse pinocytosis (Fig. 1.6).
We will now consider some further details about transfer of substances across cell membranes
Fig. 1.8: Scheme to illustrate how extracellular molecules can pass through the entire thickness of a cell (transcytosis). Caveolae are involved
- As endocytic vesicles are derived from cell membrane, and as exocytic vesicles fuse with the latter, there is a constant transfer of membrane material between the surface of the cell and vesicles within the cell.
- Areas of cell membrane which give origin to endocytic vesicles are marked by the presence of fusogenic proteins that aid the formation of endocytic vesicles. Fusogenic proteins also help in exocytosis by facilitating fusion of membrane surrounding vesicles with the cell membrane.
- When viewed by EM areas of receptor mediated endocytosis are seen as depressed areas called coated pits (Fig. 1.7). The membrane lining the floor of the pits is thickened because of the presence of a protein called clathrin. This protein forms a scaffolding around the developing vesicle and facilitates its separation from the cell membrane. Thereafter, the clathrin molecules detach from the surface of the vesicle and return to the cell membrane.
- The term transcytosis refers to a process where material is transferred right through the thickness of a cell.The process is seen mainly in flat cells (e.g., endothelium). The transport takes place through invaginations of cell membrane called caveolae. A protein caveolin is associated with caveolae (Fig. 1.8). Caveolae differ from coated pits in that they are not transformed into vesicles. Caveolae also play a role in transport of extracellular molecules to the cytosol (without formation of vesicles). (See Fig. 1.9).
Contacts between adjoining cells
In tissues in which cells are closely packed the cell membranes of adjoining cells are separated, over most of their extent by a narrow space (about 20 nm). This contact is sufficient to bind cells loosely together, and also allows some degree of movement of individual cells.
In some regions the cell membranes of adjoining cells come into more intimate contact: these areas can be classified as follows.
Classification Of Cell Contacts
These are contacts that do not show any specialized features on EM examination. At such sites adjoining cell membranes are held together as follows.
Some glycoprotein molelcules, present in the cell membrane, are called cell adhesion molecules (CAMs). These molecules occupy the entire thickness of the cell membrane (i.e., they are transmembrane proteins). At its cytosolic end each CAM is in contact with an intermediate protein (or link protein) (that appears to hold the CAM in place). Fibrous elements of the cytoskeleton are attached to this intermediate protein (and thus, indirectly, to CAMs). The other end of the CAM juts into the 20 nm intercellular space, and comes in contact with a similar molecule from the opposite cell membrane. In this way a path is established through which forces can be transmitted from the cytoskeleton of one cell to another (Fig. 1.10).
CAMs and intermediate proteins are of various types. Contacts between cells can be classified on the basis of the type of CAMs proteins present. The adhesion of some CAMs is dependent on the presence of calcium ions; while some others are not dependent on them (Fig. 1.11). Intermediate proteins are also of various types (catenins, vinculin, αactinin).
Specialized junctional structures
These junctions can be recognized by EM. The basic mode of intercellular contact, in them, is similar to that described above and involves, CAMs, intermediate proteins, and cytoskeletal elements.
Junctional areas that can be identified can be summarized as follows.
- Anchoring junctions or adhesive junctions bind cells together, They can be of the following types.
Modified anchoring junctions attach cells to extracellular material. Such junctions are seen as hemidesmosomes, or as focal spots.
- Adhesive spots (also called desmosomes, or maculae adherens).
- Adhesive belts or zona adherens.
- Adhesive strips or fascia adherens.
- Occluding junctions (zonula occludens or tight junctions). Apart from holding cells together, these junctions form barriers to movement of material through intervals between cells.
- Communicating junctions (or gap junctions). Such junctions allow direct transport of some substances from cell to cell.
Adhesion spots (Desmosomes, Maculae Adherens)
These are the most common type of junctions between adjoining cells. Desmosomes are present where strong anchorage between cells is needed e.g., between cells of the epidermis. As seen by EM a desmosome is a small circumscribed area of attachment (Fig. 1.12A). At the site of a desmosome the plasma membrane (of each cell) is thickened because of the presence of a dense layer of proteins on its inner surface (i.e., the surface towards the cytoplasm). The thickened areas of the two sides are separated by a gap of 25 nm. The region of the gap is rich in glycoproteins. The thickened areas of the two membranes are held together by fibrils that appear to pass from one membrane to the other across the gap.
We now know that the fibrils seen in the intercellular space represent CAMs (Fig. 1.13). The thickened area (or plaque) seen on the cytosolic aspect of the cell membrane is produced by the presence of intermediate (link) proteins. Cytoskeletal filaments attached to the thickened area are intermediate filaments (page 22). CAMs seen in desmosomes are integrins (desmogleins I, II). The link proteins are desmoplakins.
Adhesive Belts (Zonula Adherens)
In some situations, most typically near the apices of epithelial cells, we see a kind of junction called the zonula adherens, or adhesive belt (Fig. 1.12B). This is similar to a desmosome in being marked by thickenings of the two plasma membranes, to the cytoplasmic aspects of which fibrils are attached. However, the junction differs from a desmosome as follows:
- Instead of being a small circumscribed area of attachment the junction is in the form of a continuous band passing all around the apical part of the epithelial cell.
- The gap between the thickenings of the plasma membranes of the two cells is not traversed by filaments.
The CAMs present are cadherins. In epithelial cells zona adherens are located immediately deep to occluding junctions (Fig. 1.16).
Adhesive Strips (Fascia adherens)
These are similar to adhesive belts. They differ from the latter in that the areas of attachment are in the form of short strips (and do not go all round the cell). These are seen in relation to smooth muscle, intercalated discs of cardiac muscle, and in junctions between glial cells and nerves.
These are similar to desmosomes, but the thickening of cell membrane is seen only on one side. As such junctions the ‘external’ ends of CAMs are attached to extracellular structures. Hemidesmosomes are common where basal epidermal cells lie against connective tissue.
The cytoskeletal elements attached to intermediate proteins are keratin filaments (as against intermediate filaments in desmosomes). As in desmosomes, the CAMs are integrins.
These are also called focal adhesion plaques, or focal contacts. They represent areas of local adhesion of a cell to extracellular matrix. Such junctions are of a transient nature (e.g., between a leucocyte and a vessel wall). Such contacts may send signals to the cell and initiate cytoskeletal formation.
The CAMs in focal spots are integrins. The intermediate proteins (that bind integrins to actin filaments) are αactinin, vinculin and talin.
OCCLUDING JUNCTIONS (ZONULA OCCLUDENS)
Like the zonula adherens the zonula occludens is seen most typically near the apices of epithelial cells. At such a junction the two plasma membranes are in actual contact (Fig. 1.14A).
These junctions act as barriers that prevent the movement of molecules into the intercellular spaces. For example, intestinal contents are prevented by them from permeating into the intercellular spaces between the lining cells. Zonulae occludens are, therefore, also called tight junctions.
Recent studies have provided a clearer view of the structure of tight junctions (Fig. 1.15). Adjoining cell membranes are united by CAMs that are arranged in the form of a network that ‘stiches’ the two membranes together.
Other functions attributed to occluding junctions are as follows.
- These junctions separate areas of cell membrane that are specialized for absorption or secretion (and lie on the luminal side of the cell) from the rest of the cell membrane.
- Areas of cell membrane performing such functions bear specialized proteins. Occluding junctions prevent lateral migration of such proteins.
- In cells involved in active transport against a concentration gradient, occluding junctions prevent back diffusion of transported substances.
Apart from epithelial cells, zonulae occludens are also present between endothelial cells.
In some situations occlusion of the gaps between the adjoining cells may be incomplete and the junction may allow slow diffusion of molecules across it. These are referred to as leaky tight junctions.
Near the apices of epithelial cells the three types of junctions described above, namely zonula occludens, zonula adherens and macula adherens are often seen arranged in that order (Fig. 1.16) They collectively form a junctional complex. In some complexes the zonula occludens may be replaced by a leaky tight junction, or a gap junction (see below).
