M.

Gorky Donetsk National medical university

CYTOLOGY
AND
GENERAL
EMBRYOLOGY

Barinov E.F., Sulayeva O.N., Tereschuk B.P.,

Khlamanova L.I., Chereshneva E.V., Gatina K.I.,
Prylutskaya I.A.
Edited by
academician of the Highest School of Ukraine,
Doctor of Medical Sciences,
Professor Barinov E.F.
Donetsk
2011

METHODS OF HISTOLOGICAL INVESTIGATION

AIM:To get acquainted with a microscopic technique, main stages of preparing histological specimens
and their investigation
Rules of working with a light microscope
1. Open your drawing-pad and put your microscope on the left half of the drawing-pad in front of you.
2. Place the low magnification objective opposite the microscope table opening at the distance 1.5cm.
3. Check up the position of the small objective. Centre the small objective - turn it to the click.
4. Looking through the eyepiece, illuminate the visual field with mirror using the concave side of the mirror.
5. Put the specimen on the microscope table with coverslip upwards. Place the section opposite the
objective lens.
6. Look from the side, move the microscope tubus down, leave a minimal distance between the objective
and the specimen (up to 1cm).
7. Look through the ocular lens and slowly raise the tubus with macroscrew until the image is clear.
8. Observe the whole specimen under a low magnification.
9. Choose the part of the specimen that you need to study under a high magnification. Centre it precisely
in the visual field.
10. Raise the tubus a little, turn the objective turret and install the higher objective lens.
11. Look from the side, very carefully move the microscope tubus down until 1mm distance is left
between the lens and the specimen surface. Do not perform this operation looking through the eyepiece!
12. Look through the eyepiece and turn the microscrew until the image is clear.
13. When you have finished your work, raise the tubus by turning the macroscrew and turn the objective
turret, returning to the small objective.
14. Remove the specimen from the microscope table and put it on the desk.
Main rules of specimen investigation in light microscopy
To learn the general structure of a specimen it is necessary to study it under a low magnification. Profound
investigation of the specimen is possible while studying it under a high magnification when you can study the
shape, proportions and disposition of the cells.
Illustration includes studying a specimen and its drawing in detail.
Notice that colour and scale of reproduction of histological structures is of great importance. It should be
performed with coloured pencils in a drawing-pad with corresponding indications.
Main steps of preparing of tissue specimens for a light microscopy
1. Selection and fixation of the material for investigation (formalin, ethyl alcohol, special mixtures)
2. Dehydration (substitutions) in ethyl alcohol
3. Clearing (xylene)
4. Infiltration (ethyl alcohol, xylol)
5. Embedding (in paraffin or celloidin)
6. Sectioning with microtome (6-8mkm thick)
7. Mounting (glass slide)
8. Removal of paraffin
9. Rehydration
10. Staining and covering.
Histological dyes
1. Nuclear or alcaline: hematoxylin, carmine, saphranin.
2. Cytoplasmic or acidic: eosin, acid fushcin, picrin acid, orange.
3. Special dyes: orsein, sudan, osmic acid.
4. Heavy metal impregnation: silver, gold.

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LESSON 1
THEME: GENERAL ORGANIZATION OF CELL. PLASMA MEMBRANE.
BACKGROUND: Eukaryotic cell is an elementary living system which consists of three basic elements:
plasmolemme, cytoplasm and nucleus. Cells provide cell division, passing genetic information, body growth,
adaptation processes, physiological and reparative regeneration. Plasmolemme plays an important role in the
cell performing protective function, providing metabolism, intracellular homeostasis, shape of the cell, its ability
to move, in that way regulating life processes, creating different systems within multicellular organism by
providing morphological and informational bound in the intercellular cooperation and association of cells with
extracellular matrix. Alterations in structure and functions of plasmolemme cause development of different
pathological processes (insulin-independent diabetes mellitus and many others)
AIM OF STUDY: Be able to differentiate cells and their derivatives in histological specimens and
electron photographs. In the electron photos be able to differentiate plasmolemme, intercellular junctions.
TO ACHIEVE THIS AIM ONE HAS TO (practical procedures):
1. Differentiate cells, their components in histological sections and electron photographs.
2. Differentiate histological elements in histological sections and electronogramms.
3. Identify the structure and features of biological membrane.
4. Interpret the structure and functions of plasmolemme.
5. Differentiate intercellular junctions in the electron photographs, understand their structure and functions.
TO ACHIEVE THE AIM IT IS NECESSARY TO CENTRE AROUND THE FOLLOWING POINTS:
1. Cell as a basic form of multi-cell organism existence. Cell theory.
2. Histological elements as structural parts of human body. Types of histological elements.
3. Main principles of eukaryotic cell structure, its main parts.
4. Biological membrane: its structure, chemical characteristics and functions.
5. Plasma membrane, its structure, chemical and functional characteristics of its layers.
6. Functions of plasmolemme and their morphological representation.
7. Intercellular junctions: types, structure and functions.
REFERENCES:
1. Junqueira L. C., Carneiro J., Kelley R. O. Basic histology. A Lange medical book. Tenth edition: Appleton
& Lange, 2008.
2. Burkitt H. G., Young B., Heath J. W. Wheaters Functional Histology. A text and colour atlas. 5th edition:
Churchill Livingstone, 2005.
3. Ross M. H., Romrell L. J., Kaye G. Histology. A text and atlas. 5d ed. Baltimore: Williams & Wilkins, 2007.
WHAT MUST YOU KNOW? (INSTRUCTION FOR YOUR SELF-LEARNING)
Tissues and organs of human body consist of different histological elements.The histological elements
are stated below:
Cellular:
1. Cells.
2. Syncytium (a multinucleated protoplasmic mass formed by the secondary union of originally separate cells);
3. Symplast (a multinucleated cell that has formed by fusion of separate cells);
4. Postcellular structures - (erythrocytes, thrombocytes).
Non-cellular:
5. Extracellular matrix.
The smallest living entity of an organism is the cell. In contrast to single-celled organisms that are
independent entities, the cells of higher organisms form functional units. In accordance with their function, the
cells are differentiated by size, shape, and the degree of definition of certain characteristics. Size and shape are

3
often closely linked to a cells specific properties.
For all cells of the body there are a certain basic structure. The cell consists of the:
1. cytoplasm containing the cell organelles,
2. nucleus,
3. surrounding cell membrane (plasmolemma).
Plasmolemma (plasma membrane) consists of:
1. Glycocalix
The layer of oligosaccharides is called glycocalix. A layer of carbohydrate chains covers the outer plasma
membrane. They are part of the glycolipids and glycoproteins of the cell membrane, forming the surface coat of
the cell. The chemical structure of the glycocalyx is laid down genetically and it is specific for each cell. The
glycocalix serves as a cell-specific antigen. By this structure cells can recognize each other as self and nonself.
2. Membrane (also surrounds the cell organelles and the nucleus).
The elementary membrane consists of a double lipid layer, in which the fat-soluble components face
each other while the water-soluble parts form the inner and outer boundaries (a three-layered structure). The
structure of the plasma membrane cannot be resolved by light microscopy. In high-resolution electron micrographs,
the plasma membrane appears as a tripartite structure: two dark lines separated by a light region.
An electron-microscopic section demonstrates a three-layered structure (fig. 1.1): this includes a double
layer of lipids in which two layers of lipid molecules are arranged so that their lipid-soluble parts (fatty acids)
oppose each other (light middle line) while the water-soluble ends form the outer and inner boundaries of the
cell membrane (dark outer and inner lines).

Fig. 1.1. Electron micrograph of cell

membrane (arrow).

The outer and inner dark lines seen in electron

micrographs of plasma membranes are the result of the
deposition of osmium onto the hydrophilic portions of the lipid
molecules.
The membrane contains lipids:
1.Phospholipids
(phosphatidylcholine,
phosphatidylserine, phosphatidylethanolamine).
The lipid molecules form a lipid bilayer with an
amphipathic character (it is both hydrophobic and hydrophilic).
The fatty acid chains of the lipid molecules (tails) face each
other, making the inner portion of the membrane hydrophobic
(i.e., having no affinity for water). The surfaces of the
membrane are formed by the polar head groups of the lipid
molecules, thereby making the surfaces hydrophilic (i.e.,
having an affinity for water).

2. Cholesterol makes the membrane less fluid and more mechanically stable.
3. Glycolipids (sphingolipids): galactocerebroside and ganglioside are substances, which are responsible
for special function of nerve tissue (fig. 1.2).
The double lipid layer is infiltrated with proteins. In most membranes, protein molecules constitute
approximately half of the total membrane mass.
Most of the proteins are embedded within the lipid bilayer or pass through the lipid bilayer completely.
These proteins are called integral membrane proteins. Integral membrane proteins can move within the plane
of the membrane; this movement can be compared to the movement of icebergs floating in the ocean. The
other types of protein, called peripheral membrane proteins, are not embedded within the lipid bilayer. They
are associated with the plasma membrane by strong ionic interactions, mainly with integral proteins on both the
extracellular and infra-cellular surfaces of the membrane.

4
8
2
7
4
6b

7
3

10
9

Fig. 1.2. Plasma membrane structure.

1 membrane; 2 glycocalyx; 3 submembrane layer; 4 hydrophilic zone of phospholipid molecules; 5 hydrophobic
zone of phospholipid molecules; 6 integral and 6b - semiintegral proteins; 7 peripheral proteins; 8 carbohydrate
chains of glycocalyx; 9 microtubules; 10 microfilaments.

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

Fig. 1.3. Plasma membrane

proteins functions.
Pumps serve to transport certain ions, such as Na+, actively across membranes. Pumps also transport
metabolic precursors of macromolecules, such as amino acids and sugars, across membranes, either by themselves
or linked to the Na+ pump (fig. 1.4).
Channels allow the passage of small ions and molecules across the plasma membrane in either direction,
i.e., passive diffusion. Gap junctions formed by aligned channels in the membranes of adjacent cells permit
passage of ions and small molecules from the cytoplasm of one cell to the cytoplasm of the adjacent cells.
Receptor proteins allow recognition and localized binding of ligands (molecules that bind to the
extracellular surface of the plasma membrane) in processes such as hormonal stimulation, coated-vesicle
endocytosis, and antibody.
Linker proteins anchor the intracellular cytoskeleton to the extracellular matrix. Examples of linker

Fig. 1.4. Na-K ATPase.

proteins include the family of integrins that link

cytoplasmic actin filaments to an extracellular
matrix protein (fibronectin).
Enzymes have a variety of roles. Adenosine
triphosphatases (ATPases) have specific roles in ion
pumping: ATP synthase is the major protein of the
inner mitochondrial membrane, and digestive
enzymes such as disaccharidases and dipeptidases
are integral membrane proteins.
Structural proteins are visualized by the
freeze fracture method, especially where they
form junctions with neighboring cells. Often,
certain proteins and lipids are concentrated in
localized regions of the plasma membrane to carry
out specific functions. Examples of such regions can
be recognized in polarized cells, such as epithelial
cells.

Modified fluid-mosaic model means that integral membrane proteins may move to a different region
of the plasma membrane. The lateral diffusion of proteins is often limited by physical connections between
membrane proteins and intracellular or extracellular structures.
3. Submembrane layer (cortical layer of cytoplasm) - consists of microtubules and microfilamentes
which include contractile proteins - actin and myosin. This layer maintains the plasma membrane shape and
movement.
Properties of plasmolemma: The plasma membrane is flexible, semi-permeable, and experiences
active transport and potential differences across it.
Functions of plasmolemma are as follows:
1. Barrier, or protection of cell.
2. Selective transport of substances.
3. Antigenic determinants (cell passport)
4. Receptors of PM determines the regulation of cell by systemic and local factors (hormons,
neurotransmitters, growth factors).
5. Firm attachment to other cells or a basal lamina; membrane specializations for this are: intercellular
junctions and receptors.
6. Movement of the cell itself (migration) by pseudopodial, filipodial, or lamellipodial extensions and
the release of any firm attachments, or by flagellate activity, e.g., by sperm.
Transport of materials in and out of the cell served by (fig. 1.5):
(a) diffusion (selective) through the membrane;
Molecules move freely in aqueous solutions or in gases, and differences in concentration equilibrate by
diffusion. In this process, molecules diffuse toward the lower concentration until the concentrations even out.
Small molecules, such as the respiratory gases O2 and CO2 as well as water pass through the cell membrane
unimpeded (free diffusion). Pores through the membrane (channel proteins, membrane pores) or mobile transport
proteins (carriers) facilitate the passage (facilitated diffusion) of nutrients (e. g., glucose and amino acids in
the cells of the intestinal mucosa) and ions.
(b) active transport through the membrane;

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

Active transport is the transport of substances through the cell membrane by means of an energyconsuming transport system (transport ATPase). Such a transport process can move a substance through the
membrane against a concentration gradient These active transport processes are served by specialized proteins
in the cell membrane that can move several ions simultaneously.
(c) endocytosis, and its more scaled-up forms - pinocytosis and phagocytosis (fig. 1.6);
1
2
1

2
3
5

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

1 - plasmolemma; 2 - plasmolemma invagination (pit); 3 - endocytotic vesicle; 4 - ligand, called a receptor related
with; 5 - clathrin; 6 - endosome.

Large molecules, such as proteins, enter (endocytosis) through the cell membrane by so-called vesicular
transport. During this process, substances are attached in part to the outside of the cell by membrane-bound
receptors, enclosed by a part of the plasma membrane, and moved into the interior of the cell as a membrane
wrapped vesicle (receptor-mediated endocytosis). Depending on the size of the absorbed particle, this process

7
may also be called pinocytosis or phagocytosis.
Pinocytosis is the ingestion of fluid and small protein molecules via small vesicles, usually smaller
than 150 nm in diameter. Pinocytosis is performed by virtually every cell in the organism, and it is constitutive;
i.e., it involves a continuous dynamic formation of small vesicles at the cell surface.
Phagocytosis is the ingestion of large particles such as cell debris, bacteria, and other foreign materials.
In this process, large vesicles (larger than approximately 250 nm in diameter) called phagosomes are produced.
Receptor-mediated endocytosis allows entry of specific molecules into the cell. In this mechanism,
receptors for specific molecules, called cargo receptors, accumulate in well-defined regions of the cell membrane.
These regions eventually become coated pits. Cargo receptors recognize and bind to specific molecules that
come in contact with the plasma membrane. Clathrin molecules then assemble into a basket-like cage that helps
change the shape of the plasma membrane at that site into a vesicle-like invagination for transport into the cells.
(d) exocytosis
Large molecules, such as proteins, exit (exocytosis) through the cell membrane by so-called vesicular
transport. In exocytosis, products synthesized in the cell are enclosed in membranous vesicles and, by coalescence
of these vesicles with the inside of the plasma membrane, reach the extracellular space
In the constitutive pathway, substances designated for export are continuously delivered in transport
vesicles to the plasma membrane. Proteins that leave the cell by this process are secreted immediately after
their synthesis and exit from the Golgi apparatus.
In the regulated secretory pathway, specialized cell, such as endocrine and exocrine cells and neurons,
concentrate secretory proteins and transiently store them in secretory vesicles within the cytoplasm. In this
case, a regulatory event (hormonal or neural stimulus) must be activated for secretion to occur.
Communication and transduction. Each cell collaborates with different (adjacent and distant) cells of
human body cells for development, growth, homeostasis, regeneration, and its own particular task. The importance
of the cell membrane in receiving and sending the necessary signals is stressed by the number of examples given:
(a) The binding of hormones and transmitters to receptors on the membrane.
(b) The binding of the immune cells membrane receptor to an antigen.
(c) Chemical stimuli are transduced into nerve impulses in chemoreceptors; mechanical stimuli in
mechanoreceptors.
(d) Chemotactic agents act on phagocytic cells to attract them to their targets.
Specializations of the plasma membrane include:
1) microvilli are responsible for increase of transport area (surface),
2) cilia with basal bodies, which can move and determine the movement of material outside of the cell.
3) stereocilia - is a specialised structure of some sensor cells
4) pinocytotic vesicles reflect the activity of endocytosis;
5) infoldings often associated with mitochondria to active transport of ions through PM,
6) intercellular junctions,
Intercellular junctions (fig. 1.7)perform mechanical or chemical relations between cells.
Mechanical junction (fig. 1.9) is due to adhesion, desmosomes, hemidesmosomes, interdigitations.
The chemical relations between cells are mediated by gap junctions (nexuses) (fig. 1.8).
Chemical isolation is due to tight junctions (fig. 1.10).
Tight junction (zonula occludens) represents an area of fusion of the outer leaflets of the plasma
membranes of two adjacent cells.
Morphology: Tight junctions seal adjacent epithelial cells in a narrow band 0,1-0,5 mkm wide just
beneath their apical surface.
Molecules: This cell junction contains the adhesion molecule, E-cadherin. Tight junction an intercellular
junction at which adjacent plasma membranes are joined tightly together by interlinked rows of integral
membrane proteins - occludins, limiting or eliminating the intercellular passage of molecules.