COMMUNICATING JUNCTIONS (GAP JUNCTIONS)
At these junctions the plasma membranes are not in actual contact (as in a tight junction), but lie very close to each other, the gap being reduced (from the normal 20 nm) to 3 nm. In transmission electronmicrographs this gap is seen to contain bead-like structures (Fig. 1.14B). A minute canaliculus passing through each ‘bead’ connects the cytoplasm of the two cells thus allowing the free passage of some substances (sodium, potassium, calcium, metabolites) from one cell to the other (Also see below). Gap junctions are, therefore, also called maculae communicantes. They are widely distributed in the body.
Changes in pH or in calcium ion concentration can close the channels of gap junctions. By allowing passing of ions they lower transcellular electrical resistance. Gap junctions form electrical synapses between some neurons.
The number of channels present in a gap junction can vary considerably. Only a few may be present in which case the junctions would be difficult to identify. At the other extreme the junction may consist of an array of thousands of channels. Such channels are arranged in hexagonal groups.
The wall of each channel is made up of six protein elements (called nexins, or connexons). The ‘inner’ ends of these elements are attached to the cytosolic side of the cell membrane while the ‘outer’ ends project into the gap between the two cell membranes (Fig. 1.17). Here they come in contact with (and align perfectly with) similar nexins projecting into the space from the cell membrane of the opposite cell, to complete the channel.
We have seen that (apart from the nucleus) the cytoplasm of a typical cell contains various structures that are referred to as organelles. They include the ER, ribosomes, mitochondria, the Golgi complex, and various types of vesicles (Fig. 1.18). The cytosol also contains a cytoskeleton made up of microtubules, microfilaments, and intermediate filaments. Centrioles are closely connected with microtubules. We shall deal with these entities one by one.
The cytoplasm of most cells contains a system of membranes that constitute the endoplasmic reticulum (ER). The membranes form the boundaries of channels that may be arranged in the form of flattened sacs (or cisternae) or of tubules.
Because of the presence of the ER the cytoplasm is divided into two components, one within the channels and one outside them (Fig. 1.19). The cytoplasm within the channels is called the vacuoplasm, and that outside the channels is the hyaloplasm or cytosol.
In most places the membranes forming the ER are studded with minute particles of RNA (page 32) called ribosomes. The presence of these ribosomes gives the membrane a rough appearance. Membranes of this type form what is called the rough (or granular) ER. In contrast some membranes are devoid of ribosomes and constitute the smooth or agranular ER (Fig. 1.19).
Rough ER represents the site at which proteins are synthesized. The attached ribosomes play an important role in this process (page 35). The lumen of rough ER is continuous with the perinuclear space (between the inner and outer nuclear membranes). It is also continuous with the lumen of smooth ER.
Smooth ER is responsible for further processing of proteins synthesized in rough ER. It is also responsible for synthesis of lipids, specially that of membrane phospholipids (necessary for membrane formation).
Fig. 1.19: Schematic diagram to show the various organelles to be found in a typical cell. The various structures shown are not drawn to scale
Most cells have very little smooth ER. It is a prominent feature of cells processing lipids.
Products synthesized by the ER are stored in the channels within the reticulum. Ribosomes, and enzymes, are present on the ‘outer’ surfaces of the membranes of the reticulum.
We have seen above that ribosomes are present in relation to rough ER. They may also lie free in the cytoplasm. They may be present singly in which case they are called monosomes; or in groups which are referred to as polyribosomes (or polysomes). Each ribosome consists of proteins and RNA (ribonucleic acid) and is about 15 nm in diameter. The ribosome is made up of two subunits one of which is larger than the other. Ribosomes play an essential role in protein synthesis.
Mitochondria can be seen with the light microscope in specially stained preparations. They are so called because they appear either as granules or as rods (mitos = granule; chondrium = rod). The number of mitochondria varies from cell to cell being greatest in cells with high metabolic activity (e.g., in secretory cells). Mitochondria vary in size, most of them being 0.5 to 2 μm in length. Mitochondria are large in cells with a high oxidative metabolism.
A schematic presentation of some details of the structure of a mitochondrion (as seen by EM) is shown in Fig. 1.20. The mitochondrion is bounded by a smooth outer membrane within which there is an inner membrane, the two being separated by an intermembranous space. The inner membrane is highly folded on itself forming incomplete partitions called cristae. The space bounded by the inner membrane is filled by a granular material called the matrix. This matrix contains numerous enzymes. It also contains some RNA and DNA: these are believed to carry information that enables mitochondria to duplicate themselves during cell division. An interesting fact, discovered recently, is that all mitochondria are derived from those in the fertilized ovum, and are entirely of maternal origin.
Mitochondria are of great functional importance. They contain many enzymes including some that play an important part in Kreb's cycle (TCA cycle). ATP and GTP are formed in mitochondria from where they pass to other parts of the cell and provide energy for various cellular functions. These facts can be correlated with the observation that within a cell mitochondria tend to concentrate in regions where energy requirements are greatest.
The enzymes of the TCA cycle are located in the matrix, while enzymes associated with the respiratory chain and ATP production are present on the inner mitochondrial membrane. Enzymes for conversion of ADP to ATP are located in the intermembranous space. Enzymes for lipid synthesis and fatty acid metabolism are located in the outer membrane.
Mitochondrial DNA can be abnormal. This interferes with mitochondrial and cell functions, resulting in disorders referred to as mitochondrial cytopathy syndromes. The features (which differ in intensity from patient to patient) include muscle weakness, degenerative lesions in the brain, and high levels of lactic acid. The condition can be diagnosed by EM examination of muscle biopsies. The mitochondria show characteristic para-crystalline inclusions.
The Golgi complex (Golgi apparatus, or merely Golgi) was known to microscopists long before the advent of electron microscopy. In light microscopic preparations suitably treated with silver salts the Golgi complex can be seen as a small structure of irregular shape, usually present near the nucleus (Fig. 1.18).
When examined with the EM the complex is seen to be made up of membranes similar to those of smooth ER. The membranes form the walls of a number of flattened sacs that are stacked over one another. Towards their margins the sacs are continuous with small rounded vesicles (Fig. 1.21). The cisternae of the Golgi complex form an independent system. Their lumen is not in communication with that of ER. Material from ER reaches the Golgi complex through vesicles.
From a functional point of view the Golgi complex is divisible into three regions (Fig. 1.22). The region nearest the nucleus is the cis face (or cis Golgi). The opposite face (nearest the cell membrane) is the trans face (also referred to as trans Golgi). The intermediate part (between the cis face and the trans face) is the medial Golgi.
Material synthesized in rough ER travels through the ER lumen into smooth ER. Vesicles budding off from smooth ER transport this material to the cis face of the Golgi complex. Some proteins are phosphorylated here. From the cis face all these materials pass into the medial Golgi. Here sugar residues are added to proteins to form protein-carbohydrate complexes.
Finally, all material passes to the trans face, which performs the following functions.
- Proteolysis of some proteins converts them from inactive to active forms.
- Like the medial Golgi the trans face is also concerned in adding sugar residues to proteins.
- In the trans face various substances are sorted out and packed in appropriate vesicles. The latter may be secretory vesicles, lysosomes, or vesicles meant for transport of membrane to the cell surface.
The membranes of the Golgi complex contain appropriate enzymes for the functions performed by them. As proteins pass through successive sacs of Golgi they undergo a process of purification.
Membrane Bound Vesicles
The cytoplasm of a cell may contain several types of vesicles. The contents of any such vesicle are separated from the rest of the cytoplasm by a membrane which forms the wall of the vesicle.
Vesicles are formed by budding off from existing areas of membrane. Some vesicles serve to store material. Others transport material into or out of the cell, or from one part of a cell to another. Vesicles also allow exchange of membrane between different parts of the cell.
Details of the appearances of various types of vesicles will not be considered here. However, the student must be familiar with their terminology given below.
Solid ‘foreign’ materials, including bacteria, may be engulfed by a cell by the process of phagocytosis. In this process the material is surrounded by a part of the cell membrane. This part of the cell membrane then separates from the rest of the plasma membrane and forms a free floating vesicle within the cytoplasm.
Such membrane bound vesicles, containing solid ingested material are called phagosomes. (Also see lysosomes).
Some fluid may also be taken into the cytoplasm by a process similar to phagocytosis. In the case of fluids the process is called pinocytosis and the vesicles formed are called pinocytotic vesicles.
Just as material from outside the cell can be brought into the cytoplasm by phagocytosis or pinocytosis, materials from different parts of the cell can be transported to the outside by vesicles. Such vesicles are called exocytic vesicles, and the process of discharge of cell products in this way is referred to as exocytosis (or reverse pinocytosis).
The cytoplasm of secretory cells frequently contains what are called secretory granules. These can be seen with the light microscope. With the EM each ‘granule’ is seen to be a membrane bound vesicle containing secretion. The appearance, size and staining reactions of these secretory granules differ depending on the type of secretion. These vesicles are derived from the Golgi complex.
Other Storage Vesicles
Materials such as lipids, or carbohydrates, may also be stored within the cytoplasm in the form of membrane bound vesicles.
These vesicles contain enzymes that can destroy unwanted material present within a cell. Such material may have been taken into the cell from outside (e.g., bacteria); or may represent organelles that are no longer of use to the cell. The enzymes present in lysosomes include (amongst others) proteases, lipases, carbohydrases, and acid phosphatase. (As many as 40 different lysosomal enzymes have been identified).
Lysosomes belong to what has been described as the acid vesicle system. The vesicles of this system are covered by membrane which contains H+ATPase. This membrane acts as a H+ pump creating a highly acid environment within the vesicle (up to pH5). The stages in the formation of a lysosome are as follows.
- Acid hydrolase enzymes synthesized in ER reach the Golgi complex where they are packed into vesicles (Fig. 1.23). The enzymes in these vesicles are inactive because of the lack of an acid medium. (These are called primary lysosomes or Golgi hydrolase vesicles).
- These vesicles fuse with other vesicles derived from cell membrane (endosomes). These endosomes possess the membrane proteins necessary for producing an acid medium. The product formed by fusion of the two vesicles is an endolysosome (or secondary lysosome).
- H+ ions are pumped into the vesicle to create an acid environment. This activates the enzymes and a mature lysosome is formed.
Lysosomes help in ‘digesting’ the material within phagosomes (described above) as follows. A lysosome, containing appropriate enzymes, fuses with the phagosome so that the enzymes of the former can act on the material within the phagosome. These bodies consisting of fused phagosomes and lysosomes are referred to as phagolysosomes (Fig. 1.23).
In a similar manner lysosomes may also fuse with pinocytotic vesicles. The structures formed by such fusion often appear to have numerous small vesicles within them and are, therefore, called multivesicular bodies.
After the material in phagosomes or pinocytotic vesicles has been ‘digested’ by lysosomes, some waste material may be left. Some of it is thrown out of the cell by exocytosis. However, some material may remain within the cell in the form of membrane bound residual bodies.
Lysosomal enzymes play an important role in the destruction of bacteria phagocytosed by the cell. Lysosomal enzymes may also be discharged out of the cell and may influence adjoining structures.
Lysosomes are present in all cells except mature erythrocytes. They are a prominent feature in neutrophil leucocytes.
Genetic defects can lead to absence of specific acid hydrolases that are normally present in lysosomes. As a result some molecules cannot be degraded, and accumulate in lysosomes. Examples of such disorders are lysosomal glycogen storage disease in which there is abnormal accumulation of glycogen, and Tay-Sach's disease in which lipids accumulate in lysosomes and lead to neuronal degeneration.
These are similar to lysosomes in that they are membrane bound vesicles containing enzymes. The enzymes in most of them react with other substances to form hydrogen peroxide which is used to detoxify various substances by oxidising them. The enzymes are involved in oxidation of very long chain fatty acids. Hydrogen peroxide resulting from the reactions is toxic to the cell. Other peroxisomes contain the enzyme catalase which destroys hydrogen peroxide, thus preventing the latter from accumulating in the cell. Peroxisomes are most prominent in cells of the liver and in cells of renal tubules.
Defects in enzymes of peroxisomes can result in metabolic disorders associated with storage of abnormal lipids in some cells (brain, adrenal).
The cytoplasm is permeated by a number of fibrillar elements that collectively form a supporting network. This network is called the cytoskeleton. Apart from maintaining cellular architecture the cytoskeleton facilitates cell motility (e.g., by forming cilia), and helps to divide the cytosol into functionally discrete areas. It also facilitates transport of some constituents through the cytosol, and plays a role in anchoring cells to each other.
The elements that constitute the cytoskeleton consist of the following.
- Intermediate filaments.
These are considered below.
These are about 5 nm in diameter. They are made up of the protein actin. Individual molecules of actin are globular (G-actin). These join together (polymerise) to form long chains called F-actin, actin filaments, or microfilaments.
Actin filaments form a meshwork just subjacent to the cell membrane. This meshwork is called the cell cortex. (The filaments forming the meshwork are held together by a protein called filamin). The cell cortex helps to maintain the shape of the cell. The meshwork of the cell cortex is labile. The filaments can separate (under the influence of actin severing proteins), and can reform in a different orientation. That is how the shape of a cell is altered.
Microvilli contain bundles of actin filaments, and that is how they are maintained. Filaments also extend into other protrusions from the cell surface.
Microtubules are about 25 nm in diameter (Fig. 1.24). The basic constituent of microtubules is the protein tubulin (composed of subunits α and β). Chains of tubulin form protofilaments. The wall of a microtubule is made up of thirteen protofilaments that run longitudinally (Fig. 1.24). The tubulin protofilaments are stabilized by microtubule associated proteins (MAPs).
The roles played by microtubules are as follows.
- As part of the cytoskeleton, they provide stability to the cell. They prevent tubules of ER from collapsing.
- Microtubules facilitate transport within the cell. Some proteins (dynein, kinesin) present in membranes of vesicles, and in organelles, attach these to microtubules, and facilitate movement along the tubules. Such transport is specially important in transport along axons.
- In dividing cells microtubules form the mitotic spindle.
- Cilia are made up of microtubules (held together by other proteins).
These are so called as their diameter (10 nm) is intermediate between that of microfilaments (5 nm) and of microtubules (25 nm). The proteins constituting these filaments vary in different types of cells.
They include cytokeratin (in epithelial cells), neurofilament protein (in neurons), desmin (in muscle), glial fibrillary acidic protein (in astrocytes); lamin (in the nuclear lamina of cells), and vimentin (in many types of cells).
The role played by intermediate filaments is as follows.
- Intermediate filaments link cells together. They do so as they are attached to transmembrane proteins at desmosomes. The filaments also facilitate cell attachment to extracellular elements at hemidesmosomes.
- In the epithelium of the skin the filaments undergo modification to form keratin. They also form the main constituent of hair and of nails.
- The neurofilaments of neurons are intermediate filaments. Neurofibrils help to maintain the cylindrical shape of axons.
- The nuclear lamina (page 27) consists of intermediate filaments.
All cells capable of division (and even some which do not divide) contain a pair of structures called centrioles. With the light microscope the two centrioles are seen as dots embedded in a region of dense cytoplasm which is called the centrosome. With the EM the centrioles are seen to be short cylinders that lie at right angles to each other. When we examine a transverse section across a centriole (by EM) it is seen to consist essentially of a series of microtubules arranged in a circle. There are nine groups of tubules, each group consisting of three tubules (Fig. 1.25).