1
4
2

2
3
5

Fig.1.7. Intercellular junctions.

Scheme.

Fig. 1.8. Gap junction (nexus junction):

A - E photomicrograph 36000; Scheme.

1 - tight junction; 2 - desmosome;

3 - gap junction; 4 - adherent junction;
5 - plasmolemma.

1 gap junction; 2 connexins.

2
3
4
5

10
Fig. 1.9. Mechanical junctions between cells and cells with a basal membrane: A - E photomicrograph of
desmosome 36 000; B - scheme of desmosome structure; C - scheme of hemidesmosome structure.
1 - plasmolemma; 2 - intermediate filaments of cell cytoskeleton, 3 - cytoplasmic plate; 4 - electron-dense layer in
the desmosome, 5 - cleft between two plasmolemms, 6 - desmoplakins and desmogleins; 7 - hemidesmosome, 8 - basal
plate, 9 - anchoring filaments, 10 - reticular fibers.

1
Fig. 1.10. Scheme of tight junction
structure .
1 - plasmolemms of neighboring cells,
2 - cleft between neighboring
plasmolemms; 3 - ZO-2 protein; 4 - ZO1 protein, 5 - actin; 6 - ZO-3 protein,
7 - transmembrane domain of occludin;
8 - extracellular domain of occludin;
9 - cytoplasmic domain of occludin;
10 - claudin.

10

3
5

4
5
6

8 7

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Tight junctions perform two vital functions: They prevent the passage of molecules and ions through the
space between cells. So materials must actually enter the cells (by diffusion or active transport) in order to pass
through the tissue. This pathway provides control over what substances are allowed through.They block the
movement of integral membrane proteins between the apical and basolateral surfaces of the cell.
Adherent junction is a type of intercellular junction that links cell membranes and cytoskeletal elements
within and between cells, connecting adjacent cells mechanically. Adherens junctions provide strong mechanical
attachments between adjacent cells. They hold cardiac muscle cells, epithelial cells tightly together as the heart
expands and contracts. They seem to be responsible for contact inhibition.
Morphology: The intermediate junction (zonula adherens) is characterized by the presence of an
intercellular space 10-20 nm wide, separating areas of cytoplasmic density occurring in each of the participating
cells.
Molecules: Adherens junctions are built from: cadherins transmembrane proteins whose extracellular
segments bind to each other and whose intracellular segments bind to catenins. Catenins are connected to actin
filaments.
Desmosome (macula adherens).
Morphology: The structure of a desmosome, or macule adherens, is fairly uniform in most tissues
examined to date: within each cell, at the region of localized contact of two cells, there is a dense plaque
adjacent to the cell membrane, made up of converging cytoplasmic actin microfilaments (tonofibrils). The two
cell membranes do not appear modified. Within the intercellular substance 20-30 nm wide, there is a dense
central lamina. Very slender filaments run between the central lamina and the adjacent cell membranes.
Molecules: C-cadherin are an essential component of desmosomes. Desmosomes are biochemically
complex structures containing many different filamentous proteins, some of which are desmosome specific.
Specific adhesion proteins (adherins) have been identified in cytoplasmic plaques. Other protein components of
desmosomes are desmoplakins and desmogleins. The desmosomes also contain intermediate filaments of various
molecular weights.
Hemidesmosomes (half-desmosomes) are observed at the attachment points of epithelial basal cells to
the basement lamina. The half-desmosome is morphologically somewhat similar to the desmosome: there is a
thickening of a limited area of the cytoplasm of a basal cell adjacent to the cell membrane, upon which converge
cytoplasmic fibrils. However, the apposed basement membrane shows merely a slight thickening, which contains
slender filaments. An intermediate thickening, or membrane, is usually present within the fibrils of the
hemidesmosome. Hemidesmosomes serve as connectors between the extracellular matrix and the intermediate
filaments in the cytoplasm of the cell.
The Gap Junctions (Nexus Junctions) have multiple functions: they provide cell-to-cell communications
of essential metabolites and ions and may serve as electrical synapses.
Morphology: The Gap Junctions is a narrowed portion of the intercellular space containing channels
linking adjacent cells and through which pass ions, most sugars, amino acids, nucleotides, vitamins, hormones,
and cyclic AMP. In electrically excitable tissues, these gap junctions transmit electrical impulses via ionic
currents. The junction is composed of seven layers, three of which are electrontranslucent and are sandwiched
in between electron-dense layers. The central electron-lucent zone (or gap) is composed of small hexagonal
subunits, forming the channels of communication between adjacent cells.
Molecules: The gap junction channels are composed of a diverse family of proteins, named connexins.

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LESSON 2
THEME: CELL. CYTOPLASM. HYALOPLASM, ORGANELLES.
BACKGROUND: The vast majority of life processes is going on within the cytoplasm: catabolism and
digestion of organic substances and creation of energy, synthesis of proteins, lipids and carbohydrates specific
for the cell, their accumulation and secretion. These processes provide specific cell functions, as well as
functioning of the whole multi-cellular organism.
AIM OF STUDY: To be able to differentiate cytoplasm and its key structures hyaloplasm, organelles,
inclusions within the eukaryotic cell on light and electron microscopy. Differentiate organelles within the normal
cell for better understanding of their functions and possible changes under condition of pathology.
TO ACHIEVE THIS AIM ONE HAS TO (practical procedures):
1. Differentiate the basic structures of cytoplasm: hyaloplasm, organelles, inclusions.
2. Distinguish hyalopasm in histological specimen and electronograms.
3. Distinguish organelles in histological specimen and electronogramme, interpret their structure and functions.
4. Analyze cell function on basis of the structure of cytoplasm and organelles interaction.
TO ACHIEVE THE AIM IT IS NECESSARY TO CENTRE AROUND THE FOLLOWING POINTS:
1. Main components of the cell cytoplasm hyaloplasm, organelles, inclusions.
2. Hyaloplasm definition, basic elements, features, importance.
3. Organelles definition, classification.
4. Membrane organelles ( rough and smooth endoplasmic reticulum, Golgi apparatus, lysosomes, peroxisomes,
mitochondria), their structure and functions.
5. Non-membrane organelles (ribosomes, centrioles, microtubules, microfilaments), their structure, functions.
6. Organelles with specific functions (microvilli, cilia, tonofibrills, miofibrills, neurofibrills).
REFERENCES:
1. Junqueira L. C., Carneiro J., Kelley R. O. Basic histology. A Lange medical book. Tenth edition: Appleton
& Lange, 2008.
2. Burkitt H. G., Young B., Heath J. W. Wheaters Functional Histology. A text and colour atlas. 5th edition:
Churchill Livingstone, 2005.
3. Ross M. H., Romrell L. J., Kaye G. Histology. A text and atlas. 5d ed. Baltimore: Williams & Wilkins, 2007.
WHAT MUST YOU KNOW? (INSTRUCTION FOR YOUR SELF-LEARNING)
CYTOPLASM
The cytoplasm is the component of the cell, located between the nucleus and the cell membrane. Depending
on the type and origin of the cell, the cytoplasm may present a variegated light microscopic appearance. Its
shape, size, and staining properties vary greatly and will be described in detail for the various tissues and organs.
In living cells, there is an intense movement of particles within the cytoplasm.
Cytoplasm consists of three components - hyaloplasm (cytomatrix), organelles and inclusions.
Hyaloplasm (cytomatrix) is the so-called soluble phase of the cell, consisting mostly of water, dissolved
solutes, and larger molecules in suspension tending to link repetitively with covalent bonds giving the cytoplasm
a dense, viscous colloidal sol or gel consistency. It contains molecules of different size and shape (e.g., electrolytes,
metabolites, RNA, and synthesized proteins). In most cells, it is the largest single compartment.
Organelles are the obligatory components of cytoplasm which determine the specification of cell.
There are several types of organelles according to their significance, morphological features and functions.
Classification of organelles:
1. According to the significance it has been divided all organelles on (into) two types:
- organelles of general significance
- specific organelles which present only in certain types of cells. For example, in muscle fibres we can

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see myofilaments or myofibrils. Nervous cell has neurofibrils and chromatophilic substance. Sperm cells include
tail.
2. According to the morphplogical features organelles have been designed as
- membranous - which are bounded by a membrane. This type includes: endoplasmic reticulum (rough
and smooth), Golgi complex, mitochondria, lysosome, peroxisome.
- unmembranous which are not bounded by a membrane. They are as follows: ribosomes, centrioles,
microtubules, filaments.
3. According to function it has been shown several functional apparatus as follows:
- apparatus of protein synthesis and lipid and carbohydrates metabolism: endoplasmic reticulum, Golgi
complex, ribosomes.
- apparatus of intracellular digestion: lysosomes.
- apparatus of cell movement and maintain of cell shape cytoskeleton, plasma membrane.
- energy-requiring and energy-generating apparatus: mitochondria
and many others specialised apparatus in certain types of cells.
Characteristics of membranous organelles:
1. Endoplasmic reticulum.
Morphological features: It is a cytoplasmic closed system of unit membranes forming tubular canals and
flattened sacs or cisternae that subdivide the cytoplasm into a series of compartments (fig. 2.1). This system
leads towards the Golgi complex.
2

1
3

Fig. 2.1. Structure of the endoplasmic

retuculum. Scheme: A - granular;
B - agranular.
1 - membranes; 2 - the internal cavity of
cisterns, 3 - ribosomes.

B
1

There are two varieties of endoplasmic reticulum:

1) Granular/rough has the membranes of the endoplasmic reticulum that are covered with numerous
attached granules composed of ribonucleic acid (RNA) and proteins (RNP granules or ribosomes, l5 nm in
diameter. RER forms a closed system not open (closed) to cell exterior but continuous with outer surface of
nuclear envelope. In general, rough endoplasmic reticulum is abundant in cells with marked synthesis of proteins
for exportfor instance, in the pancreas or the salivary glands. In light microscopy, the RNA-rich cytoplasmic
areas stain bluish with hematoxylin and the cell is said to be basophilic (its cytoplasm stained by hematoxylin
and has blue colour). This feature is commonly observed in metabolically active cells.
Function: Granular endoplasmic reticulum takes place in secreting protein synthesis. It is a site of mRNA,
tRNA and amino acids interaction. This organelle functions in the synthesis of proteins destined to be transported
out of the cell, of proteins that comprise lysosomes, and of the proteins in the plasma membrane. Proteins
synthesized on the rough ER are transported to the Golgi complex in transport vesicles. In the Golgi, the proteins
destined for export out of the cell, or those that end up in the plasma membrane or in lysosomes, are sorted and
sent to their appropriate destination.

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2) Agranular/smooth is more tubular and consists of small, interconnecting channels or tubules within
the cell and lacks ribosomes on its cytosolic surface. The cytoplasm of cells which have a lot of Smooth ER is
stained by eosin and has red or pink colour. This is said to be eosinophilic or acidophilic. Smooth ER is
especially abundant in steroid hormone secreting cells.
Function: The smooth ER is the site for the synthesis and metabolism of fatty acids and phospholipids,
steroid hormones, and cholesterol, for the detoxification of drugs in the liver, adds lipid portion of lipoproteins
(liver) and for the storage of calcium in muscle.
2. Golgi body/complex/apparatus.
Location: This usually takes one area near to the nucleus and often in a specific place, e.g., supra-nuclear
in cuboidal epithelial cells.
Light microscopic images of cells active in protein synthesis and secretion can exhibit an unstained area
of cytoplasm that corresponds to the Golgi in electron micrographs. Plasma cells secrete antibody molecules
(immunoglobulins) into the extracellular matrix. The region of their cytoplasm adjacent to the nucleus appears
clear and unstained, whereas the remainder of the
cytoplasm is basophilic as a result of the presence
of stacks of rough endoplasmic reticulum. The
unstained area of the plasma cells marks the
location of the Golgi complex.
Morphological features: It consists of a
complex of stacked smooth flattened cisternae
with dilated ends, tubules, and vesicles of various
sizes (fig. 2.2).
Cisternae segregated into convex (cis),
medial (middle), and concave (trans)
compartments. The Golgi is polarized so that the

cis region faces the endoplasmic reticulum

(forming face) and the trans region faces the
secretory vesicles in cells manufacturing proteins
2
for export. Small transitional vesicles are near
convex face. Large condensing vacuoles located
1
near concave face. Cis cisternae stain with
osmium, medial saccules stain for nicotinamide
adenine dinucleotide phosphatase (NADPase),
trans saccules react with thiamine
pyrophosphatase (TPPase)
3
Identification: In LM after special silver
staining, the Golgi apparatus may be seen as a
tangled network. With routine haematoxylin
B
staining in certain cells, e.g., active osteoblasts or
Fig. 2.2. Ultramicroscopic structure of Golgi
plasmocytes, the juxta-nuclear vacuole reveals
complex : A - scheme; B - E photomicrograph
the site of the Golgi structure as a pale negative
10000.
image.
Functions: The Golgi plays important roles
1 - flat cisterns, 2 - vacuoles; 3 - transport vesicles.
in sorting plasma membrane and intracellular
membrane traffic, in the modification of
carbohydrates on proteins (e.g., acylation and
sulfation), and in the packaging of proteins that
will be secreted by the cell.
3. Mitochondria.
Location: Because mitochondria generate ATP, they are more numerous in cells that use large amounts

13
of energy, such as striated muscle cells and cells engaged in fluid and electrolyte transport. Mitochondria also
localize at sites in the cell where energy is needed, as in the middle piece of the sperm, the intermyofibrillar
spaces in striated muscle cells.
Morphological features: Mitochondria are small, usually elongated structures, usually less than 0.5 m in
width and less than 7 m in length. Each mitochondrion is composed of two membranes, located one within the
other (fig. .2.3).

Fig. 2.3. Scheme of ultramicroscopic structure of

mitochondria with plate cristae.
1
2
3
5

- the outer mitochondrial membrane;

- the inner mitochondrial membrane;
- intermembrane space, 4 - matrix;
- cristae.

Outer and inner membranes separated by ~20 nm space. The outer shell of the mitochondrion is a
continuous, closed-unit membrane. Running parallel to the outer membrane is a morphologically similar inner
membrane that forms numerous crests or invaginations (cristae mitochondriales), subdividing the interior of the
organelle into a series of communicating compartments. Cristae may be tubular but are usually shelf-like. A
homogeneous material or mitochondrial matrix fills the interior of the organelle. Matrix contains dense matrix
granules, mitochondrial DNA, tRNA, rRNA and mRNA.
Identification: In LM they can be stained with special methods and appear as coloured rods or granules.
In EM their shape tends to be tubular or spheroid.
Functions: The key role of the mitochondria within the cell is that of carriers of energy-producing complex
enzyme systems. Several oxidative systems have been identified within the mitochondria: Krebs cycle enzymes,
fatty acid cycle enzymes, and the enzymes of the respiratory chain, including the cytochromes. Most importantly,
the formation of energy-producing adenosine triphosphate (ATP) from phosphorus and adenosine diphosphate
(ADP) takes place within the mitochondria. The ATP is exported into the cytoplasm where it serves as an
essential source of energy for the cell. They may also store calcium. Tubular cristae prevalent in steroid
synthesizing cells and include specific enzymes for steroidogenesis.
The mitochondria possess their own DNA that is independent of nuclear DNA and is responsible for
independent protein synthesis and for the mitochondrial division cycle. This supports the concept that the
mitochondria are quasi-independent organelles, living in symbiosis with the host cell, which they supply with
energy. Two genetic systems exist within a cell, one vested in the mitochondria and the other in the nucleus. The
two systems are interdependent.
4. Lysosomes.
Morphological features:
In electron microscopic preparations, the lysosomes may be identified as spherical or oval structures of
heterogeneous density and variable diameter (fig. 2.4). Lysosomes contain a collection of hydrolytic or digestive
enzymes, acid phosphatase being the first one identified, that serve to digest the phagocytized material, and are
surrounded by a unique membrane that resists hydrolysis by their own enzymes. There are more then 50
hydrolytic enzymes. The lysosomal membrane possesses highly glycosylated specific membrane proteins that
protect the membrane from digestion by lysosomal enzymes. These lysosome-specific proteins are synthesized

14
in the rER, transported to the Golgi apparatus. In
addition, lysosomes contain proton (H+) pumps
that transport H+ ions into the lysosomal lumen,
maintaining a low pH (-4.7).
There are primary and secondary types of
lysosomes. Primary lysosomes are the newly
1
formed lysosomes, which arise as complete and
2
functional organelles budding from the Golgi
apparatus. Primary lysosomes are round, TEMlucent vesicles that have not participated in a
digestive event. Secondary lysosomes formed from
fusion of primary lysosome with phagosome (see
below).
Identification: In the greater majority of
Fig. 2.4. Ultramicroscopic structure of
cells, lysosomes cannot visualized by light
lysosomes. 19 000.
microscopy. Exceptions to this statement are the
granules of two types of white blood cells,
1 - primary lysosome, 2 - secondary lysosome
neutrophils and eosinophils, which can be seen by
(fagolysosome).
light microscopy.
Functions:
The lysosomes, or cell disposal units, are the organelles participating in the removal of phagocytized
foreign material. Occasionally, the lysosomes also digest obsolete fragments
of cytoplasm and organelles, such as mitochondria, for which the cell has no further use.
Depending on the nature of the digested material, different pathways deliver material for digestion within
the lysosomes. In the digestion process, most of the digested material comes from endocytotic processes;
however, the cell also uses lysosomes to digest its own obsolete parts, nonfunctional organelles, and unnecessary
molecules. Three pathways for digestion exist:
Extracellular large particles such as bacteria, cell debris, and other foreign materials are engulfed in
the process of phagocytosis. A phagosome, formed as the material is internalized within the cytoplasm,
subsequently fuses with a lysosome to create a phagolysosome (secondary lysosome).
Extracellular small particles such as extracellular proteins, plasma membrane proteins, and ligandreceptor complexes are internalized by endocytosis and receptor-mediated endocytosis. These particles follow
the endocytotic pathway through early and late endosomal compartments and are finally delivered to lysosomes
for degradation.
lntracellular particles such as entire organelles, cytoplasmic proteins, and other cellular components
are isolated from the cytoplasmic matrix by endoplasmic reticulum membranes, transported to lysosomes, and
degraded in the process called autophagy (removal of cytoplasmic components, particularly membrane-bounded
organelles, by digesting them within lysosomes).
In addition, some cells (e.g., osteoclasts involved in bone resorption and neutrophils involved in acute
inflammation) may release lysosomal enzymes directly into the extracellular space to digest components of the
extracellular matrix.
More than 20 diseases are due to lysosomes altering and storage of a heterogeneous material in the
cytoplasm.
5. Peroxisome.
Morphological features: Peroxisomes (peroxide + soma) are membrane-bound spheres of moderate
electron density with 0,5-1,2 mkm in diametre. The matrix of peroxisome contains some specific enzymes, such
as catalase, peroxidase D- fnd L-aminooxydase. In some species, but not humans, a crystalline nucleoid is
present that is composed of urate oxidase (fig. 2.5).
Functions:
1) The key enzyme of peroxisome is catalasa, an enzyme that decomposes hydrogen peroxide to water
and oxygen (2 H202>2 H2O + 02). Thus, peroxisomes protect the cell from the effects of hydrogen peroxide,

15
which could cause irreversible damage to many important cellular constituents.
2) Anoter inportant function of peroxisome is detoxification (the elimination of a variety of toxic
compounds).
3) Moreover peroxisomes contain enzymes involved in lipid metabolism and take place in b-oxidation of
fatty acids
4) Peroxisomes also contain certain enzymes that take place in the formation of the bile acids in hepar
cells.

3
2

Fig. 2.5. Ultramicroscopic structure of

peroxisome. 33 000.
1 - peroxisome; 2 - crystalloid core;
3 - glycogen inclusions.

Characteristics of unmembranous organelles:

1. The Ribosome.
Location: In the cytoplasm, the ribosomes may be either floating free or they may be attached to the
outer surface of the endoplasmic reticulum. Morphological features: The ribosomes are submicroscopic particles
and are composed of RNA and proteins in approximately equal proportions. They are ubiquitous and have been
identified in practically all cells of animal and plant origin. Each ribosome is composed of two, approximately
round subunits of unequal size (fig 2.6).
Ribosomes may be joined together by strands
of
messenger
RNA (mRNA) to form aggregates or
1
polyribosomes that thus resemble a string of beads.
3
The string may be either open or closed. Ribosomes
4
are attached to the membranes of the endoplasmic
2
reticulum by the larger subunit.
Functions: It appears likely that the two types
5
of ribosomes exercise different functions: the free
ribosomes are primarily engaged in the production of
proteins for the cells own use, whereas attached
Fig . 2.6. The structure of the ribosome. Scheme.
ribosomes are responsible for protein production for
export. A marked concentration of ribosomes (and
1 - small subunit 2 - large subunit 3 - mRNA 4 - tRNA,
hence proteins) confers upon the cytoplasm a
5 - a signal peptide.
basophilic staining.

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

16
The cytoskeleton acts as a track on which cells can move organelles, chromosomes and other things.
Some examples are:
- Vesicle movement between organelles and the cell surface, frequently studied in the squid axon.
- Cytoplasmic streaming
- Movement of pigment vesicles for protective coloration
- Discharge of vesicle content for water regulation in protozoa
- Cell divisioncytokinesis
- Movement of chromosomes during mitosis and meiosis
Cells have protein motors that bind two molecules, and using ATP as energy, cause one molecule to shift
in relationship to the other. Two types of these protein motors are myosin and actin, and dynein or kinesin and
microtubules (fig. 2.7).
endocytotic vesicle
receptor

kinesins
(+ end)

(- end)
lysosome
dyneins

mitochondria (energy of ATP)

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

These families of proteins all have a motor end, but may have several kinds of different molecular
structures on the binding end. When these proteins bind, they can cause many different molecules, organelles,
etc. to move. To the right is an example of the different binding ends found in the kinesin family of motors.
When linked to other microtubules, protein motors can cause motion if the ends are fixed or extend the
lengths of the fiber bundles if the ends are free.
Broken motors: In healthy individuals, the protein dystrophin is part of the linkage between the cellular
cytoskeleton and the adhesive proteins on the outside of the cell. In Duchenne Muscular Dystrophy, however,
the gene that codes for dystrophin is defective, resulting in muscle degeneration and finally death. This disease
is X-linked recessive and occurs in 1 out of every 3,500 males.
Cytoskeleton
The skeleton of the cells and, hence, the structures maintaining their physical shape, facilitating their
motion, and providing structural support to all cell functions, is provided by a family of fibrillar proteins. The
cytoskeleton is fundamentally composed of three types of fibrillar proteins, initially classified by their diameter
in electron microscopic photographs: the actin filaments (microfilaments, tonofilaments), intermediate filaments,
and microtubules.
Intermediate Filaments
Intermediate filaments are about 10 nm diameter and provide tensile strength for the cell, they are larger
than actin microfilaments and smaller than microtubules (fig. 2.8). Several subspecies of intermediate filaments
proteins have been identified, differing from each other by relative molecular mass and anatomic distribution.
Perhaps the best known of the intermediate filaments are the keratins, which have been extensively studied in
the epidermis of the skin. There are several subfamilies of keratin filaments (proteins) forming pairs, each
composed of one basic and one acidic protein. Each type of squamous epithelium (skin, cornea, other epithelia)
may be represented by a special pair of proteins of high relative molecular mass. With the change of epithelial

17
type from a single layer to multilayer epithelium, different keratin genes, producing proteins of increasing molecular
mass are activated. This mechanism may be important in understanding the change known as squamous
metaplasia.
Examples of the cytoskeleton: in epithelial cells (skin), cells of the intestine, all three types of fibers are
present. Microfilaments project into the villi, giving shape to the cell surface. Microtubules grow out of the
centrosome to the cell periphery. Intermediate filaments
connect adjacent cells through desmosomes.
Microfilaments
The ubiquitous actin filaments, measuring 5 to 7 nm in
diameter, are observed in all cells of all vertebrate species. In
1
electron microscopy, they can be recognized as bundles of
longitudinal cytoplasmic filaments (fig. 2.9) crisscrossing the
cytoplasm and often converging on specific targets such as
desmosomes. The actin filaments are found within virtually
2
1
all structural cell components and interact with many other
proteins that regulate their length. The fundamental structure
of these elongated fibrillar proteins is helical, with two different
ends: this latter feature allows the filaments to attach to two
3
different molecules and function as an intermediary polarized
link. The actin filaments are easily polymerized (i.e., they
form structures composed of several actin units). This is
3
probably the mechanism that allows actin filaments to form
tight meshworks in conjunction with other proteins. Among
the latter, it is important to mention the links of actin filaments
Fig. 2.8 The structures of the apical cytoskeleton to a contractile protein, myosin, accounting for motion and
contractility of cells. Other linkages occur with transmembrane
in epithelial cells. 47 000.
proteins, such as spectrin, ensuring the communications
1 - actin filaments, 2 - terminal network, between the cell membrane and cell interior. Thus, actin
3 - intermediate filaments.
microfilaments perform several essential functions within cells
as linkage filaments coordinating the activity of divergent cell
components. Microfilaments can also carry out cellular movements including gliding, contraction, and cytokinesis.
Microtubules
Microtubules (fig. 2.9) are an integral component of cilia, flagella, and centrioles.

2
1
1
2

Fig. 2.9. Microtubules and microfilaments: A - scheme; B - Electron micrograph, 39 000.

1 - microtubules, 2 - microfilaments.

18
Microtubules, measuring between 22 and 25 nm in diameter, have long been recognized and identified by
light microscopy as the constituents of the mitotic spindle. Microtubules are hollow, tube-like structures, which
appear to be universally present in all cells, and are synthesized from precursor molecules of tubulin. They are
composed of subunits of the protein tubulin-these subunits are termed alpha and beta. Microtubules are polarized,
they have one minus and one plus end; hence, they can be attached to two different molecules and form a
bridge between them.
Microtubules act as a scaffold to determine cell shape, and provide a set of tracks for cell organelles
and vesicles to move on. The principal role for microtubules and associated proteins is their participation in
cellular events requiring motion. Cilia and flagella are a good example of this function in which microtubules
perform a sliding movement in association with a protein, dynein, and an energy-producing system, adenosine
triphosphate (ATP).
The mitotic spindle is synthesized by the cells undergoing mitosis from molecules of tubulin. During cell
division, the centrioles serve as an organizing center for the mitotic spindle. From the centrioles, located at the
opposite poles of the cell, the microtubules attach to the condensed double chromosomes arranged at the
metaphase plate and participate in the migration of the single chromosomes into the two daughter cells. Once
the mitosis is completed, the spindle microtubules are depolarized and redistributed in the cytoplasm.
3. The Centriole.
The centrioles are cytoplasmic organelles that play a key role during cell division. Each interphase animal
cell contains a pair of centrioles, short tubular structures, usually located in the vicinity of the concave face of
the Golgi complex. As the cell is about to enter mitosis, another pair of centrioles appears, and each pair travels
to the opposite poles of the cell and becomes the anchoring point of the mitotic spindle. The origin of the second
pair of centrioles has not been fully clarified; apparently it is synthesized de novo from precursor molecules in
the cytoplasm. This event is induced and directed in an unknown fashion by the original pair of centrioles. Each
pair of centrioles is surrounded by a clear zone, the centrosome, which, in turn, is surrounded by a slightly
denser area or the astrosphere. Within each pair, the centrioles are placed at right angles to each other. Thus, in
a fortuitous electron micrograph (fig. 2.10), one centriole will appear in a longitudinal section and the other in
cross section. In the cross section, each centriole appears as a cylindrical structure with a clear center and nine
triplets or groups of three microtubules.

Fig. 2.10. Ultramicroscopic

structure of the cell center.
Electronic micrography, 30 000.

1
3

1 - centrioles in cross section;

2 - longitudinal section of centrioles;
3 - triplets of microtubules.

Another derivatives of microtubules complex are ciliae and flagella.

Each cilium or flagellum contains 11 microtubules, of which two are single and located within the center,
and nine are double (doublets) and located at the periphery. Dynein arms attached to the microtubules serve
as the molecular motors (fig. 2.11). Defective dynein arms cause male infertility and also lead to respiratory
tract and sinus problems.

19

Fig. 2.11. Cilia and flagella structure.

20
LESSON 3
THEME: CELL INCLUSIONS.
BACKGROUND: Inclusions are one of the component of cytoplasm that reflect some specific features
(metabolism, age, functional activity, the stage of secretory cycle and others) of different cells in normal and
pathological conditions. Several diseases have been shown to be caused by defective metabolic steps, resulting
in appearance or increase of certain inclusions. This fact is often used to diagnostics in biopsies of tissue taken
from patients with diseases that store iron (eg, hemochromatosis, hemosiderosis), glycogen (glycogenosis),
glycosaminoglycans (mucopolysaccharidosis), and sphingolipids (sphingolipidosis) in tissues.
AIM OF STUDY: To determine cell inclusions, their type and functional significance. To link the quantity
and quality of different inclusions with organelles activity.
TO ACHIEVE THIS AIM ONE HAS TO (practical procedures):
1. Determine the cell inclusions in different cells stained with different dyes.
2. Differentiate storage inclusions in different cells.
3. Identify the stages of secretory cycle by the secretory inclusions.
4. Explain the structural and functional links between inclusion and different organelles.
TO ACHIEVE THE AIM IT IS NECESSARY TO CENTRE AROUND THE FOLLOWING POINTS:
1. What is inclusion as the component of the cell cytoplasm.
2. Morphofunctional types of inclusions.
3. Methods of inclusions identification.
4. Diagnostic significance of different inclusions quantity.
REFERENCES:
1. Junqueira L. C., Carneiro J., Kelley R. O. Basic histology. A Lange medical book. Tenth edition: Appleton
& Lange, 2008.
2. Burkitt H. G., Young B., Heath J. W. Wheaters Functional Histology. A text and colour atlas. 5th edition:
Churchill Livingstone, 2005.
3. Ross M. H., Romrell L. J., Kaye G. Histology. A text and atlas. 5d ed. Baltimore: Williams & Wilkins, 2007.
WHAT MUST YOU KNOW? (INSTRUCTION FOR YOUR SELF-LEARNING)
Cell inclusions - These are usually transitory and non-obligatory components of the cytoplasm. (Nonliving, non-participating, poorly structured cell elements, often seen in cytoplasm).
Cell inclusions may be different in shape, size and thin structure:
Examples:
Fat droplets appearing as vacuoles in ordinary light microscopy preparation, but usually preserved by
electron microscopy procedures as dark rounded bodies.
Lamellar bodies contain lipids to be secreted.
Glycogen granules visible as dark l5-30 nm wide granules in EM, e.g., in cardiac muscle and liver cells.
Secretion granules, e.g. zymogen, in pancreatic cells.
Pigments, e.g., melanin (skin cells), lipofuscin (old neurons), haemosiderin (natural), carbon (exogenous
- from outside the body).
Crystalls, e.g., in testicular interstitial cells.
Bacteria and viral inclusion bodies (pathological).
INCLUSIONS CLASSIFICATION
Thus, cell inclusions form a wide variety in morphology, functions and chemical composition, group, that
according to functional significance has been divided on four types:
1. Storage inclusions (storage of calories/energy)

21
2. Secretory granules
3. Excretory inclusions
4. Pigments.
1. Storage of calories/energy.
This type of inclusions usually composed of accumulated metabolites, such as:
a) lipids
b) carbohydrates
c) yolk inclusions are typical only for egg cell.
Characteristics of lipids inclusions:
Morphological features: lipids inclusions usually form droplets.
Routine processing methods for microscopy generally extract lipid from tissues and therefore lipid droplets
within cells appear as unstained vacuoles.
The selective method for lipid demonstration (identification) is histochemical staining of frozen sections
with osmium (Fig. 3.1 - lipids are visible as black droplets) or sudan (lipids have orange colour)
1

Fig. 3.1. The inclusion of lipids in liver cells (impregnation osmic acid and safranin):
1 - liver cells; 2 - a drop of fat; 3 - cytoplasm; 4 - nucleus; 5 - nucleolus.