Fig. 1.25: Transverse section acros a centriole (near its base). Note nine groups of tubules, each group having three microtubules
Centrioles play an important role in the formation of various cellular structures that are made up of microtubules. These include the mitotic spindles of dividing cells, cilia, flagella, and some projections of specialized cells (e.g., the axial filaments of spermatozoa). It is of interest to note that cilia, flagella and the tails of spermatozoa all have the 9 + 2 configuration of microtubules that are seen in a centriole.
Projections from the Cell Surface
Many cells show projections from the cell surface. The various types of projections are described below.
These can be seen, with the light microscope, as minute hair-like projections from the free surfaces of some epithelial cells (Fig. 1.26). In the living animal cilia can be seen to be motile. Details of their structure, described below, can be made out only by EM. A scanning EM view is shown in Fig. 1.27.
The free part of each cilium is called the shaft. The region of attachment of the shaft to the cell surface is called the base (also called the basal body, basal granule, or kinetosome). The free end of the shaft tapers to a tip.
Each cilium is 0.25 μm in diameter. It consists of (a) an outer covering that is formed by an extension of the cell membrane; and (b) an inner core (axoneme) that is formed by microtubules arranged in a definite manner. The arrangement of these tubules, as seen in a transverse section across the shaft of a cilium is shown in Fig. 1.28. It has a striking similarity to the structure of a centriole (described above). There is a central pair of tubules that is surrounded by nine pairs of tubules. The outer tubules are connected to the inner pair by radial structures (which are like the spokes of a wheel). Other projections pass outwards from the outer tubules.
As the tubules of the shaft are traced towards the tip of the cilium it is seen that one tubule of each outer pair ends short of the tip so that near the tip each outer pair is represented by one tubule only. Just near the tip, only the central pair of tubules is seen (Fig. 1.29).
At the base of the cilium one additional tubule is added to each outer pair so that here the nine outer groups of tubules have three tubules each, exactly as in the centriole.
Microtubules in cilia are bound with proteins (dynein and nexin). Nexin holds the microtubules together. Dyenin molecules are responsible for bending of tubules, and thereby for movements of cilia.
Functional significance of cilia
The cilia lining an epithelial surface move in co-ordination with one another the total effect being that like a wave. As a result fluid, mucous, or small solid objects lying on the epithelium can be caused to move in a specific direction. Movements of cilia lining the respiratory epithelium help to move secretions in the trachea and bronchi towards the pharynx. Ciliary action helps in the movement of ova through the uterine tube, and of spermatozoa through the male genital tract.
In some situations there are cilia-like structures that perform a sensory function. They may be non-motile, but can be bent by external influences. Such ‘cilia’ present on the cells in the olfactory mucosa of the nose are called olfactory cilia: they are receptors for smell. Similar structures called kinocilia are present in some parts of the internal ear. In some regions there are hair-like projections called stereocilia: these are not cilia at all, but are large microvilli (see below).
Abnormalities of cilia
Cilia can be abnormal in persons with genetic defects that interfere with synthesis of ciliary proteins. This leads to the immotile cilia syndrome. As secretions are not removed from respiratory passages the patient has repeated and severe chest infections. Women affected by the syndrome may be sterile as movement of ova along the uterine tube is affected. Ciliary proteins are present in the tails of spermatozoa, and an affected male may be sterile because of interference with the motility of spermatozoa.
Ciliary action is also necessary for normal development of tissues in embryonic life. Migration of cells during embryogenesis is dependent on ciliary action, and if the cilia are not motile various congenital abnormalities can result.
These are somewhat larger processes having the same basic structure as cilia. In the human body the best example of a flagellum is the tail of the spermatozoon. The movements of flagella are different from those of cilia. In a flagellum, movement starts at its base. The segment nearest the base bends in one direction. This is followed by bending of succeeding segments in opposite directions, so that a wave like motion passes down the flagellum. When a spermatozoon is suspended in a fluid medium this wave of movement propels the spermatozoon forwards (exactly in the way a snake moves forwards by a wavy movement of its body).
Fig. 1.30: Microvilli as seen in longitudinal section. The regular arrangement of microvilli is characateristic of the striated border of intestinal abssorptive cells
Microvilli & Basolateral folds
Microvilli are finger-like projections from the cell surface that can be seen by EM (Fig. 1.30). Each microvillus consists of an outer covering of plasma membrane and a cytoplasmic core in which there are numerous microfilaments (actin filaments). The filaments are continuous with actin filaments of the cell cortex. Numerous enzymes, and glycoproteins, concerned with absorption have been located in microvilli.
With the light microscope the free borders of epithelial cells lining the small intestine appear to be thickened: the thickening has striations perpendicular to the surface. This striated border of light microscopy (Fig. 1.31) has been shown by EM to be made up of long microvilli arranged parallel to one another.
In some cells the microvilli are not arranged so regularly. With the light microscope the microvilli of such cells give the appearance of a brush border (Fig. 1.32).
Microvilli greatly increase the surface area of the cell and are, therefore, seen most typically at sites of active absorption e.g., the intestine, and the proximal and distal convoluted tubules of the kidneys. Modified microvilli called stereocilia are seen on receptor cells in the internal ear, and on the epithelium of the epididymis.
In some cells the cell membrane over the basal or lateral aspect of the cell shows deep folds (basolateral folds). Like microvilli, basolateral folds are an adaptation to increase cell surface area.
Basal folds are seen in renal tubular cells, and in cells lining the ducts of some glands. Lateral folds are seen in absorptive cells lining the gut.
The nucleus constitutes the central, more dense, part of the cell. It is usually rounded or ellipsoid. Occasionally it may be elongated, indented or lobed. It is usually 4–10 μm in diameter. The nucleus contains inherited information that is necessary for directing the activities of the cell as we shall see below.
In usual class-room slides stained with haematoxylin and eosin, the nucleus stains dark purple or blue while the cytoplasm is usually stained pink. In some cells the nuclei are relatively large and light staining. Such nuclei appear to be made up of a delicate network of fibres: the material making up the fibres of the network is called chromatin (because of its affinity for dyes). At some places (in the nucleus) the chromatin is seen in the form of irregular dark masses that are called heterochromatin. At other places the network is loose and stains lightly: the chromatin of such areas is referred to as euchromatin. Nuclei which are large and in which relatively large areas of euchromatin can be seen are referred to as open-face nuclei. Nuclei that are made up mainly of heterochromatin are referred to as closed-face nuclei (Fig. 1.33).
In addition to the masses of heterochromatin (which are irregular in outline), the nucleus shows one or more rounded, dark staining bodies called nucleoli (See below). The nucleus also contains various small granules, fibres and vesicles (of obscure function). The spaces between the various constituents of the nucleus described above are filled by a base called the nucleoplasm.
With the EM the nucleus is seen to be surrounded by a double layered nuclear membrane or nuclear envelope. The outer nuclear membrane is continuous with endoplasmic reticulum. The space between the inner and outer membranes is the perinuclear space. This is continuous with the lumen of rough ER. The inner layer of the nuclear membrane provides attachment to the ends of chromosomes (see below). Deep to the inner membrane there is a layer containing proteins and a network of filaments: this layer is called the nuclear lamina. Specific proteins present in the inner nuclear membrane give attachment to filamentous proteins of the nuclear lamina. These proteins (called lamins) form a scaffolding that maintains the spherical shape of the nucleus. At several points the inner and outer layers of the nuclear membrane fuse leaving gaps called nuclear pores. Each pore is surrounded by dense protein arranged in the form of eight complexes. These proteins and the pore together form the pore complex.
Nuclear pores represent sites at which substances can pass from the nucleus to the cytoplasm and vice versa (Fig. 1.19). The nuclear pore is about 80 nm across. It is partly covered by a diaphragm that allows passage only to particles less than 9 nm in diameter. A typical nucleus has 3000 to 4000 pores.
It is believed that pore complexes actively transport some proteins into the nucleus, and ribosomes out of the nucleus.
Nature and Significance of Chromatin
In recent years there has been a considerable advance in our knowledge of the structure and significance of chromatin. It is made up of a substance called deoxyribonucleic acid (usually abbreviated to DNA); and of proteins.
The structure of DNA is described on page 30. It is in the form of a long chain of nucleotides. Most of the proteins in chromatin are histones. Some non-histone proteins are also present.
Filaments of DNA form coils around histone complexes. The structure formed by a histone complex and the DNA fibre coiled around it is called a nucleosome. Nucleosomes are attached to one another forming long chains (Fig. 1.34). These chains are coiled on themselves (in a helical manner) to form filaments 30 nm in diameter. These filaments constitute chromatin.
Fig. 1.34: Scheme to show the structure of a chromatin fibre. The DNA fibril makes two turns around a complex formed by histones to form a nucleosome. Nucleosomes give the chromatin fibre the appearance of a beaded string. The portion of the DNA fibre between the nucleosomes is called linker-DNA
Filaments of chromatin are again coiled on them selves (supercoiling), and this coiling is repeated several times. Each coiling produces a thicker filament. In this way a filament of DNA that is originally 50 mm long can be reduced to a chromosome only 5 μm in length. (A little calculation will show that this represents a reduction in length of 10,000 times!).
Some details of the formation of a histone complex are shown in Fig. 1.35. Five types of histones are recognized. These are H1, H2A, H2B, H3 and H4. Two molecules each of H2A, H2B, H3 and H4 join to form a granular mass, the nucleosome core. The DNA filament is wound twice around this core, the whole complex forming a nucleosome. The length of the DNA filament in one nucleosome contains 146 nucleotide pairs. One nucleosome is connected to the next by a short length of linker DNA. Linker DNA is made up of about 50 nucleotide pairs.
Heterochromatin represents areas where chromatin fibres are tightly coiled on themselves forming ‘solid’ masses. In contrast euchromatin represents areas where coiling is not so marked. During cell division the entire chromatin within the nucleus becomes very tightly coiled and takes on the appearance of a number of short, thick, rod-like structures called chromosomes. Chromosomes are made up of DNA and proteins. Proteins stabilize the structure of chromosomes.
Chromosomes are considered in detail on page 29. The structure of DNA is considered on page 30. Also see sex-chromatin (page 44).
We have seen that nuclei contain one or more nucleoli. These are spherical and about 1–3 μm in diameter. They stain intensely both with haematoxylin and eosin, the latter giving them a slight reddish tinge. In ordinary preparations they can be distinguished from heterochromatin by their rounded shape. (In contrast masses of heterochromatin are very irregular). Nucleoli are larger and more distinct in cells that are metabolically active.
Using histochemical procedures that distinguish between DNA and RNA it is seen that the nucleoli have a high RNA content. With the EM nucleoli are seen to have a central filamentous zone (pars filamentosa) and an outer granular zone (pars granulosa) both of which are embedded in an amorphous material (pars amorphosa) (Fig. 1.36).
Nucleoli are formed in relationship to the secondary constrictions of specific chromosomes (page 37). These regions are considered to be nucleolar organizing centres. Parts of the chromosomes located within nucleoli constitute the pars chromosoma of nucleoli.
Nucleoli are sites where ribosomal RNA is synthesized. The templates for this synthesis are located on the related chromosomes. Ribosomal RNA is at first in the form of long fibres that constitute the fibrous zone of nucleoli. It is then broken up into smaller pieces (ribosomal subunits) that constitute the granular zone. Finally, this RNA leaves the nucleolus, passes through a nuclear pore, and enters the cytoplasm where it takes part in protein synthesis as described on page 33.
Haploid and Diploid Chromosomes
We have seen that during cell division the chromatin network in the nucleus becomes condensed into a number of thread-like or rod-like structures called chromosomes. The number of chromosomes in each cell is fixed for a given species, and in man it is 46. This is referred to as the diploid number (diploid = double). However, in spermatozoa and in ova the number is only half the diploid number i.e., 23: this is called the haploid number (haploid = half).
Autosomes and Sex Chromosomes
The 46 chromosomes in each cell can again be divided into 44 autosomes and two sex chromosomes. The sex chromosomes may be of two kinds, X or Y. In a man there are 44 autosomes, one X chromosome, and one Y chromosome; while in a woman there are 44 autosomes and two X chromosomes in each cell. When we study the 44 autosomes we find that they really consist of 22 pairs, the two chromosomes forming a pair being exactly alike (homologous chromosomes). In a woman the two X chromosomes form another such pair; but in a man this pair is represented by one X and one Y chromosome. We shall see later that one chromosome of each pair is obtained (by each individual) from the mother, and one from the father.
As the two sex chromosomes of a female are similar the female sex is described as homogametic; in contrast the male sex is heterogametic.
Significance of Chromosomes
Each cell of the body contains within itself a store of information that has been inherited from precursor cells. This information (which is necessary for the proper functioning of the cell) is stored in chromatin. Each chromosome bears on itself a very large number of functional segments that are called genes. Genes represent ‘units’ of stored information which guide the performance of particular cellular functions, which may in turn lead to the development of particular features of an individual or of a species. Recent researches have told us a great deal about the way in which chromosomes and genes store and use information.
The nature and functions of a cell depend on the proteins synthesized by it. Proteins are the most important constituents of our body. They make up the greater part of each cell and of intercellular substances. Enzymes, hormones, and antibodies are also proteins.
It is, therefore, not surprising that one cell differs from another because of the differences in the proteins that constitute it. Individuals and species also owe their distinctive characters to their proteins. We now know that chromosomes control the development and functioning of cells by determining what type of proteins will be synthesized within them.
Chromosomes are made up predominantly of a nucleic acid called deoxyribonucleic acid (or DNA), and all information is stored in molecules of this substance. When the need arises this information is used to direct the activities of the cell by synthesizing appropriate proteins. To understand how this becomes possible we must consider the structure of DNA in some detail.
Basic Structure of DNA
DNA in a chromosome is in the form of very fine fibres. If we look at one such fibre it has the appearance shown in Fig. 1.37. It is seen that each fibre consists of two strands that are twisted spirally to form what is called a double helix. The two strands are linked to each other at regular intervals. (Note the dimensions shown in Fig. 1.37).
Each strand of the DNA fibre consists of a chain of nucleotides. Each nucleotide consists of a sugar, deoxyribose, a molecule of phosphate and a base (Fig. 1.38). The phosphate of one nucleotide is linked to the sugar of the next nucleotide (Fig. 1.39). The base that is attached to the sugar molecule may be adenine, guanine, cytosine or thymine. The two strands of a DNA fibre are joined together by the linkage of a base on one strand with a base on the opposite strand (Fig. 1.40).
This linkage is peculiar in that adenine on one strand is always linked to thymine on the other strand, while cytosine is always linked to guanine. Thus the two strands are complementary and the arrangement of bases on one strand can be predicted from the other.
The order in which these four bases are arranged along the length of a strand of DNA determines the nature of the protein that can be synthesized under its influence. Every protein is made up of a series of amino acids; the nature of the protein depending upon the amino acids present, and the sequence in which they are arranged. Amino acids may be obtained from food or may be synthesized within the cell. Under the influence of DNA these amino acids are linked together in a particular sequence to form proteins.
Fig. 1.41: Codes for some amino acids made up of the bases adenine (A), cytosine (C), guanine (G), and thymine (T) on a DNA molecule. When this code is transferred to messenger RNA, cytosine is formed opposite guanine (and vice versa), adenine is formed opposite thymine, while uracil (U)is formed opposite adenine
Further Details of DNA Structure
In the preceding paragraphs the structure of DNA has been described in the simplest possible terms. We will now consider some details.
- The structure of the sugar deoxyribose is shown in Fig. 1.42. Note that there are five carbon atoms; and also note how they are numbered.