Under the electron microscope lipids droplets are of variable size and electron density and are not
bounded by a membrane. Usually these droplets are associated with organelles that take place in lipid metabolism:
smooth endoplasmic reticulum and mitochondria.
Location: You can see lipid droplets in:
a) adipose tissue or
b) liver cell, where lipid droplets are used as fatty acids and glycerine storage.
c) adrenal cortex cells, where lipid droplets are used as storage of cholesterol, that are needed for steroid
hormones synthesis. In this case lipid droplets are associated with specific mitochondria - with tubular crists
and smooth endoplasmic reticulum.
Characteristics of glycogen inclusions:
Morphological features: glycogen inclusions usually form granules.
Routine processing methods for microscopy generally extract glycogen from tissues and these inclusions
are not visible.
The selective method for glycogen demonstration (identification) is histochemical staining with PAS
glycogen granules become magenta (Fig. 3.2).

22
1

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

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

Electron microscopy: After impregnation with lead salts, this substance appears as collections of coarse,
irregular electrondense particles. They often are associated with smooth endoplasmic reticulum cisterns.
Location: Glycogen appears as granules in different cells, mostly in hepatocytes, muscles.
2. Secretive inclusions.
Secretive inclusions are typical for cells with secretive abilities: for example, cells of endocrine and
exocrine glands (pancreas, parathyroid gland and others), blood granulocytes, mast cells and others.
In brief, secretive inclusions are secretive product of certain cells. This type of inclusion is resulted for
Golgi apparatus activity and reflects accumulation in the cell secretive products. They may be different in
chemical composition, but always include proteins:
- proteins,
- lipoproteins,
- glycoproteins.
Morphological features: Secretive inclusions form membrane-bound granules with rounded shape, which
well are visible under the electron microscope. The size and thin structure of granules in cells are different and
specific for certain cell types. It can be used for morphological diagnostic (determination) of these cells. In
addition, the dynamics of secretive granules amount permits to detect the secretive activity and the stage of
secretive cycle. It increases during accumulation of secretive products and decreases when secret emerge
outside the cell (in extracellular medium).
Under the light microscope secretive products may appear in different forms, according to their chemical
composition and quantity. They can react both with acid and base dyes, or can be identified by histochemical
methods.
3. Pigment inclusions
Deposits of coloured substances - pigments - are often found in different cells. They may be synthesized
by the cell (eg, in the skin melanocytes) or come from outside the body (eg, carotene).
This group of inclusions includes a wide variety of chemical substances which have different colours.
They are:
- melanin - have black or dark brown colour. This type of inclusions form dense intracellular membranelimited granules in cells of the epidermis of the skin and in the pigment layer of the retina
- Lipofuscin - have yellowish-brown colour (Fig. 3.3).
- Haemoglobin and myoglobin (iron-contained pigments that can link oxygen)

23
1

4
2

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

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

Diagnostic significance: One of the most common pigments is lipofuscin present mainly in permanent
cells (eg, neurons, cardiac muscle) that increases in quantity with age. Its chemical constitution is complex. It is
believed that granules of lipofuscin derive from secondary lysosomes and represent deposits of undigestible
substances.
Pathological forms of inclusion. The development of some disease is due to altering of methabolic
processes in several cells. Lysosomes play an important role in the metabolism of several substances in the
human body and consequently many diseases have been ascribed to deficiencies of lysosomal enzymes. In
metachromatic leukodystrophy, there is an intracellular accumulation of sulfated cerebrosides caused by lack of
lysosomal sulfatase. In most of these diseases, a specific lysosomal enzyme is absent or inactive, and the
digestion of certain substances (glycogen, cerebrosides, gangliosides, sphingomyelin, glycosaminoglycans, etc)
does not occur. As a result, these substances accumulate in different cell types, interfering with their normal cell
function. This diversity of affected cell types explains the variety of clinical symptoms observed in these diseases.

24
LESSON 4
THEME: THE CELL NUCLEUS.
BACKGROUND: Nucleus is the main part of living cell. It performs many functions and controls
realisation of different processes, such as cell division, differentiation, adaptation, regeneration and others.
AIM OF STUDY: To find and analyze the cell nucleus under light and electron microscopy, interpret
functional state of cells and determine pathological features.
TO ACHIEVE THIS AIM ONE HAS TO: (practical procedures):
1. Find out the cell nucleus in fixed cells stained with different dyes.
2. Detect the main components of the cell nucleus in a histological specimen under the light microscope
and interpret their chemical composition.
3. Determine the main components of the cell nucleus under the electron microscopy.
4. Estimate the relation between functional state of cell nucleus and cytoplasm.
5. Explain the functional state of the cell on the basis of structural organisation of its components.
TO ACHIEVE THE AIM IT IS NECESSARY TO CENTRE AROUND THE FOLLOWING
THEORETICAL POINTS:
1. The nucleus: its general morphological characteristics and functions.
2. The nucleus components, their chemical composition and morphological features.
3. Chromatin: its chemical composition, levels of condensation, chromatin types and their functions.
4. Morphological, functional and chemical characteristics of nucleolus.
5. Nuclear matrix, the organisation and role of karyoskeleton.
6. Nucleus envelope: structural basis of transport processes between nucleus and cytoplasm, pore
apparatus.
REFERENCES:
1. Junqueira L. C., Carneiro J., Kelley R. O. Basic histology. A Lange medical book. Tenth edition: Appleton
& Lange, 2008.
2. Burkitt H. G., Young B., Heath J. W. Wheaters Functional Histology. A text and colour atlas. 5th edition:
Churchill Livingstone, 2005.
3. Ross M. H., Romrell L. J., Kaye G. Histology. A text and atlas. 5d ed. Baltimore: Williams & Wilkins, 2007.
WHAT MUST YOU KNOW? (INSTRUCTION FOR YOUR SELF-LEARNING)
The nucleus is the main part of the living cell. Its functions include: storage, realisation and reproduction
of the genetic information throughout whole cell life.
Cell nuclei of different tissues vary greatly in size, shape, location in cell and intensity of staining (fig.
1.4). These features depend on cell function and level of activity with regard to protein synthesis.
In most cells under the light microscope the cell nucleus appears as a rounded, elongated or irregular
shape basophilic structure, usually located in the centre of the cell. In mammalian tissues its diameter varies
between 5 and 10 mkm. Even in a single tissue you can detect cells with nuclei different in size and morphologic
features (fig. 4.1).
Using light and/or electron microscopy you can identify structural components of the cell nucleus.
The nucleus comprises the nuclear envelope, chromatin, nucleolus, and nuclear matrix (fig. 4.2).
Each cell type has its specific nuclear morphology. In general, the degree of activity of any cell may be
estimated by the structural appearance of its nucleus. For this purpose you have to analyze all structural
components of cell nucleus and their chemical composition.

25

Fig. 4.1. In this picture you can see variety of the nucleus sizes and intensity of staining (A), shape
(picture B) and location in the cells (picture C).
- The cells of the nervous tissue, 480.
B - The blood cells. Different leucocytes have nuclei varying in shape.
C - The white adipocyte, in which small dark elongated nucleus is located at the periphery of the cell.

Fig. 4.2. How does the cell nucleus appear under the light and electron microscope? In this pictures you
can see structural components of the nucleus under the light (A) and electrone microscope (B).

CHROMATIN is the main part of nucleus.

The main chemical component of chromatin is DNA which carries most of the genetic information the information coded for different protein structure. Besides, DNA comprises less than 20% of nucleus mass.
The DNA codes for numerous different proteins that are not synthesised simultaneously in a cell. Moreover, the
total length of DNA molecule is very long. That is why DNA needs packing with certain genes selective
activation.
The nucleoproteins play the key role in regulation of DNA arrangement and gene activity regulation .
There are two types DNA-binding proteins: histones and nonhistones.
1) histones are relatively low molecular weight proteins with a high content of positively charged amino
acids. Functional significance: histones are involved in the packing and regulation of gene activity.
2) Nonhistones include enzymes responsible for DNA and RNA synthesis. These DNA-associated
proteins may be involved in gene activity regulation.
All nuclear proteins are synthesised in the cytoplasm and imported into the nucleus.
In addition, chromatin can include RNA, that represents newly synthesised messenger RNA which has
not passed into the cytoplasm yet.

26
HOW DOES CHROMATIN PACKING GET? (how is chromatin packed)
There are several stages of chromatin packing:
Firstly, the basic structural unit of chromatin is the nucleusome. Nucleosome consists of a core of
eight histones and a short segment of DNA double helix (fig. 4.3 A) coiled around histones (fig. 4.3 B). This
organization of chromatin has been referred to as beads-on-a-string and corresponds to morphological
form of euchromatin.

30

Fig. 3.4. Stages of chromatin packing.

The nucleosomes form aggregates (fig. 4.3 C). The next higher order of chromatin organization is the
30-nm fibers. In this structure, nucleosomes coiled an axis (with six nucleosome per turn) to form the chromatin
fiber that organizes loops (looks like solenoid). (fig. 4.3 D). These forms of condensing chromatin organization
correspond to heterochtomatin.
The final stage of chromatin parking is a chromosome, which may be visible only during mitosis and
meiosis.
ESTIMATION of CHROMATIN PACKING AND ACTIVITIES BY MORPHOLOGICAL
FEATURES
The degree of DNA coiling varies during different stages of cell activity. According to the intensity of
chromatin packing there are two (morphofunctional types of chromatin.
Structural and functional states of the chromatin: Except the cell division process you can see two
different types of the chromatin, which can be distinguished with both the light and the electron microscopes.

27
They are euchromatin (or active chromatin) and heterochromatin (or inactive chromatin).
Under the light microscopy euchromatin corresponds to a lightly stained zones (areas) in the
nucleus. The genetic information, which are coded in these zones, is accessible for reading during such process
as transcription (the first stage of protein synthesis) and replication (during S-period of cell cycle). That is why
highly active cell has a big light nucleus. But the structural organization of euchromatin is visible only in the
electron microscope.
Heterochromatin is visible in the light microscope and appears as basophilic clumps which in
electron micrograph corresponds to coarse granules. Heterochromatin tends to be clamped around the periphery
of the nucleus, but also forms irregular clamps throughout the nucleus. This type of chromatin dominates in
nuclei of relatively inactive cells.
Diagnostic possibilities: The proportions between eu- and heterochramatin reflects the level of functional
activity. Relatively inactive cells have small dark nuclei in which heterochromatin dominates. In highly active
cells there are big light nuclei in which there is a big amount of active dispersed chromatin.
CAN YOU (How to) DETERMINE WHETHER CELL is MALE OR FEMALE ORGANISM FROM?
The reply is: Yes! For this aim you have to know What the sex chromatin is.
SEX CHROMATIN: Careful study of the chromatin of mammalian cell nuclei reveals a heterochromatin
mass that is frequently observed in female cells but not in male cells. This chromatin clump is the sex chromatin
and is one of the pair of X chromosomes that is visible in female cells during interphase. It remains tightly
coiled and visible, while the other X chromosome is uncoiled and not visible. The male has one X chromosome
and one Y chromosome as sex determinants; the X chromosome is uncoiled, and therefore no sex chromatin is
visible.
Morphological features: In human epithelial cells, sex chromatin appears as a small granule attached to
the nuclear envelope. The cells lining the internal surface of the cheek are frequently used to study sex chromatin.
Blood smears are also often used, in which case the sex chromatin appears as a drumstick-like appendage to
the nuclei of the neutrophilic leukocytes (fig. 4.4).

Fig. 4.4. Sex chromatin

inthe neutrophilic leukocyte
nucleus (arrow).
Diagnostic significance: The study of sex chromatin has wide applicability to medicine, because it
permits determination of genetic sex in patients whose external sex organs do not permit diagnosis of gender, as
in hermaphroditism and pseudohermaphroditism. It is essential for the study of other anomalies involving the
sex chromosomes, for examples, Klinefelters syndrome, in which testicular abnormalities, azoospermia, and
other symptoms are associated with the presence of XXY chromosomes in the cell.
NUCLEOLUS
The function of the nucleolus are synthesis of ribosomal RNA (rRNA) and ribosome assembly.
Chemical composition of nucleolus includes: 1) special zones of DNA that is known as nucleolar
organiser, 2) high amount of ribosomal RNA and 3) proteins, which are organized in complexes with rRNA
molecules to form ribosome subunits.

28
Morphologic features: Under the light microscope nucleolus appears as a spherical structure, up to 1
mm in diameter, and rich in rRNA and proteins. It is usually basophilic when stained with hematoxylin and eosin.
As seen with the electron microscope, the nucleolus consists of three distinct components. They are:
1) The amorphous part, that looks as one or several pale-staining regions composed of nucleolar organiser
DNA sequences of bases that code for rRNA (fig. 5.4). In the human genome, five pairs of chromosomes
(13, 14, 15, 21, 22) contain nucleolar organizers. That is why the maximal amount of nucleoli in mammalian cells
nucleus is five. In this zone the transcription is realised.
2) The part fibrosa, that closely associated with the nucleolar organizers and consists of densely packed
5-10-nm ribonucleoprotein fibers. This part is composed of primary transcripts of rRNA genes.
3) The third component of the nucleolus is the pars granulosa, consisting of 15-20-nm granules (fig. 4.5),
presented by ribosome subunits.

1
1

2
2

Fig. 4.5 Morphology of the nucleolus under the electron

microscopy.
1 granular component; 2 fibrillar component.

You have to know that many different ribosomal proteins are synthesised in the cytoplasm, imported in
the nucleus and then become associated with rRNAs in the nucleolus to form different ribosomal subunits.
After that ribosome subunits migrate into the cytoplasm, where subunits being assembled into the fully active
ribosomes.
Diagnostic possibilities: The size and amount of nucleolus reflects the intensity of protein synthesis.
Large nucleoli are encountered in embryonic cells during their proliferation, in cells that are actively synthesizing
proteins, and in rapidly growing malignant tumors. The nucleolus disperses during cell division but reappears in
the telophase stage of mitosis.
NUCLEAR MATRIX
The nuclear matrix is the component that fills the space between the chromatin and the nucleoli in the
nucleus.
It is composed mainly of proteins, metabolites, and ions.
There are two types of nuclear matrix proteins. They are enzymes and fibrillar proteins which in order to
maintain structural stability and specific shape of cell nucleus arrange a supporting framework that is known
as karyoskeleton (nucleoskeleton). This framework is connected with nuclear envelope by fibrous lamina, that
also is a part of the nuclear matrix. The nucleoskeleton probably contributes to the formation of a protein base

29
to which DNA loops are bound.
Thus nuclear matrix arranges the supports of nuclear morphology, controls DNA and nuclear RNA
space organisation and regulates general nucleus methabolism.
NUCLEAR ENVELOPE
All cells nuclei are bounded by a membrane system, called the nuclear envelope.
Functional significance: The nuclear envelope serves as dynamic interface that mediates the continuous
exchange between nucleus and cytoplasm.
Morphological features: Under the light microscope you can observe a border of nucleus as a thin
basophilic line surrounding the nucleus. This is a thin layer of heterochromatin that lines and binds to the
internal surface of the nuclear envelope (fig. 4.2). But a single nuclear envelope (alone) can be observed
only under the electron microscopy.
Electron microscopy shows that the nucleus is actually surrounded by two parallel unit membranes
separated by a narrow (40-70-nm) space called the perinuclear cisterna. Together, the paired membranes
and the intermembranous space make up the nuclear envelope (fig. 4.6).
1
7
6
2
3
4
5
4
7

2
3
Fig. 4.6. Ultramicroscopic structure of the nuclear envelope. Electronic micrography. Mag. 80 000.
1 - nucleus, 2 - outer nuclear membrane, 3 - perinuclear space (cisterna), 4 - inner nuclear membrane,
5 - heterochromatin, 6 - ribosomes on the external surface of the nuclear membrane;
7 - cisterns of rough endoplasmic reticulum. Arrows indicate nuclear pores.

The internal membrane of the nuclear envelope is closely associated with a fibrous lamina (appearing
as electron dense layer) - the part of nucleoskeleton, which varies in thickness from 80 to 300 nm, depending on
the cell examined. The fibrous lamina is composed of three main polypeptides, called laniins which bound on
membrane proteins and are linked with underlying chromatin.
The outer membrane is studded with ribosomes and polyribosomes. This portion of the nuclear envelope
is sometimes continuous with the rough endoplasmic reticulum (fig. 4.6). When covered with polyribosomes,
the nuclear envelope functions as rough endoplasmic reticulum, synthesizing polypeptide chains and segregating
them in the perinuclear cistern between its two membranes.
The perinuclear space is continuous with cisterns of the endoplasmic reticulum.

30
The nuclear envelope contains numerous nuclear pores,(a) sites where the inner and outer membranes
become continuous to form circular gaps (fig. 4.7 A, B), that provide pathways between the nucleus and the
cytoplasm. Nuclear pores have an average diameter of 70 nm and are composed of eight subunits. The pores
are not open but are bridged by an electrondense membrane that forms a singlelayered diaphragm of protein.
This structure is thinner than the membranes that constitute the nuclear envelope. While the permeability of the
nucleus to molecules is variable, all pores are permeable to some macromolecules (mRNA, cytoplasmic proteins).