- Next observe, in Fig. 1.43, that C-3 of one sugar molecule is linked to C-5 of the next molecule through a phosphate linkage (P). It follows that each strand of DNA has a 5’ end and a 3’ end.
- Next observe that although the two chains forming DNA are similar they are arranged in opposite directions. In Fig. 1.43 the 5’ end of the left chain, and the 3’ end of the right chain lie at the upper end of the figure.Fig. 1.42: Diagram to show the structure of deoxyribose. Note the numbering of carbon atoms. From Fig. 1.43 you can see that C3 of one molecule is attached to C5 of the next molecule through a phosphate bond.The two chains of nucleotides are, therefore, said to be antiparallel.
- The C-1 carbon of deoxyribose give attachment to a base. This base is attached to a base of the opposite chain as already described.
- The reason why adenine on one strand is always linked to thymine on the other strand is that the structure of these two molecules is complementary and hydrogen bonds are easily formed between them. The same is true for cytosine and guanine.
Ribonucleic Acid (RNA)
In addition to DNA, cells contain another important nucleic acid called ribonucleic acid or RNA. The structure of a molecule of RNA corresponds fairly closely to that of one strand of a DNA molecule, with the following important differences.
- RNA contains the sugar ribose instead of deoxyribose.
- Instead of the base thymine it contains uracil.
RNA is present both in the nucleus and in the cytoplasm of a cell. It is present in three main forms namely messenger RNA (mRNA), transfer RNA (tRNA) and ribosomal RNA. Messenger RNA acts as an intermediary between the DNA of the chromosome and the amino acids present in the cytoplasm and plays a vital role in the synthesis of proteins from amino acids.
Some forms of RNA are confined to nuclei. The small nuclear RNAs (SnRNA) are concerned with RNA splicing (page 35).
Synthesis of Protein
We have seen that a protein is made up of amino acids that are linked together in a definite sequence. This sequence is determined by the order in which the bases are arranged in a strand of DNA. Each amino acid is represented in the DNA molecule by a sequence of three bases (triplet code). It has been mentioned earlier that there are four bases in all in DNA, namely adenine, cytosine, thymine and guanine. These are like letters in a word. They can be arranged in various combinations so that as many as sixty four code ‘words’ can be formed from these four bases. There are only about twenty amino acids that have to be coded for so that each amino acid has more than one code. The code words for some amino acids are shown in Fig. 1.41. The code for a complete polypeptide chain is formed when the codes for its constituent amino acids are arranged in proper sequence. That part of the DNA molecule that bears the code for a complete polypeptide chain constitutes a structural gene or cistron.
Fig. 1.44: Simplified scheme to show how proteins are synthesized under the influence of DNA. The process is actually more complex as explained in the text
At this stage it must be emphasized that a chromosome is very long and thread-like. Only short lengths of the fibre are involved in protein synthesis at a particular time.
The main steps in the synthesis of a protein may now be summarized as follows (Fig. 1.44).
- The two strands of a DNA fibre separate from each other (over the area bearing a particular cistron) so that the ends of the bases that were linked to the opposite strand are now free.
- A molecule of messenger RNA is synthesized using one DNA strand as a guide (or template), in such a way that one guanine base is formed opposite each cytosine base of the DNA strand, cytosine is formed opposite guanine, adenine is formed opposite thymine, and uracil is formed opposite adenine. In this way the code for the sequence in which amino acids are to be linked is passed on from DNA of the chromosome to messenger RNA. This process is called transcription. [Transcription takes place under the influence of the enzyme RNA polymerase.] That part of the messenger RNA strand that bears the code for one amino acid is called a codon.
- This molecule of messenger RNA now separates from the DNA strand and moves from the nucleus to the cytoplasm (passing through a nuclear pore).
- In the cytoplasm the messenger RNA becomes attached to a ribosome.
- As mentioned earlier the cytoplasm also contains another form of RNA called transfer RNA. In fact there are about twenty different types of transfer RNA each corresponding to one amino acid. On one side transfer RNA becomes attached to an amino acid. On the other side it bears a code of three bases (anticodon) that are complementary to the bases coding for its amino acid on messenger RNA. Under the influence of the ribosome several units of transfer RNA, along with their amino acids, become arranged along side the strand of messenger RNA in the sequence determined by the code on messenger RNA. This process is called translation.
- The amino acids now become linked to each other to form a polypeptide chain. From the above it will be clear that the amino acids are linked up exactly in the order in which their codes are arranged on messenger RNA, which in turn is based on the code on the DNA molecule (but also see below). Chains of amino acids formed in this way constitute polypeptide chains. Proteins are formed by union of polypeptide chains.
The flow of information from DNA to RNA and finally to protein has been described as the “central dogma of molecular biology”.
Some Further Details About Genes and Protein Synthesis
In addition to the protein coding sequences (of bases) DNA also bears other regions that have a controlling function. These regions provide signals for initiation and termination of the process of transcription, or for the control of the process in other ways. The DNA sequence that provides the signal for initiation of transcription is called the promoter. Binding of RNA polymerase to the promoter causes the DNA fibre to uncoil, and thus makes it possible for RNA polymerase to reach the fibre and to begin the process of transcription. Transcription continues up to the region of the DNA fibre that bears a code that gives a signal for termination of transcription.
Apart from initiating the process of transcription, the promoter also determines the rate of transcription.
Although all cells contain a complete complement of DNA all of it is not used for transcription in every cell. The region of DNA that is to be transcribed (in a particular cell) is determined by gene regulatory proteins present in the nucleus. These proteins bind to sites on DNA that are called enhancers. This binding is necessary before transcription can take place. Enhancers also control the rate of transcription by determining the number of RNA polymerase molecules that are engaged in transcription of the same length of DNA. Apart from sites on DNA that act that act as enhancers, there are others that act as repressors.
Messenger RNA formed as described above is modified before it is used for protein synthesis. Long chains of mRNA are broken up into short lengths. This process is called splicing. Some of the pieces formed by splicing again join together (in modified sequence) to form a new chain that is used for protein synthesis. Other short lengths are destroyed. Combination of the short lengths in different ways can enable formation of different proteins under the influence of the same DNA sequences. It will be obvious that only some of the DNA sequences used for transcription will actually be used for protein synthesis. These sequences are referred to as exons. Those not used are called introns. The process of RNA splicing may be regarded as a method to get rid of introns. Why introns are created in the first place is not clear, but they probably perform some regulatory function.
The initially formed mRNA (or primary transcript) is modified as follows.
- The mRNA has two ends. The end at which transcription begins is the 5’ end (the other being the 3’ end). A molecule of methylguanine gets attached to the 5’ end (and is called the methylguanine cap). The cap is responsible for protecting mRNA from degradation.The opposite (3’) end is referred to as the poly(A) tail. This tail is also concerned with stability of mRNA.
- As mentioned above, the process of removal of introns from the primary transcript is called splicing. Splicing takes place while mRNA is still within the nucleus. The mechanism of splicing is complex and we will not consider it here. Apart from removal of introns splicing allows exons to be united in sequences different from the original.
- After entry into cytoplasm most mRNAs have a short life (a few minutes to a day) after which they are destroyed. The first region to undergo destruction is the poly(A) tail and this exposes the rest of mRNA to the action of ribonuclease (which is responsible for destruction of mRNA).
Formation of proteins by translation of mRNA is controlled by various factors. Newly synthesized protein often needs modification before it is in its final form. The modifications may include:
- A process of folding which may require the presence of accessory proteins.
- Addition of other molecules (sugar, phosphate).
- Cleavage of the originally formed protein to generate an active form.
Role of Ribosomes in Protein Synthesis
Ribosomes play an essential part in protein synthesis. They ‘read’ the code on mRNA and help to arrange units of tRNA in proper sequence. The two subunits of ribosomes (large and small) play different roles in protein synthesis. The smaller unit (40s unit) is concerned with the process of translation. The larger unit (60s unit) is responsible for release of new protein into the vacuoplasm within the cisternae of endoplasmic reticulum.