47 nm
84 nm
115 nm

4 5

Fig. 4.7. This scheme demonstranes the structure of nuclear pore complex. Find in this picture the membranes
of the nuclear envelope (2, 3) that are continuous in sites of the pore complex, perinuclear spase (1), peripheral (4)
and central (5) granules of the nuclear pore complex and the fibrilles of pore diaphragm (6).

THERE ARE SEVERAL MECHANISMS OF THE SUBSTANCES EXCHANGE BETWEEN

NUCLEUS AND CYTOPLASM.
1. Low molecular substances are transported across the nuclear envelope according to regularities of
membrane transport in both directions.
2. Macromolecules can be exchanged through nuclear envelope by:
a) perinuclear space;
b) bulk transport;
c) pore complex.
3. Universal mechanism of nuclear-cytoplasm exchange - during cell division associated with nuclear
envelope disappearance.

31
LESSON 5
THEME: CELL CYCLE AND REPLICATION. CELL DIFFERENTIATION. CELL DEATH.
BACKGROUND: Even in a full-developed organism, many cells keep dividing because of cell
regeneration. New cells production is necessary for keeping the tissue integrity or for the organs restoration
after its alteration (wounds healing, reparation of the altered tissue etc.). Uncontrolled cell division is the
reason of malignant tumors outcome. Decrease of mitotic properties of cells can be a reason of growth and
regeneration altering in some organs.
In some tissues division may take place throughout all life, in others cells divide only under certain
conditions. To explain processes which take place in an organism for supporting cell amount (populations), it is
necessary to determine dependence between the degree of cell specialisation and its mitotis activity.
AIM OF STUDY: To determine cell life period by morphological features. To know the possible ways
of regulation of mitotic cell activity and cell death. To prognose the development of pathological changes in
cells, tissues, organs and possibilities of the cell reparation.
TO ACHIEVE THIS AIM ONE HAS TO (practical procedures):
1. Determine the period of cell life.
2. Differentiate cell cycle periods by morphological features.
3. Identify kinds of cell division, their phases and essence.
4. Determine the structure features and role of chromosomes during the interphase and mitosis.
5. Define a level of cell differentiation
6. Explain the functional state of the cell on the basis of structural organisation of the cells components.
7. Detect cell death and its type.
TO ACHIEVE THE AIM IT IS NECESSARY TO FOCUS ON THE FOLLOWING
THEORETICAL POINTS:
1. The cell life periods, their characteristics.
2. Cell replication. Mitosis, its phases.
3. Cell cycle, its periods and their morphological reflections.
4. Cell differentiation (specialisation): its morphological features.
5. Morphological characteristics of different functional types of mature cells.
6. Cell death: its types, morphological signs and mechanisms.
REFERENCES:
1. Junqueira L. C., Carneiro J., Kelley R. O. Basic histology. A Lange medical book. Tenth edition: Appleton
& Lange, 2008.
2. Burkitt H. G., Young B., Heath J. W. Wheaters Functional Histology. A text and colour atlas. 5th edition:
Churchill Livingstone, 2005.
3. Ross M. H., Romrell L. J., Kaye G. Histology. A text and atlas. 5d ed. Baltimore: Williams & Wilkins, 2007.
WHAT MUST YOU KNOW? (INSTRUCTION FOR YOUR SELF-LEARNING):
During embryogenesis a complex multicellular organism is developed from a single, fertilized egg cell zygota. This process involves cellular division, growth and progressive morphofunctional specialisation.
During emryogenesis and early postnatal period human tissues containe immature (i.e. relatively
unspecialised) cells. During human organism development cells believed to be capable of differentiation into all
types of cells found in mature organism. However some immature cells remain in fully mature tissues and
provide a source of cells required for replacement or repairment of tissues.
Thus in the adult human organism there are many different cells. Some of them perform individually
specialised functions. These cells are known as highly differentiated (or specialised) ones. In addition, to
produce restoration of specialised cells in fully developed tissues there is a certain amount of immature cells.

32
The turnover rate of the cells varies greatly from one tissue to anotherrapid in the epithelium of the alimentary
canal (tract) and the epidermis, slow in the pancreas and thyroid. The life time period is different for particular
cells. By the end of their life period cells become old (aged) and then die.
Thus whole cell life time can be divided into different periods or states (fig. 5.1) as follows:
1) When cells are immature. These cells have ability to replication and pass through cell cycle.
2) When cells undergo differentiation and become highly specialised to perform individual functions.
3) Old cells which have lower functional activities.
4) Cells that undergo cell death.

Fig. 5.1. Life cycle of cell.

1. Immature cells.
The morphological features and functional properties of immature cells.
The key morphological features of unspecialized cells are big nucleus and a small amount of cytoplasm,
that designated high nuclear-cytoplasm index (fig. 5.2).

A
B
Fig. 5.2. Morphological characteristics of unspecialised cells under light (A) and electron (B) microscopes.
The proportion of nucleus and cytoplasm volume called nuclear-cytoplasm index is known as the main criterion of
cell maturity detection.

33

Under the electron microscope you can see a little amount of membrane organelles, as well as a lot of
free ribosomes in cytoplasm.
Immature cells are able to replication .
Replication is necessary for increase of cell amount. This process occures not only during embryogenesis
but also in a fully mature organs and tissues.
Division of these cells is necessary for realisation of physiological and reparative regeneration (provide
the maintenance of cellular amount in different tissues in normal conditions and after injury/alteration).
The mechanism of cells replication is known as mitosis.
MITOSIS
Mitosis (or mitotic division) of a single cell results in the formation of two daughter cells. Each of the
daughter cells receives a chromosomal karyotype identical to that of the parent cell. For realisation of this
process it is necessary to duplicate genetic information in parent cell and to distribute it identically to the
daughter cells. The time interval during which the cell does not undergo division is called interphase. During
interphase, chromosomes are not visible and exist as an unravelled mass within the nucleus. This arrangement
may facilitate genes expression.
The process of mitosis is dynamic and continuous. It is traditionally divided into four phases. They are:
prophase, metaphase, anaphase and telophase (fig. 5.3).

Prophase
(21 hours)

Metaphase
(< 1 hour)

Anaphase
(< 1/2 hour)

Telophase
(minutes)

Fig. 5.3. Phases of mitosis.

Morphological features of each phases of mitosis can be observed with the light microscope. Cell division
requires the mitotic apparatus which comprises coupled centrioles and microtubules. The mitotic apparatus
is visible only during mitosis, when it forms specific morphological pattern.
PROPHASE: The prophase is characterised by the gradual coiling of nuclear chromatin, giving rise to
several chromosomes (which become visible within the nucleus as individualised rod or hairpin-shaped bodies).
During early prophase the nuclear envelope remains unaltered. As prophase continues the chromosomes become
increasingly condensed and shortened and the nucleoli disappear. Dissolution of the nuclear envelope marks
the end of prophase.
In cytoplasm the disaggregation of cytoskeleton takes place during prophase. The centrioles separate
and migrate toward opposite poles of the cell. At that time the microtubules of the mitotic spindle appear
between the two pairs of centrioles. The key morphological feature is intranuclear condensation of chromosomes.
METAPHASE: During metaphase, the nuclear envelope is disintegrated. The chromosomes migrate to
the equatorial plane of the cell, where each divides longitudinally to form two chromatids. This pattern is known
as metaphase plate.
The duplicated chromosomes become attached to the microtubules of the mitotic spindle (Figure 5-4).
The region of attachment of each chromatide to the spindle plaquelike, electron dense region, is known as
centromere (Gr. kentron, center, + meros, part), or kinetochore (Gr. kinetos, moving, + chora, central region).
The key morphological features are individualisation of chromosomes; initiation of mitotic spindles, formation
of metaphase plate.
ANAPHASE: This phase of mitosis is detected by the separation of the centromere and sister chromatids
from each other. These chromatids migrate toward the opposite poles of the cell, following the direction of the
spindle microtubules. This pattern looks like two anaphase stars.

34
It has been shown by immunofluorescence that microtubules of mitotic spindle include some proteins:
tubulin, actin and myosin. These proteins participate in the process of chromosome migration to the cell poles.
During this process the mitotic spindle stretches tubules to its pole. The chromatids are drawn by the pole-tokinetochore tubules to opposite ends of the spindle to achieving an exact division of the duplicated genetic
material. By the end of anaphase two equal groups of chromosomes are located at opposite poles of the cell.
The key morphological feature is two anaphase stars - chromosomes aggregate at the opposite poles.
TELOPHASE is the final stage of mitosis, during which chromosomes begin to uncoil and to regain their
interphase morphology. This phase is marked by the reappearance of nuclei in the daughter cells and reformation
of nuclear envelop. Described process is called as karyokinesis. Then the division of parent cell cytoplasm
(cytokinesis) takes place. The plane of cytoplasmic division is defined by the position of the spindle equator.
During cytokinesis the plasma membrane around this place becomes indented to form a circumferential furrow
- the cleavage furrow, which progressively constricts the cell until it is divided into two daughter cells. It has
been suggested that cytokinesis occurs as a result of contraction of numerous microfilamentes containing both
actin and myosin which form filamentous ring just beneath the surface of cleavage furrow.
By the end of telophase two cells of equal size are formed.
DIAGNOSTIC SIGNIFICANCE: Frequency of mitosis pattern varies in different tissues and in different
periods of ontogenesis. Rapidly growing tissues (eg, intestinal epithelium) frequently demonstrate cells in mitosis,
the opposite is seen in slowly growing tissues The increased number of mitotic figures and abnormal mitoses in
tumors is an important characteristic distinguishing malignant from benign tumors
Moreover, mitosis can be used for investigation of humans chromosomes and their genes. This method
is calls karyotyping.
KARYOTYPING: the study of chromosomes of humans. The best phase of mitosis for chromosome
investigation is metaphase. For karyotyping it is necessary to induce cells division, arrest mitotic cells during
metaphase, and subsequently cause cellular rupture.
The pattern of chromosomes obtained with a human cell after staining. In addition to the X and Y sex
chromosomes, it is customary to group the remaining chromosomes, according to their morphologic characteristics,
in 22 successively numbered pairs.
DIAGNOSTIC SIGNIFICANCE: The number and characteristics of chromosomes encountered in an
individual are known as the karyotype (fig. 5.4).

B
Fig. 5.4. Mitotic chromosomes (human
karyotype).

35
The study of karyotypes has revealed chromosomal alterations associated with tumors, leukemias, and
several types of genetic diseases.
Until recently, recognition of individual chromosomes was difficult, because different chromosomes of
the same karyotype often had the same general morphology. The development of techniques that reveal
segmentation of chromosomes in transverse, differentially stained bands enabled not only the identification of
individual chromosomes but also the detailed study of genetic deletion or translocation and the chromosomal
localisation of several genes
THE CELL CYCLE
Mitosis is the visible manifestation of cell division, but there are other processes, not so easily observed
with the light microscope, that play a fundamental role in cell multiplication. These processes are realised during
interphase (period between two subsequent mitosis) which together with mitosis form cell cycle (fig. 5.5).

Prophase
(21 hours)

G2
S (8 hours)

G2 +
(2,5 3 hours)

G1 (25 hours)

Metaphase
(< 1 hour)

Anaphase
(< 1/2 hour)
Telophase
(minutes)
G0

INTERPHASE

MITOSIS

Fig. 5.5. Cells life periods: G 1 presynthetic period; S synthetic period; G 2 postsynthetic period;
G0 exit of the cycle, cell death; M - mitosis.

Depending upon the cell type and also on growth conditions, the various phases may be of different
lengths. S-phase, G2-phase, and M-phase usually last approximately 10 hours while the lengths of the G1phases may vary considerably.
CHARACTERISTICS OF INTERPHASE: Interphase is itself divided into three periods (Figure 58): G1-period (presynthesis or postmitosis), S-period (DNA synthesis), and G2-period (post-DNA duplication
or premitosis).
Periods of the cell cycle. The G1- period is variable and depends on many factors. The S-period lasts
about 8 hours and include DNA-replication. The G2-period lasts 2,5-3 hours then cell enters to mitosis.
The G1-period (postmitosis) is characterised by mitotic spindle desaggregation and beginning of active
synthesis of mRNA and proteins for cell growth. It results in the cell volume, previously reduced to one-half by
mitosis, is restored and daughter cells increase their size to one of parent cell before division. The time involved
in G1-period determines the whole cell cycle time.
Synthesis and replication of DNA and centrioles take place in the S period. This is the main period of

36
interphase. The enzyme that controls DNA-synthesis is known as DNA-polymerasa-a. If some alterations of
DNA-replication occur the DNA-synthesis is stopped (arrested) and reparation of altering DNA points begins.
If DNA-restoration is impossible damaged cell undergo programmed cell death - apoptosis (see below).
Processes that occur during the G2 period are the production and accumulation of energy to be used
during mitosis and the synthesis of tubulin to be assembled in microtubules during mitosis.
Resting cells, are non-dividing cells. Normally these cells are arrested somewhere before the G1period before the initiation of DNA synthesis. Some epithelial cells may be arrested also in the G2-period.
Important examples of resting cells are stem cells.
REGULATION OF THE CELL CYCLE: The organism has elaborate regulatory systems that control
cell reproduction, either stimulating or inhibiting mitosis.
The individual stages of the cycle are also under strict control and the exact and unperturbed temporal
order of associated gene activities is an absolute prerequisite for successful completion of the cycle. A so-called
CDC genes (cell cycle genes) have been identified. Mutations in any of these genes lead to the arrest of the cell
at a different phases of cell cycle. Cell cycle regulatory proteins include a special family of proteins, designated
cyclins. The name cyclins for these internal regulators of the cell cycle derives from the observation that in all
cells these proteins accumulate periodically in a cell cycle phase-specific manner (fig. 5.6).
disassembly
of spindle

cyclin 2 telophase and cytotomia

anaphase

metaphase

R3

restriction
point R2

G1

M
G2

R2

cyclin 1

S-period

cyclin

restriction point R1
The passage of this
point occurs after
cyclins activating
R1
(cyclins D1, D2, D3)
and cyclin-dependent
kinase (CDK-C)
CDK-C complex
activates DNA
replication

DNA replication

Fig. 5.6. Regulation of cell cycle.

There are external factors that regulate (stimulate or inhibit) cells proliferation. These proteins are known
as growth factors (GF), for example nerve growth factor, epithelial growth factor, fibroblast growth factor and
others.
Cell proliferation is usually regulated by precise mechanisms that can, when necessary, stimulate or
retard mitosis according to the needs of the organism. Several factors (eg chemical substances, certain types
of radiation, viral infections) can induce abnormal cell proliferation that bypasses normal regulation mechanisms
for controlled growth and forms tumors.
Many observations suggest that the regulation of cell proliferation and the progression of cells through

37
the cell cycle is linked intimately to the control of cell death (apoptosis). Manipulation of the cell cycle may
either prevent or induce an apoptotic response. Thus, cell cycle and Apoptosis control act to preserve homeostasis
and developmental morphogenesis. Control of both processes is achieved, in part, by coupling the process of
cell cycle progression and apoptosis through the controlled expression of a shared set of factors. This linkage
has been recognized for tumor suppressor genes such as P53 and RB, the dominant apoptogenes, c-Myc, and
several cyclins-depending kinases (Cdks) and their regulators.
The more or less exact knowledge of processes regulating the cell cycle has provided the prerequisites
for a type of tumor therapy known as cell cycle therapy. It is based essentially on the treatment of resting cells
with genes to induce their entry into the S-phase. This effectively renders them vulnerable to the action of Sphase-specific drugs. In addition, this knowledge can be used also to design treatment regimens that specifically
protect non-cycling stem cells.
2. CELL DIFFERENTIATION
Then cells become increasingly specialised to carry out of different specific functions. The process
during which cells become specialised is called differentiation (cells come to G0-phase).
During differentiation the cytoplasm volume is increased by organelles that perform specialised function,
thus nuclear-cytoplasm index is decreased (fig. 5.7).
4
3
4
1
5
2

1
4

1
5

B
3

Fig. 5.7. Cells of fibroblasts differon in different periods of life cycle: A - undifferentiated cells; B - poorly
differentiated cell, C - mature (functionally active) cell, D - fibrocyte - inactive cell.
1 - nucleus; 2 - Golgi complex; 3 - mitochondria; 4 - granular endoplasmic reticulum; 5 - collagen fibrills.