Messenger RNA entering the cytosol meets a free ribosome. Messenger RNA has an initial part that bears the code for a signal sequence. This signal sequence tells the ribosome about the nature of the protein to be formed, and determines how the ribosome will behave.
- If the signal is for a protein that is to remain in the cytosol, the ribosome does not get attached to ER. The protein synthesized is released into the cytosol.
- If the signal is for a membrane protein, or for a protein to be secreted, the ribosome attaches to the surface of ER. Membrane proteins synthesized get incorporated into ER membrane. They are transferred from rough ER to smooth ER and, thereafter, to the Golgi complex, and then to the cell surface.
Proteins that are to form secretions enter the lumen of rough ER. They pass into the lumen of smooth ER, and then (through vesicles) to the cis face of the Golgi complex. After being appropriately processed in the Golgi complex they are packaged into vesicles and are discharged from the cell by exocytosis.
Duplication of Chromosomes
One of the most remarkable properties of chromosomes is that they are able to duplicate themselves. From the foregoing discussion on the structure of chromosomes it is clear that duplication of chromosomes involves the duplication (or replication) of DNA. This takes place as follows (Fig. 1.45).
- The two strands of the DNA molecule to be duplicated unwind and separate from each other so that their bases are ‘free’.
- A new strand is now synthesized opposite each original strand of DNA in such a way that adenine is formed opposite thymine, guanine is formed opposite cytosine, and vice versa. This new strand becomes linked to the original strand of DNA to form a new molecule. As the same process has taken place in relation to each of the two original strands, we now have two complete molecules of DNA. It will be noted that each molecule has one strand that belonged to the original molecule and one strand that is new. It will also be noted that the two molecules formed are identical to the original molecule.
Structure of Fully Formed Chromosomes
Each chromosome consists of two parallel rod-like elements that are called chromatids (Fig. 1.46). The two chromatids are joined to each other at a narrow area that is light staining and is called the centromere (or kinetochore). In this region the chromatin of each chromatid is most highly coiled and, therefore, appears to be thinnest. The chromatids appear to the ‘constricted’ here and this region is called the primary constriction.
Fig. 1.46: Diagram to show the terms applied to some parts of a typical chromosome. Note that this chromosome is submetacentric
Fig. 1.47: Nomenclature used for different types of chromosomes, based on differences in lengths of the two arms of each chromatid
Typically the centromere is not midway between the two ends of the chromatids, but somewhat towards one end. As a result each chromatid can be said to have a long arm and a short arm. Such chromosomes are described as being submetacentric (when the two arms are only slightly different in length); or as acrocentric (when the difference is marked) (Fig. 1.47). In some chromosomes the two arms are of equal length: such chromosomes are described as metacentric. Finally, in some chromosomes the centromere may lie at one end: such a chromosome is described as telocentric.
Differences in the total length of chromosomes, and in the position of the centromere are important factors in distinguishing individual chromosomes from each other. Additional help in identification is obtained by the presence in some chromosomes of secondary constrictions. Such constrictions lie near one end of the chromatid. The part of the chromatid ‘distal’ to the constriction may appear to be a rounded body almost separate from the rest of the chromatid: such regions are called satellite bodies. (Secondary constrictions are concerned with the formation of nucleoli and are, therefore, called nucleolar organizing centres). Considerable help in identification of individual chromosomes is also obtained by the use of special staining procedures by which each chromatid can be seen to consist of a number of dark and light staining transverse bands.
We have noted that chromosomes are distinguishable only during mitosis. In the interphase (between successive mitoses) the chromosomes elongate and assume the form of long threads. These threads are called chromonemata (Singular = chromonema).
Fig. 1.48: Chromosome showing dark and light bands revealed by staining with the Giemza method. The pattern of banding is specific for each chromosome, but different patterns are obtained with different staining methods
Using the criteria described above it is now possible to identify each chromosome individually and to map out the chromosomes of an individual. This procedure is called karyotyping. For this purpose a sample of blood from the individual is put into a suitable medium in which lymphocytes can multiply. After a few hours a drug (colchicin, colcemide) that arrests cell division at a stage when chromosomes are most distinct is added to the medium. The dividing cells are then treated with hypotonic saline so that they swell up. This facilitates the proper spreading out of chromosomes. A suspension containing the dividing cells is spread out on a slide and suitably stained. Cells in which the chromosomes are well spread out (without overlap) are photographed. The photographs are cut up and the chromosomes arranged in proper sequence. In this way a map of chromosomes is obtained, and abnormalities in their number or form can be identified. In many cases specific chromosomal abnormalities can be correlated with specific diseases.
(For details of chromosomal abnormalities see the author's HUMAN EMBRYOLOGY).
In recent years greater accuracy in karyotyping has been achieved by use of several different banding techniques, and by use of computerized analysis.
It has been estimated that the total DNA content of a cell (in all chromosomes put together) is represented by about 6 × 109 nucleotide pairs. Of these 2.5 × 108 are present in chromosome 1 (which is the largest chromosome). The Y-chromosome (which is the smallest chromosome) contains 5 × 107 nucleotide pairs.
In the region of the centromere the DNA molecule is specialized for attachment to the spindle. This region is surrounded by proteins that form a mass. This mass is the kinetochore. The ends of each DNA molecule are also specialized. They are called telomeres.
Multiplication of cells takes place by division of pre-existing cells. Such multiplication constitutes an essential feature of embryonic development. Cell multiplication is equally necessary after birth of the individual for growth and for replacement of dead cells.
We have seen that the chromosomes within the nuclei of cells carry genetic information that controls the development and functioning of various cells and tissues and, therefore, of the body as a whole. When a cell divides it is essential that the whole of the genetic information within it be passed on to both the daughter cells resulting from the division.
In other words the daughter cells must have chromosomes identical in number and in genetic content to those in the mother cell. This type of cell division is called mitosis.
A different kind of cell division called meiosis occurs during the formation of gametes. This consists of two successive divisions called the first and second meiotic divisions. The cells resulting from these divisions (i.e., the gametes) differ from other cells in the body in that:
- the number of chromosomes is reduced to half the normal number, and
- the genetic information in the various gametes produced is not identical.
Many cells of the body have a limited span of functional activity at the end of which they undergo division into two daughter cells. The daughter cells in turn have their own span of activity followed by another division. The period during which the cell is actively dividing is the phase of mitosis. The period between two successive divisions is called the interphase.
The greater part of interphase is called the G1 stage, which may last from a few hours to many years. During this period the cell carries out its ‘normal’ functions. About 12 hours before the onset of mitosis the synthesis of DNA takes place and is completed in about 7 hours: this period is called the S stage (S for synthesis). The last five hours before mitosis are utilized for synthesis of proteins required for cell division. This is called the G2 stage of interphase. Cells at G2 stage have a double complement of DNA.
Mitosis is conventionally divided into a number of stages called prophase, metaphase, anaphase and telophase. The later part of prophase is also called prometaphase. The sequence of events of the mitotic cycle is best understood by starting with a cell in telophase. At this stage each chromosome consists of a single chromatid (Fig. 1.49G). With the progress of telophase the chromatin of the chromosome uncoils and elongates and the chromosome can no longer be identified as such. However, it is believed to retain its identity during the interphase (which follows telophase). This is shown diagrammatically in Fig. 1.49A. During the S stage of interphase the DNA content of the chromosome is duplicated so that another chromatid identical to the original one is formed: the chromosome is now made up of two chromatids (Fig. 1.49B).
When mitosis begins (i.e., during the prophase) the chromatin of the chromosome becomes gradually more and more coiled so that the chromosome become recognizable as a thread-like structure that gradually acquires a rod-like appearance (Fig. 1.49C). Towards the end of prophase the two chromatids constituting the chromosome become distinct (Fig. 1.49D) and the chromosome now has the typical structure described above.
While the changes described above are occurring in the chromosomes a number of other events are taking place. The two centrioles separate and move to opposite poles of the cell. They produce a number of microtubules that pass from one centriole to the other and form a spindle. Tubules radiating from each centriole create a star like appearance or aster. The spindle and the two asters collectively form the diaster (also called amphiaster or achromatic spindle). Meanwhile the nuclear membrane breaks down and the nucleoli disappear (Fig. 1.49D). With the formation of the spindle the chromosomes move to a position midway between the two centrioles (i.e., at the equator of the cell) where each chromosome becomes attached to microtubules of the spindle by its centromere. This stage is referred to as metaphase (Fig. 1.49E). The plane along which the chromosomes lie during metaphase is the equatorial plate.
In the anaphase the centromere of each chromosome splits longitudinally into two so that the chromatids now become independent chromosomes. At this stage the cell can be said to contain 46 pairs of chromosomes. One chromosome of each such pair now moves along the spindle to either pole of the cell (Fig. 1.49F). This is followed by telophase in which two daughter nuclei are formed by appearance of nuclear membranes around them. The chromosomes gradually elongate and become indistinct. Nucleoli reappear. The centriole is duplicated at this stage or in early interphase (Fig. 1.49G).
The division of the nucleus is accompanied by the division of the cytoplasm. In this process the organelles are presumably duplicated and each daughter cell comes to have a full complement of them. The cleavage into two separate cells is referred to as cytokinesis.
The rate of cell division varies from tissue to tissue, being greatest in those epithelia which lose cells because of friction (e.g., the epidermis and the lining cells of the intestine). The rate varies with demand becoming much greater during repair after injury. The rate is precisely controlled to correlate with demand. Failure of such control results in uncontrolled growth leading to formation of tumours. Abnormalities in mitosis may be produced by exposure to various radiations, the most important being nuclear radiation. Mitosis can be arrested by chemicals. One of them is colchicin (or colcemide). It stops mitosis at metaphase and allows us to study chromosomes at this stage.
Some cells do not undergo mitosis (neurons, cardiac muscle cells). They are said to be in the G0 phase. Some cells (e.g., those of the liver) do not normally divide. This may divide to replace cell damage by disease.
As already stated meiosis consists of two successive divisions called the first and second meiotic divisions. During the interphase preceding the first division duplication of the DNA content of the chromosomes takes place as in mitosis.
First Meiotic Division
The prophase of the first meiotic division is prolonged and is usually divided into a number of stages as follows.
- Leptotene: The chromosomes become visible (as in mitosis). Although each chromosome consists of two chromatids these cannot be distinguished at this stage (Fig. 1.50A). At first the chromosomes are seen as threads bearing bead-like thickenings (chromomeres) along their length. One end of the thread is attached to the nuclear membrane. During leptotene the chromosomes gradually become thicker and shorter.
- Zygotene: We have seen that the 46 chromosomes in each cell consist of 23 pairs (the X and Y chromosomes of the male being taken as a pair). The two chromosomes of each pair come to lie parallel to each other, and are closely apposed. This pairing of chromosomes is also referred to as synapsis or conjugation. The two chromosomes together constitute a bivalent (Fig. 1.50B).
- Pachytene: The two chromatids of each chromosome become distinct. The bivalent now has four chromatids in it and is called a tetrad. There are two central and two peripheral chromatids, one from each chromosome (Fig. 1.50C). An important event now takes place. The two central chromatids (one belonging to each chromosome of the bivalent) become coiled over each other so that they cross at a number of points. This is called crossing over. For sake of simplicity only one such crossing is shown in Fig. 1.50D. At the site where the chromatids cross they become adherent: the points of adhesion are called chiasmata.
- Diplotene: The two chromosomes of a bivalent now try to move apart. As they do so the chromatids ‘break’ at the points of crossing and the ‘loose’ pieces become attached to the opposite chromatid. This results in exchange of genetic material between these chromatids. A study of Fig. 1.50E will show that as a result of this crossing over of genetic material each of the four chromatids of the tetrad now has a distinctive genetic content.
The metaphase follows. As in mitosis the 46 chromosomes become attached to the spindle at the equator, the two chromosomes of a pair being close to each other (Fig. 1.51A).
Fig. 1.52: Daughter cells resulting from the second meiotic division. The daughter cells are not alike because of the crossing over during the first division
The anaphase differs from that in mitosis in that there is no splitting of the centromeres. One entire chromosome of each pair moves to each pole of the spindle (Fig. 1.51B). The resulting daughter cells, therefore, have 23 chromosomes, each made up of two chromatids (Fig. 1.51C).
The telophase is similar to that in mitosis.
The first meiotic division is followed by a short interphase. This differs from the usual interphase in that there is no duplication of DNA. Such duplication is unnecessary as the chromosomes of the cells resulting from the first meiotic division already possess two chromatids each (Fig. 1.51C).
Second Meiotic Division
The second meiotic division is usually said to be similar to mitosis, as there is no reduction in chromosome number. However, as explained in Chapter 18, the DNA content of the daughter cells is reduced to half. Because of the crossing over that has occurred during the first division, the daughter cells are not identical in genetic content (Fig. 1.52). These reasons make the second meiotic division different from a typical mitosis.
At this stage it may be repeated that the 46 chromosomes of a cell consist of 23 pairs, one chromosome of each pair being derived from the mother and one from the father.
During the first meiotic division the chromosomes derived from the father and those derived from the mother are distributed between the daughter cells entirely at random. This, along with the phenomenon of crossing over, results in thorough shuffling of the genetic material so that the cells produced as a result of various meiotic divisions (i.e., ova and spermatozoa) all have a distinct genetic content. A third step in this process of genetic shuffling takes place at fertilization when there is a combination of randomly selected spermatozoa and ova. It is, therefore, not surprising that no two persons (except identical twins) are alike.
CHROMOSOMAL SEX AND SEX CHROMATIN
We have seen that each cell of a human male has 44+X+Y chromosomes; and that each cell of a female has 44+X+X chromosomes. We have also seen that during the formation of gametes by meiosis the chromosome number is reduced to half. As a result all ova contain 22+X chromosomes. Spermatozoa are of two types. Some have the chromosomal constitution 22+X and the others have the constitution 22+Y. If an ovum is fertilized by a sperm bearing an X-chromosome the resulting child has (22+X)+(22+X) = 44+X+X chromosomes and is a girl. On the other hand if an ovum is fertilized by a sperm bearing a Y-chromosome the child has (22+X) +(22+Y) = 44+X +Y chromosomes and is a boy.
Of the two X-chromosomes in a female only one is functionally active. The other (inactive) X-chromosome forms a mass of hetero-chromatin that lies just under the nuclear membrane. This mass of heterochromatin can be identified in suitable preparations and can be useful in determining whether a particular tissue belongs to a male or a female. Because of this association with sex this mass of heterochromatin is called the sex chromatin. It is also called a Barr-body after the name of the scientist who discovered it. In some cells the sex chromatin occupies a different position from that described above. In neurons it forms a rounded mass lying very close to the nucleolus and is, therefore, called a nucleolar satellite. In neutrophil leucocytes it may appear as an isolated round mass attached to the rest of the nucleus by a narrow band, thus resembling the appearance of a drumstick.
Rarely, some individuals may have more than two X-chromosomes. In these cases, only one X-chromosome is active (and hence euchromatic) while others are represented by masses of heterochromatin. Thus in a person with three X-chromosomes two masses of sex chromatin are seen.
In some cases the sex of an individual may not be clear (at birth) because of abnormalities in the genital organs. In such cases the true sex of the individual may be determined by looking for the sex chromatin. Methods are also available for identifying the Y-chromosome in cells. However, the best thing to do is to make a karyotype.
For a description of common chromosomal abnormalities see the author's HUMAN EMBRYOLOGY.