In fully developed organism there are many different functional types of cells. In this text only some of
them have been discussed. They are:
1) A cell which specialised for the synthesis and secretion of proteins (for example, hormones or extra
cellular matrix) has following morphological features (fig. 5.8):
1.1. Low nuclear-cytoplasm index because of a large volume of cytoplasm.
1.2. Its nucleus has predominantly euchromatine, that facilitates transcription during proteins synthesis,
one or several nucleoli and a large surface of pore apparatus at nuclear envelope.
1.3. Basophilic under the light microscope cytoplasm.
1.4. Cytoplasme under the electron microscope contain a lot of rough endoplasmic reticulum cisterns
and developed Golgi apparatus, that can forms secretory granules.

38

Fig. 5.8. The fibroblast of

connective tissue. This cell produces
different component of extracellular
matrix. Try to find (detect) in this
picture
described
above
morphological features.

1) A cell specialised for the lipid metabolism, for example for steroid hormones synthesis.
2.1. Low nuclear-cytoplasm index because of a large volume of cytoplasm.
2.2. Its nucleus has predominantly euchromatine, that is necessary for enzymes synthesis for
steroidogenesis, one or several nucleoli and a large surface of pore apparatus at nuclear envelope.
2.3. Oxyphilic cytoplasm under the light microscope with a large amount of unstained vacuoles.
2.4. Cytoplasm under the electron microscope has a lot of cisterns of smooth endoplasmic reticulum,
Golgi appatatus, mitochondria with tubular cristae and lipid droplets (fig. 5.9).

1
2

3
1

Fig. 5.9 Ultramicroscopic structure of zona fasciculata cell in adrenal cortex. In the cytoplasm you can see
developed smoth endoplasmic reticulum (1), there are numerous fat droplets (2) and mitochondria with tubulovesicular cristae (3).
2) A cell specialised for transport of different substances (fig. 5.10).
The exchange of molecules between the internal and external environment is mediated with plasma
membrane.
In order to increase exchange surface plasma membrane forms microvilli on one and deep folds on
opposite cell surface.
For active ion transport realisation plasma membrane is closely associated with numerous mitochondriae.
As a result of endocytosis, there are a lot of vesicles in the cell (vacuoles).

3) Highly phagocytic cell has following morphological features (fig. 5.11):

- numerous folds and microvilly of plasma membrane.

39
-

A lot of lysosomes in cytoplasm.

Phagosomes or endocytotic vesicles.

Fig .5.10. Ultramicroscopic structure of cell of kidney

proximal tubules. In the apical pole of cell you can see
microvilli that increase the area of transport, and
pinocytotic vesicles. In the basal pole there are many
deep folds of plasmolemma surrounded by mitochondria
- a zone of active transport of electrolytes.

Fig. 5.11 Ultramicroscopic structure of

macrophage. Plasmolemma of cell forms
numerous folds, protrusions and invaginations,
many vacuoles in the cytoplasm, phagosomts,
lysosomes.

Most tissues undergo a constant turnover because of continuous cell division and the ongoing death of
cells. There are two types of cell death. They are:
1) Apoptosis - the programmed cell death. In brief apoptosis is cellular self-destruction regulated by own
genes and proteins.
2) Necrosis caused by action of harm factors.
Apoptosis.
Apoptosis (from a Greek word meaning the falling of leaves from a tree) is a term referring to the
cytologically observable changes associated with a process of active, gene-directed, cellular self-destruction
observed in all eukaryotes. The process has been termed also programmed cell death or active cell death.
Apoptosis is intentionally unobtrusive, to remove single cells without provoking inflammation or upsetting
tissue function.
SIGNIFICANCE OF APOPTOSIS:
1) Apoptosis of cells is needed to balance cell proliferation during the normal turnover of tissues, such as
blood and epithelia.
2) Apoptotic processes are observed during embryonal development (morphogenesis). One strategy of
development is to overproduce cells, then select, e.g., for the survival of correctly connected neurons.
3) Apoptosis plays important role in regulation of immune defense. Cellular self-destruction plays a
decisive role in the elimination of self-recognizing T-lymphocytes in the thymus. Apoptosis also provides a

40
defense mechanism against viruses by reducing virus spread through the rapid death of virus-infected cells.
4) Apoptosis occurs in endocrine tissue atrophy or if an organ cannot work properly (for example, if a
gland has a blocked main duct many cells die by apoptosis.
5) Apoptosis is observed during tumor regression.
6) The disruption of normal processes leading to apoptosis results in illegitimate cell survival and can
cause developmental abnormalities and facilitate cancer development.
MORPHOLOGICAL FEATURES of cells, died by the way of apoptosis.
With light microscopy only the increasing nuclear density and cell shrinkage are noticeable, unless special
cytochemical methods to detect apoptotic events are used. The cell and internal membranes bleb out. Finally
the shrunken cell is phagocytosed by macrophages. With electron microscopy appears the following changes
(fig. 5.12).

Fig. 5.12. Ultramicroscopic signs of apoptosis - cell wrinkling (a), fragmentation of

nuclei (b) and cytoplasm (c)

MECHANISMS AND CYTOCHEMICAL MARKERS OF APOPTOSIS :

For apoptosis, endonucleases are activated which break up the chromatin, chopping up the DNA,
transcription slows and stops, organelles clump, and the endoplasmic reticulum cisterns dilate. Caspases (Caspases
- cysteinyl aspartate-specific proteinases) digest relevant cellular materials and structures.
Comparison of cell death by necrosis and apoptosis.
Theses two processes differ in various aspects (fig. 5.13).
Cellular necrosis is usually caused by cell damage and does not require further gene activity. The
membrane is the major site of damage and, among other things, loses its ability to regulate osmotic pressure.
Eventually cell contents are released and elicit inflammatory reactions.
Apoptotic cell death requires gene activity. It can be prevented by increased expression of some
genes or by external signals. Apoptotic cells eventually break up into apoptotic cell bodies. Cell contents are
not released and there is no inflammation. Apoptosis allows selective elimination and swift clearance by
phagocytosis of cells from a proliferating cell population and is an evolutionarily conserved process for killing
unwanted cells in multicellular organisms.

41

B
Fig. 5.13. Two ways of cell
death - apoptosis (A) and
necrosis (B).

Cell death can be triggered by a variety of stimuli, including gamma irradiation, cytotoxic lymphocytes,
glucocorticoids, and various cytolytic cytokines, for example, TNF-a. In thymocytes apoptosis can be induced
by treatment with glucocorticoids or irradiation.

42
LESSON 6
THEME: EMBRYOLOGY OF BIRDS AND MAMMALS.
BACKGROUND: The study of general embryology begins with embryogenesis of birds and placental
mammals, because their development has common features with human embryogenesis. A basic knowledge of
comparative embryology help to understand the general biological laws of evolution of vertebrates (biogenetic
law, the theory of germ layers, etc.), phylogenetic principles of human development. The fundamentals of
vertebrates embryology and patterns of embryo development are necessary for understanding of histo- and
organogenesis, determine the causes of anomalies and malformations, which may be the subject of medical
practice.
AIM OF STUDY: To be able to interpret the essence of the period of embryonic development of birds
and mammals, which is necessary for understanding the evolutionary patterns of vertebrates development and
the use of this knowledge during the study of human embryogenesis.
TO ARCHIVE THIS AIM ONE HAS TO (practical skills):
1. Interpret the essence of the period of embryogenesis of birds and mammals, understand the biological
processes that underlie it.
2. Determine the types of eggs, understand the dependence of their structure on the conditions of embryo
development, their role in determining the features of fragmentation in birds and mammals.
3. Interpret mechanisms and ways of gastrulation, the nature and role of implantation in mammals.
4. Determine the germinal layers of embryos of birds and mammals, the axial complex primordia, interpret their
differentiation and the formation of various organs and systems.
5. Distinguish extraembryonic organs of birds and mammals, explain their formation and functions.
TO ACHIVE THE AIM IT IS NECESSARY TO CENTRE AROUND THE FOLLOWING POINTS:
1. Concept of onto- and phylogenesis.
2. Periods of embryogenesis.
3. Types of eggs, Eggs characteristics in birds and mammals.
4. Fertilization and its biological meaning.
5. Cleavage and its features in birds and mammals, dependence to the type of egg. Blastula characteristics in
different animal classes.
6. Gastrulation in birds and mammals. Ecto- and entoderm formation. Mesoderm and notochord formation.
7. Implantation in mammals. Its stages and meaning.
8. Germ layers differentiation, germ organs axial complex formation, their further development.
9. Development, structure and functions of provisional organs in mammals.
REFERENCES:
1. Medical Embryology. Langman J. Baltimore. Wilkins Co. 1969. P. 37-54.
2. Conception of Human Anatomy and Physiology. Kent M., Van De Graaff, Stuart Ira Fox. Wm. C. Brown
Publishers, 1995. P. 904-908.
3. Study Guide and Review Manual of Human Embriology. Keight L. Moore. W. B. Saunders Company.
Philadelphia. London. Toronto. 1975. P. 21-45.
4. An Atlas of Ultrastructure. Johanes, A.Y. and Rhodin M.D. Philadelphia, London, Saunders Co. 1963.
WHAT MUST YOU KNOW? (INSTRUCTION FOR YOUR SELF-LEARNING)
Before studying the patterns of embryogenesis in vertebrates, revise the essence of the biological processes
underlying the development of the embryo: the division, migration, growth, interaction, cell death. Pay attention
to the concept of embryonic induction, consider the nature of the processes of determination and cell
differentiation.
You should start by studying the periods of embryogenesis. Embryogenesis precedes the development of

43
the gametes - sperm and ova, which takes place during progenesis.
Learn the types of eggs, paying attention to the features of their structure in birds and mammals and to
the link between the structure of the egg and condions of embryo development. Make sure you understand the
essence of the process of fertilization, cleavage period, determine its nature and relationship to the type of egg
(fig. 1.6).

3
4

5
Fig. 1.6. Types of eggs according to number and location of the yolk (top figure) and their corresponding types of
fragmentation (lower figure): A - oligo- and isoletsital egg of mammals and humans - cleavage completely irregular,
B - poly- and thelolecital egg of birds - cleavage incomplete irregular ; B - poly- and centrolecital amphibian egg cleavage incomplete irregular.
1 - the nucleus of egg, 2 - yolk inclusion, 3 - animal pole, 4 - vegetative pole, 5 - blastomeres.

Embryonic development starts from the moment of fertilization and the formation of single-cell embryo
(zygote) and finishes with formation of a multicellular organism capable of independent feeding. Embryogenesis
lasts until birth or egg hatching .
There are 4 periods in the birds embryogenesis :
1 period - fertilization and the formation of a zygote. Fertilization is the process of fusion of the male and
female reproductive cells, which results in the restoration of the diploid set of chromosomes specific for this
organism, and formation a zygote. Fertilization in birds is internal, as occurs in the genital tract of the maternal
organism. The newly formed zygote, moving on oviduct, enters the period of cleavage, coats in protein, under
eggshell envelope and eggshell.
2 period - cleavage. It is characterized by repeated mitotic division of the zygote, during which formed
cells, called blastomeres, do not reach the size of the parent cells and start a new division. Consequently, the
number of blastomeres increases and their size decreases. As a result, a multicellular embryo forms, which size
is almost the same as zygote size.The type of cell division depends on the type of egg cells. Birds have polycetal
egg (with more than one yolk ) because the fetus develops outside the mothers organism and it needs an
adequate supply of trophic inclusions. The yolk is located at one pole of the egg - vegetative, so the egg is called
thelolecital. On the opposite side, the animal pole, there is little yolk as it is the location of the nucleus. In the
cleavage process only a portion of a zygote is involved - the animal pole, whereas the vegetal pole is undivisible.
Because of this cleavage period in birds is called partial (meroblastic, incomplete). At first, the number of
blastomeres is growing exponentially, but with time this order is violated. This is due to the fact that the blastomeres
differ in size and fission rate, so the cleavage of birds is called uneven. At the end of cleavage discoblastula is
formed, which is the germinal disc located on the yolk (fig. 2.6).

44

2
1

5
3

6
4

Fig. 2.6. The scheme of splitting embryo of mammals:

A - stage of light and dark blastomeres, B, C - stage fouling dark blastomeres light; D - morula stage, E, F - the
blastocyst stage.
1 - dark blastomeres; 2 - light blastomeres; 3 - cavity of the blastocyst; 4 - fertilization membrane; 5 - embryoblast;
6 - trophoblast.

3 period - gastrulation. Gastrulation is a complex process of chemical and morphogenetic changes

accompanied by reproduction, growth, directed motion and differentiation of cells, resulting in formation of
germ layers - an outer layer (ectoderm), middle (mesoderm) and internal (endoderm) - sources of tissues and
organs germs. At that time, notochord and neural plate are formed. Gastrulation is divided into two phases.
Gastrulation in birds begins with delamination of germinal disc plate, resulting in the epiblast and hypoblast
formation, between which blastocoel lies (primary body cavity). Later on the cells from peripheral zones of
hypoblast leave it, migrate towards the yolk and form extraembryonic endoderm, which is involved in the
formation of the yolk sac wall.
The main morphogenetic events occur in the epiblast, where cells rapidly and unevenly divide and migrate.
Directed movement of streams of cells leads to the formation of primitive streak and the primary (Gensen)
nodule (fig. 3.6, 4.6).
Then the cells of the primary nodule shift inwards in the shape of bundle towards the blastocoel, under
the ectoderm, and grow to the front end of the embryo, forming the notochord germ. Primitive streak cells also
migrate inwards integrating into hypoblast and forming embryo endoderm, and to the both sides of notochord,
forming two wings - the germ of embryonic and extraembryonic mesoderm. In the ectoderm over notochord
outgrowth neural plate appears in the shape of the thickened strip. Its development is induced by notochord.
4 period - histo-and organogenesis. From embryonic germs by further differentiation tissues and organs
are formed.
As a result of the inductive activity of notohord neural plate bends and forms the neural groove. Neural
ridges along the edges of the groove converge and coalesce, the groove closes creating the neural tube. This
process is called neurulation. There are medial and lateral parts in the mesodermal wings. Medial part is
segmented into somites. There are 33-34 pairs of somites (Fig. 5.6, 6.6).

45

1
2

2
3

3
4
5

5
4
6
9
7

6
7

6 7

10

10

Fig. 3.6. Schematic diagramm of primitive streak formation and migration of cells during the
formation of germ layers: A - formation of primitive streak and primary nodule, B - formation of
notochordomesodermal germ; C - formation of notochord (sagittal section); D - formation of
mesoderm (sagittal section). Solid arrows indicate the direction of movement of the material in
the outer layer (before immersion), dashed - in the middle layer (after immersion).
1 - epiblast; 2 - the material of the future neural plate, 3 - material of the future notochord;
4 - the primary nodule, 5 - the primary hole, 6 - primitive streak, and 7 - primary fissure;
8 - notochord; 9 - mesoderm, 10 - endoderm.

2
4
5

2
4
3

Fig. 4.6. Chicken embryo of 16 hours of incubation. Total specimen: A - mag. 2; B - mag. 20.
1 - germinal disc; 2 - the embryonic shield, 3 - primitive streak; 4 - the primary nodule, 5 - mesoderm formation
(wings).

46
1

2
3
6

Fig. 5.6. Segmentation of the chick

embryo mesoderm (30 hours of
incubation, 8 pairs of somites).

5
7
3

4
1 - nerve rollers, 2 - neural
groove; 3 - notochord 4 - somites,
5 - parietal and visceral layers of
splanchnotom 6 - foregut, 7 intestinal groove (rudiment of the
middle portion of bowel), 8 - blood
islands; 9 - residue primitive streak.

8
9
10
6

7
2

3
4

11
12

Fig. 6.6. Chicken embryo of 36

hours of incubation. Total
specimen. mag. 20.
1 - embryo disc; 2 - the embryonic
shield, 3 - bright field; 4 - dark field,
5 - the edge of fouling, 6 - neural
tube, 7 - somites, 8 - telencephalon,
9 - amniotic fold; 10 - germ of the
heart; 11 - the remainder of the
primary strip; 12 - blood islands.

47
Lateral part, which is not segmented, forms splanchnotom, which is divided by secondary cavity (coelom)
to parietal (somatopleure) and visceral (splanchnopleure) layers (fig. 7.6, 8.6).
1
1

12
10

14

8
15
5

15

13

11 7
2

Fig. 7.6. Cross-section of the chicken embryo of 42 hours of incubation at the stage of differentiation
of embryonic layers and axial germ complex formation. Histological specimen.
Mag. 100.
1 - ectoderm: 1 a - extraembryonic, 1 b - embryonic; 2 - embryonic endoderm; 3 - neural tube;
4 - notochord; 5 - mesoderm; 6 - somite; 7 - splanchnotome; 8 - visceral layer; 9 - parietal layer;
10 - coelom; 11 - nephrogonotome, 12 - dermatome; 13 - sclerotome; 14 - myotome; 15 - germ of
heart.

The part of the mesoderm which connects somites with the splanchnotom at great length of body is also
segmented, forming the so-called stalks or nephrogonotoms. Somites in turn, begin to differentiate into three
parts: the dermatome, sclerotome and myotom. You should pay attention to the ways and the results of
differentiation of germ layers. It should be noted that this period is the longest one, because during the
embryogenesis tissues and organs development is not completed, and continues in the postembryonic period.
Separtion of the embryos body from extraembryonic material occurs with the help of the trunk folds.
They are formed along the edges of the embryonic shield with their crests directed towards the yolk, under the
embryo. Because of the growth and convergence of the trunk folds the embryos body rises above the yolk and
curls into a tube. This process begins at the head end of the embryo and spreads in the caudal direction. Fusion
of the edges of the trunk folds leads to the closure of primary colon, breast and abdomen of the fetus.
Study the extraembryonic organs (membranes) formation and their functions. Extraembryonic (provisional)
organs are the temporary structures, which develop outside the body of the embryo and function only during
embryogenesis. These organs in birds are amnion, serosa, the yolk sac, allantois.Extraembryonic organs develop
from extraembryonic parts of the disk (light and dark field), isolated from the embryo by the trunk folds. Pay
attention to the fact that in birds the embryo material development precedes development of provisory organs,
except for the yolk sac (due to its trophic and hematopoietic functions). Each extraembryonic organ develops
from two layers: the amnion and serosa - from the extraembryonic ectoderm and parietal layer of extraembryonic
mesoderm (figure 8.6).

48
4
3
2

7
5

Fig. 8.6. Chicken embryo at the stage of the trunk and the amniotic folds formation. Histological
specimen. Mag. 100.
1 - trunk fold; 2 - wall of the amnion; 3 - embryonic ectoderm; 4 - neural tube; 5 - notochord;
6 - embryonic entoderm; 7 - extraembryonic ecto-, meso-and entoderm; 8 - germ of the heart;
9 - blood vessels.

Yolk sac develops earlier than other extraembryonic organs. Extraembryonic endoderm and visceral
layer of extraembryonic mesoderm, expanding uniformly in all directions to unfragmented part of the zygote,
surround the yolk, forming a wall of the yolk sac. Yolk sac has a trophic function, being also the first organ of
hematopoiesis.
Amnion and serosa are created simultaneously. Previously, extraembryonic ectoderm and parietal layer
of extraembryonic mesoderm form amniotic folds, which grow upward and toward each other, grow together
and surround the embryo. Thus, two membranes are created: the inner and outer. Inner is called amniotic or
fluid. It creates the amniotic cavity and produces amniotic fluid with a more or less constant chemicalstructure.
Function of the amnion is to create the necessary water environment for the developing embryo to protect it
against mechanical impacts and gravity field of the Earth. The outer membrane is called serosa, it is the most
superficial, adjacent to the inner films and the shell and provides embryo respiration.
Allantois develops simultaneously with the amnion and serosa. Endoderm of posterior archenteron with
the surrounding visceral mesoderm forms a protrusion like a sac, which during its development is growing, filling
the crevices between the serous membrane, on the one hand, and the yolk sac and amnion on the other.
Allantois performs the function of excretory organ, products of nitrogen metabolism are accumulated in its
cavity. In addition, the wall of the allantois forms umbilical vessels, which connect the the embryo with provisional
respiratory organ - the serous membrane.
While studying the features of embryogenesis of mammals make sure you understand the periods of
embryogenesis, comparing them to birds periods, the type of egg, its structure, the link between the structure of
the egg and conditions of embryogenesis.
In the embryogenesis of mammals, like birds, there are 4 periods: fertilization and a zygote formation,
cleavage, gastrulation, histo- and organogenesis. During the evolution eggs of placental mammals have acquired
the characteristics of isolecital and oligolecital: they contain a small amount of yolk granules, more or less
evenly distributed in the cytoplasm. This is due to the transition to fetal development, in which the fetus is fed by
the mothers organism. Fertilization in mammals as in birds, occurs in the genital tract of the female body, i.e. is
internal.
Due to these features of mammal eggs cleavage is complete, uneven. The zygote by the first meridional

49
cleavage furrow is divided completely (goloblastically) to 2 blastomeres. During the first division we can distinguish
two types of blastomeres (fig. 2.6): smaller, lighter, with the hydrated cytoplasm and larger, dark, rich in RNA.
Light blastomeres divide more quickly than the dark ones(this means uneven cleavage). Gradually the light
blastomeres surround dark ones and close up on them (morula stage). Light blastomeres form the trophoblast
(feeding bud ), which gets in contact with the tissues of the oviduct and is able to suck their secret for
nutrients. It performs trophic and protective functions. Dark blastomeres form embryoblast (the inner cell
mass), from which the embryo will develop. Initially embryoblast has the form of nodules.Because of absorption
of nutrients by trophoblast blastocyst cavity creates, which gradually increases and the fluid accumulated as a
result of trophoblast cells activity and the processes of diffusion from the uterus, shifts embryoblast to the
upper part of the nucleus, where it acquire the shape of the disk. Embryo disc of mammals corresponds embryo
disc of birds. Thus, at the end of cleavage a blastocyst is formed - embryo vesicle, which consists of embryoblast,
trophoblast and the cavity.
Intrauterine character of mammals development determines the essential features of the processes of
embryogenesis. Simultaneously with the beginning of the period of early gastrulation, the embryo is implanted
(embedded) in the endometrium. Trophoblast is actively involved in this prosses. Its cells produce hystolytical
enzymes that dissolve mucosa of the uterus where the embryo is immersed. Trophoblast grows vigorously,
forming outgrowths - the primary villi, which increase its suction surface. Next mesoderm grows into the
primary villi of trophoblast and forms the secondary villi, in which mesoderm differentiates into mesenchyme.
Secondary villi develop on the side of trophoblast that face the endometrium. Later blood vessels grow into the
mesenchyme and thus tertiary villi are formed. Implantation is important in the further development of the
embryo, providing optimal conditions for feeding, respiration and allocation at the expense of the maternal
organism.
Mammals gastrulation proceeds in two phases. In the first phase epiblast and hypoblast are being formed
by delamination, where extraembryonic sheet and three extraembryonic organs - amnion, yolk sac and chorion
are formed. Chorion is unique to mammals, its wall consists of trophoblast and extraembryonic mesoderm.
This organ is the basis for the formation of the placenta, which connects the fetus and the mothers body,
carries out trophic, protective, and in the second half of embryogenesis, endocrine function (fig. 9.6).
In the second phase of gastrulation embryonic endoderm, mesoderm, ectoderm and notochord develop.
All processes are similar to those that occur in birds - due to primitive streak formation and primary nodule with
subsequent migration of cells.
Differentiation of embryonic layers and germ organs axial complex development happen as in birds.
Further development of embryonic germs is also similar to them.

50

13
9

10
11

11

14

12
12

B
4

3
5

2
6
7

D
Fig. 9.6. Scheme of extraembryonic organs
in mammals: A - process of fouling of the
blastocyst cavity of endoderm and
mesoderm; B - formation of a closed
endodermic bubble - the lining of the yolk
sac; C - beginning of the formation of
amniotic folds; D - formation of trunk folds
- the isolation of body parts of the embryo
from the extraembryonic; E - closure of
amniotic folds, beginning of the allantois
development; F - closed amniotic cavity
developed allantois, development of
chorionic villi.

5
2
3

7
8
6

1 - body of the embryo; 2 - archenteron; 3 - trunk fold;

4 - amniotic fold; 5 - amniotic membrane; 6 - chorion;
6a - smooth chorion; 6b - villiferous chorion, and 7 - wall
of the yolk sac; 8 - allantois; 9 - trophoblast; 10 - epiblast;
11 - extraembryonic mesoderm; 12 - extraembryonic
endoderm (hypoblast); 13 - neural tube; 14 - notochord.

51
LESSON 7
THEME: HUMAN EMBRYOLOGY. FERTILIZATION. CLEAVAGE.
BACKGROUND Embriology forms the scientific background for such sciences as gynecology, obstetrics
and pediatrics. Studying the stages of human embryology creates the theoretical background for the medical
practice of a doctor focusing on:
- Supervise the process of creation and development of gametes, determine the reasons of gametopathies,
perform the profilactics for preventing the infertility.
- Determene the stages of cleavage, its blastomere differentiation, reasons of development of single eggtwins.
- Determene the term and stage of implantation which is important during the extracorporal embryo
development.
AIM OF STUDY: (general): To be able to identify periods of embryogenesis, their patterns, the phases
and mechanisms of fertilization and cleavage, which determine the early events of development.
TO ARCHIVE THIS AIM ONE HAS TO (practical skills):
1. Differentiate the periods of embryogenesis.
2. Interprete the structure of human gametes.
3. Define the phases and essence of fertilization.
4. Interpret the essence and peculiarities of cleavage.
TO ACHIVE THE AIM IT IS NECESSARY TO CENTRE AROUND THE FOLLOWING POINTS:
1. Peryods of humanembryogenesis.
2. Progenesis.
3. Structure of spermatozoon.
4. Structure of oocyte.
5. Fertilization. Phases, mechanisms, regulation.
6. Cleavage. Phases, peculiarities, mechanisms.
REFERENCES:
1. Medical Embryology. Langman J. Baltimore. Wilkins Co. 1969.
2. Conception of Human Anatomy and Physiology. Kent M., Van De Graaff, Stuart Ira Fox. Wm. C. Brown
Publishers, 1995.
3. Study Guide and Review Manual of Human Embriology. Keight L. Moore. W. B. Saunders Company.
Philadelphia. London. Toronto. 1975.
4. An Atlas of Ultrastructure. Johanes, A.Y. and Rhodin M.D. Philadelphia, London, Saunders Co. 1963.
WHAT MUST YOU KNOW? (INSTRUCTION FOR YOUR SELF-LEARNING)
Fertilization is the process whereby two sex cells (gametes) fuse together to create a new individual
with genetic potentials derived from both parents. Fertilization accomplishes two separate ends: sex (the combining
of genes derived from the two parents) and reproduction (the creation of new organisms). Thus, the first
function of fertilization is to transmit genes from parent to offspring, and the second is to initiate in the egg
cytoplasm those reactions that permit development to proceed.
A complex dialogue exists between egg and sperm. The egg activates the sperm metabolism that is
essential for fertilization, and the sperm reciprocates by activating the egg metabolism needed for the onset of
development. But before we investigate these aspects of fertilization, we need to consider the structures of the
sperm and egg the two cell types specialized for fertilization.
SPERM STRUCTURE
Each sperm consists of (fig.1.7):

52
1
2

I. Head

Fig. 1.7. Scheme of structure of human

sperm: I - head; II - tail.

B. The intermediate part

II. Tail A, B, C, D

1 - receptors of glycocalix; 2 - acrosome; 3 - outer

and inner acrosome membranes; 4 - proximal
centrioles, 5 - mitochondrion, 6 - layer of elastic
fibrils, 7 - axonema, 8 - distal centrioles; 9 - fibrous
vagina (longitudinal pillars and circular fibrils,
which consist of keratin filaments).

Nucleus: 22 + autosomes
+ 1 sex chromosome
(X or Y)
. Cervix (binding part)

5
6
7
8
C. The main part
9
D. The end part

1) a head with haploid nucleus, and a sac of enzymes (acrosoma) that enable the nucleus to enter the
egg. Most of the sperms cytoplasm is eliminated during maturation, leaving only certain organelles that are
modified for spermatic function. During the course of sperm maturation, the haploid nucleus becomes very
streamlined, and its DNA becomes tightly compressed. In front of this compressed haploid nucleus lies the
acrosomal vesicle, or acrosome, which is derived from the Golgi apparatus and contains enzymes that digest
proteins and complex sugars; thus, it can be considered a modified secretory vesicle. These stored enzymes are
used to lyse the outer coverings of the egg.
2) a tail. Each sperm is able to travel long distances by whipping its flagellum. Flagella are complex
structures. The major motor portion of the flagellum is called the axoneme. It is formed by microtubules
emanating from the centriole at the base of the sperm nucleus. The core of the axoneme consists of two central
microtubules surrounded by a row of nine doublet microtubules. Actually, only one microtubule of each doublet
is complete, having 13 protofilaments; the other is C-shaped and has only 11 protofilaments. Although tubulin is
the basis for the structure of the flagellum, other proteins are also critical for flagellar function. The force for
sperm propulsion is provided by dynein, a protein that is attached to the microtubules. Dynein hydrolyzes
molecules of ATP and can convert the released chemical energy into the mechanical energy that propels the
sperm. This energy allows the active sliding of the outer doublet microtubules, causing the flagellum to bend.
The importance of dynein can be seen in individuals with the genetic syndrome called the Kartagener triad.
These individuals lack dynein on all their ciliated and flagellated cells, rendering these structures immotile.
The ATP needed to whip the flagellum and propel the sperm comes from rings of mitochondria located in
the neck region of the sperm.
THE OOCYTE (fig.2.7):
All the material necessary for the beginning of growth and development must be stored in the mature egg
(the ovum). Whereas the sperm has eliminated most of its cytoplasm, the developing egg (called the oocyte
before it reaches the stage of meiosis at which it is fertilized) not only conserves its material, but is actively
involved in accumulating more. The meiotic divisions that form the oocyte conserve its cytoplasm (rather than
giving half of it away), and the oocyte either synthesizes or absorbs proteins, such as yolk, that act as food
reservoirs for the developing embryo.
Proteins. It will be a long while before the embryo is able to feed itself or obtain food from its mother.
The early embryonic cells need a supply of energy and amino acids. In many species, this is accomplished by

53
accumulating yolk proteins in the egg. Many of the yolk proteins are made in other organs (liver, fat body) and
travel through the maternal blood to the egg.

Ovotsyt surrounded by a zona

pellucida - ZR and a layer of
follicular cells

Between follicular cells - gap

junctions (GJ). Processes of
cells penetrate the ZR and form
GJ with oocyte

Cortical granules
containing
proteases.
Their release
under conditions
of increasing Ca2+
degrade
Zp2 and Zp3

Perivitelline space

OM nhibitor of
oocyte maturation.
O M secrete by

OM
2+

follicular cells and

blocking meiosis
SM

Golgi
complex

S stimulator of
Cdc2

cyclin

Granular ER

Glycoproteins of zona
pellucida (Zp1, Zp2, Zp3)
produced by oocyte

meiosis - a complex of
cyclin B and cyclindependent kinase
(Cdc2)

Fig. 2.7. Scheme of secondary oocyte structure.

Ribosomes and tRNA. The early embryo needs to make many of its own proteins, and in some species,
there is a burst of protein synthesis soon after fertilization. Protein synthesis is accomplished by ribosomes and
tRNA, which exist in the egg. The developing egg has special mechanisms to synthesize ribosomes, and certain
amphibian oocytes produce as many as 1012 ribosomes during their meiotic prophase.
Messenger RNA. In most organisms, the instructions for proteins made during early development are
already packaged in the oocyte. It is estimated that the eggs of sea urchins contain 25,000 to 50,000 different
types of mRNA. This mRNA, however, remains dormant until after fertilization.
Morphogenetic factors. Molecules that direct the differentiation of cells into certain cell types are
present in the egg. They appear to be localized in different regions of the egg and become segregated into
different cells during cleavage.
Protective chemicals. The embryo cannot run away from predators or move to a safer environment,
so it must come equipped to deal with threats. Many eggs contain ultraviolet filters and DNA repair enzymes
that protect them from sunlight; some eggs contain molecules that potential predators find distasteful; and the
yolk of bird eggs even contains antibodies. Within this enormous volume of cytoplasm resides a large nucleus.
Plasma membrane of oocyte must regulate the flow of certain ions during fertilization and must be
capable of fusing with the sperm plasma membrane. Outside the plasma membrane is the vitelline envelope
zona pellucida, which forms a essential for the species-specific binding of sperm. The human oocyte is also
surrounded by a layer of cells called the cumulus, which is made up of the ovarian follicular cells that were

54
nurturing the egg at the time of its release from the ovary. Human sperm have to get past these cells to fertilize
the egg. The innermost layer of cumulus cells, immediately adjacent to the zona pellucida, is called the corona
radiata.
The zona pellucida plays two major roles during fertilization: it binds the sperm, and it initiates the
acrosomal reaction after the sperm is bound. The binding of sperm to the zona is relatively, but not absolutely,
species-specific. Speciesspecific gamete recognition is not a major problem when fertilization occurs internally.
FERTILIZATION
Fertilization occurs in the ampullary region of the uterine tube. This is the widest part of the tube and is
close to the ovary. Spermatozoa may remain viable in the female reproductive tract for several days. Only 1%
of sperm deposited in the vagina enter the cervix, where they may survive for many hours.
There are 3 phases of fertilization (fig.3.7):
3
1
2

5c

1
1
4

5d

3
5b
5b

1
3
6
7
11

9
11

10
3
e

Fig. 3.7. Scheme of human fertilization.

1 - egg cell (secondary oocyte); 2 - uterine tube lumen; 3 - zona pellucida; 4 - corona radiata; 5a - sperm during
capacitation; 5b - acrosome reaction; 5c - penetration of zona pellucida, hit head and tail of spermatozoa in oocyte),
plasma membrane of spermatozoa remains outside oocyte surface; 5d - spermatozoon in the cytoplasm of oocyte;
e - formation of male pronucleus; f - pronucleuses merger; g-zygote, 1 division of cleavage; 6 - female pronucleus; 7 - male
pronucleus; 8 - chromosome of pronucleuses; 9 - mitotic spindle; 10 - reduce of sperm tail; 11 - polar body.

1. Distant phase - Action at a distance

2. Contact phase
3. Fusion of gametes - Syngamy.
Each phase is realized by different mechanisms and processes.
Action at a distance
This phase includes interaction between sperm in uterus and oocyte in ampula of uterine tube. Movement
of sperm from the cervix to the uterine tube occurs primarily by their own propulsion. The process of activation
of their activity and movement is known as capacitation.
Of the 280 106 human sperm normally ejaculated into the vagina, only about 200 reach the ampullary

55
region of the oviduct, where fertilization takes place. Since fewer than 1 in 10,000 sperm get close to the egg,
it is difficult to assay those molecules that might enable the sperm to swim toward the egg and become activated.
There is a great deal of controversy concerning the mechanisms underlying the translocation of mammalian
sperm to the oviduct, the possibility that the egg may be attracting the sperm through chemotaxis, and the
capacitation and hyperactivation reactions that appear necessary for some species sperm to bind with the egg/
Translocation and Capacitation
The reproductive tract of female mammals plays a very active role in the mammalian fertilization process.
While sperm motility is required for mouse sperm to encounter the egg once it is in the oviduct, sperm motility
is probably a minor factor in getting the sperm into the oviduct in the first place. Sperm are found in the oviducts
of humans within 30 minutes of sperm deposition in the vagina, a time too short to have been attained by even
the most Olympian sperm relying on their own flagellar power. Rather, the sperm appear to be transported to
the oviduct by the muscular activity of the uterus. By whatever means, mammalian sperm pass through the
uterus and oviduct, interacting with the cells and secretions of the female reproductive tract as they do so.
Newly ejaculated mammalian sperm are unable to undergo the acrosomal reaction without residing for
some time in the female reproductive tract. The set of physiological changes that allow the sperm to be competent
to fertilize the egg is called capacitation.
Capacitation is a period of conditioning in the female reproductive tract that in the human lasts
approximately 7 hours. Much of this conditioning, which occurs in the uterine tube, entails epithelial interactions
between the sperm and mucosal surface of the tube. During this time, a glycoprotein coat and seminal plasma
proteins are removed from the plasma membrane that overlies the acrosomal region of the spermatozoa. Only
capacitated sperm can pass through the corona cells and undergo the acrosome reaction.
Direction of sperm movement
Sperm are attracted toward eggs of their species by chemotaxis, that is, by following a gradient of a
chemical secreted by the oocyte Sperm-activating peptides cause dramatic and immediate increases in
mitochondrial respiration and sperm motility. The sperm receptor for resact is a transmembrane protein, and
when it binds resact on the extracellular side, a conformational change on the cytoplasmic side activates the
receptors enzymatic activity. This activates the mitochondrial ATP-generating apparatus as well as the dynein
ATPase that stimulates flagellar movement in the sperm
Contact phase Close interaction between oocyte and sperm
A second interaction between sperm and egg is realized by the acrosomal reaction. The acrosomal
reaction has two components: the fusion of the acrosomal vesicle with the sperm plasma membrane (an exocytosis
that results in the release of the contents of the acrosomal vesicle) and the extension of the acrosomal process.
The acrosomal reaction is initiated by contact of the sperm with the corona radiate. Contact with follicular
epithelial cell causes the exocytosis of the sperms acrosomal vesicle and the release of proteolytic enzymes
that can disintegrate the layer of epithelial cells and digest a path through the zona pellucida. The acrosomal
reaction is thought to be initiated by a fucose-containing polysaccharide and ZP3 in the Zona pellucida that
binds to the sperm and allows calcium to enter into the sperm head. The exocytosis of the acrosomal vesicle is
caused by the calcium-mediated fusion of the acrosomal membrane with the adjacent sperm plasma membrane.
The second part of the acrosomal reaction involves the extension of the acrosomal process. This protrusion
arises through the polymerization of globular actin molecules into actin filaments
Species-specific recognition and fusion of gamets
Recognition of sperm by the zona pellucida is followed by the lysis of that portion of the envelope or zona
in the region of the sperm head by the acrosomal enzymes. This lysis is followed by the fusion of the sperm
plasma membrane with the plasma membrane of the egg.
Sperm-egg binding appears to cause the extension of several microvilli to form the fertilization cone.
The initial adhesion of sperm to the oocyte is mediated in part by the interaction of integrins on the oocyte and
their ligands, disintegrins, on sperm. After adhesion, the plasma membranes of the sperm and egg fuse. In
mammals, the fertilin proteins in the sperm plasma membrane are essential for sperm membrane-egg membrane
fusion. fertilin appears to bind the sperm plasma membrane to the egg plasma membrane and then to fuse the
two of them together. Because the plasma membrane covering the acrosomal head cap disappears during the

56
acrosome reaction, actual fusion is accomplished between the oocyte membrane and the membrane that covers
the posterior region of the sperm head The sperm nucleus and tail pass through the resulting cytoplasmic
bridge, which is widened by the actin polymerization.
The prevention of polyspermy
As soon as one sperm has entered the egg, the fusibility of the egg membrane, which was so necessary
to get the sperm inside the egg, becomes a dangerous liability. In normal monospermy, in which only one
sperm enters the egg, a haploid sperm nucleus and a haploid egg nucleus combine to form the diploid nucleus of
the fertilized egg (zygote), thus restoring the chromosome number appropriate for the species. The centriole,
which is provided by the sperm, will divide to form the two poles of the mitotic spindle during cleavage.
The entrance of multiple sperm polyspermy leads to disastrous consequences in most organisms.
Fertilization by two sperm results in a triploid nucleus, in which each chromosome is represented three times
rather than twice. Worse, since each sperms centriole divides to form the two poles of a mitotic apparatus,
instead of a bipolar mitotic spindle separating the chromosomes into two cells, the triploid chromosomes may be
divided into as many as four cells. Because there is no mechanism to ensure that each of the four cells receives
the proper number and type of chromosomes, the chromosomes would be apportioned unequally. Some cells
receive extra copies of certain chromosomes and other cells lack them.
The most common way is to prevent the entry of more than one sperm into the egg. The human egg has
two mechanisms to avoid polyspermy: a fast reaction, accomplished by an electric change in the egg plasma
membrane, and a slower reaction, caused by the exocytosis of the cortical granules.
The eggs depolarization of plasma membrane induces the relies of cortical granules - cortical granule
reaction, which make a mechanical block to polyspermy Directly beneath the egg plasma membrane are about
15,000 cortical granules, each about 1 m in diameter. Upon sperm entry, these cortical granules fuse with the
egg plasma membrane and release their contents into the space between the plasma membrane and the fibrous
mat of vitelline envelope proteins.
Several proteins are released by this cortical granule exocytosis. The first are proteases. These enzymes
dissolve the protein posts that connect the vitelline envelope proteins to the cell membrane, and they clip off the
bindin receptor and any sperm attached to it. Mucopolysaccharides released by the cortical granules produce
an osmotic gradient that causes water to rush into the space between the plasma membrane and the vitelline
envelope, causing the envelope to expand and become the fertilization envelope. A third protein released by
the cortical granules, a peroxidase enzyme, hardens the fertilization envelope by crosslinking tyrosine residues
on adjacent proteins. Finally, a fourth cortical granule protein, hyalin, forms a coating around the egg. The egg
extends elongated microvilli whose tips attach to this hyaline layer. This layer provides support for the blastomeres
during cleavage.
Resumption of the second meiotic division.
The oocyte finishes its second meiotic division immediately after entry of the spermatozoon. One of the
daughter cells, which receives hardly any cytoplasm, is known as the second polar body; the other daughter cell
is the definitive oocyte. Its chromosomes (22 plus X) arrange themselves in a vesicular nucleus known as the
female pronucleus.
Metabolic activation of the egg.
The activating factor is probably carried by the spermatozoon. Postfusion activation may be considered
to encompass the initial cellular and molecular events associated with early embryogenesis.
The spermatozoon, meanwhile, moves forward until it lies close to the female pronucleus. Its nucleus
becomes swollen and forms the male pronucleus; the tail detaches and degenerates. Morphologically, the male
and female pronuclei are indistinguishable, and eventually, they come into close contact and lose their nuclear
envelopes. During growth of male and female pronuclei (both haploid), each pronucleus must replicate its
DNA. If it does not, each cell of the two-cell zygote has only half of the normal amount of DNA. Immediately
after DNA synthesis, chromosomes organize on the spindle in preparation for a normal mitotic division. The 23
maternal and 23 paternal (double) chromosomes split longitudinally at the centromere, and sister chromatids
move to opposite poles, providing each cell of the zygote with the normal diploid number of chromosomes and
DNA. As sister chromatids move to opposite poles, a deep furrow appears on the surface of the cell, gradually
dividing the cytoplasm into two parts.

57
The main results of fertilization are as follows:
Restoration of the diploid number of chromosomes, half from the father and half from the mother.
Hence, the zygote contains a new combination of chromosomes different from both parents.
Determination of the sex of the new individual. An X-carrying sperm produces a female (XX) embryo,
and a Y-carrying sperm produces a male (XY) embryo. Hence, the chromosomal sex of the embryo is determined
at fertilization.
Initiation of cleavage. Without fertilization, the oocyte usually degenerates 24 hours after ovulation.
Cleavage
After fertilization, the development of a multicellular organism proceeds by a process called cleavage, a
series of mitotic divisions whereby the enormous volume of egg cytoplasm is divided into numerous smaller,
nucleated cells (fig.4.7).

2
1

2
1

Fig. 4.7. Cleavage of human embryo: A - two blastomere stage; B - three-stage blastomere; C - four blastomere
stage; D - morula stage. Histological specimens, 500.
1 - blastomere, 2 - shell of fertilization.

These cleavage-stage cells are called blastomeres. In most species (mammals being the chief exception),
the rate of cell division and the placement of the blastomeres with respect to one another is completely under
the control of the proteins and mRNAs stored in the oocyte by the mother. The zygotic genome, transmitted by
mitosis to all the new cells, does not function in early-cleavage embryos. Few, if any, mRNAs are made until
relatively late in cleavage, and the embryo can divide properly even when chemicals are used experimentally to
inhibit transcription.
During cleavage, however, cytoplasmic volume does not increase. Rather, the enormous volume of
zygote cytoplasm is divided into increasingly smaller cells. First the egg is divided in half, then quarters, then

58
eighths, and so forth. This division of egg cytoplasm without increasing its volume is accomplished by abolishing
the growth period between cell divisions (that is, the G1 and G2 phases of the cell cycle).
The transition from fertilization to cleavage is caused by the activation of mitosis promoting factor
(MPF). MPF was first discovered as the major factor responsible for the resumption of meiotic cell divisions in
the ovulated frog egg. It continues to play a role after fertilization, regulating the biphasic cell cycle of early
blastomeres. Blastomeres generally progress through a cell cycle consisting of just two steps: M (mitosis) and
S (DNA synthesis)
Human eggs have sparse, equally spaced yolk and are thus isolecithal (Greek, equal yolk). In these
species, cleavage is holoblastic (Greek holos, complete). meaning that the cleavage furrow extends through
the entire egg. These embryos must have some other way of obtaining food. Most will generate a voracious
larval form, while mammals get their nutrition from the placent.
Characteristics of human cleavage:
- compleate
- irregular
- asynchronic
During cleavage two types of blastomeres are formed: light (small with hydratating cytoplasm rich in
lysosomes and fast division) and dark (biger and slowly dividing cells, which cytoplasm include numerous
ribosomes).
Until the eight-cell stage, they form a loosely arranged clump. After the third cleavage, blastomeres
maximize their contact with each other, forming a compact ball of cells held together by tight junctions. This
process, Compaction, segregates inner cells, which communicate extensively by gap junctions, from outer cells.
Approximately 3 days after fertilization, cells of the compacted embryo divide again to form a 16-cell morula
(mulberry). Inner cells of the morula constitute the Inner cell mass dark blastomeres, and surrounding cells
compose the Outer cell mass layer of light blastomeres. The inner cell mass gives rise to tissues of the
embryo proper and known as Embryoblast, and the outer cell mass forms the Trophoblast, which later contributes
to the placenta.
Blastocyst Formation (fig.5.7).

3
1

B
Fig. 5.7. Cleavage of human embryos. Cavitation, blastocyst stage:
A - 58-cell blastocyst (4-4,5 days of fetal development);
B - 107-cell blastocyst (5-5,5 days of prenatal development).
Histological specimens, 600.
1 - trophoblast; 2 - embrioblast; 3 - blastocyst cavity; 4 - shell fertilization.

About the time the morula enters the uterine cavity, fluid begins to penetrate through the zona pellucida
into the intercellular spaces of the inner cell mass. Gradually, the intercellular spaces become confluent, and

59
finally, a single cavity, the blastocele, forms. At this time, the embryo is a blastocyst. Cells of the inner cell
mass, now called the embryoblast, are at one pole, and those of the outer cell mass, or Trophoblast, flatten and
form the epithelial wall of the blastocyst.
Inner cell mass of the blastocyst is the source of embryonic stem cells (ES cells). Because these cells
are pluripotent and can form virtually any cell or tissue type, they have the potential for curing a variety of
diseases, including diabetes, Alzheimer and Parkinson diseases, anemias, spinal cord injuries, and many others.
Using animal models research with stem cells has been encouraging. For example, mouse ES cells in culture
have been induced to form insulin secreting cells, muscle and nerve stem cells, and glial cells. In whole animals,
ES cells have been used to alleviate the symptoms of Parkinson disease and to improve motor ability in rats with
spinal cord injuries.
ES cells may be obtained from embryos after in vitro fertilization, a process called reproductive cloning.
This approach has the disadvantage that the cells may cause immune rejection, since they would not be genetically
identical to their hosts. However, the cells could be modified to circumvent this problem. Another issue with this
approach is based on ethical considerations, since the cells are derived from fertilized viable embryos.

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LESSON 8
THEME: HUMAN EMBRYOLOGY. IMPLANTATION AND GASTRULATION.
BACKGROUND: A feature of the human embryo is the early separation and the development of
extraembryonic organs, which creates optimal conditions for the development of the embryonic material. Studies
of the mechanisms of formation of germ layers and their subsequent differentiation of embryonic induction
events, education, tissues and organs are needed for physicians to understand the principles of the morphogenesis
of organs and tissues, which determines their spatial parameters (size, shape, position), and structure. In addition,
fetal ontogeny and the processes of determination (genetically fixed properties), migration and differentiation
underlie the formation of neurohumoral imprinting, which determines the features of structural and functional
organization of organs and systems, the variability of phenotypic features, implementation details and limits of
adaptation and compensatory reactions.
AIM OF STUDY: (general): To interpret the phases and mechanisms of implantation and gastrulation,
their chronology, mechanisms of histo- and organogenesis for the interpretation of possible violations, abnomalies
and malformations of the embryo development in the process of further learning to solve practical problems of
medicine in the prevention and diagnosis of antenatal pathology.
TO ACHIEVE THIS AIM ONE HAS TO (practical skills):
1. Determine the phases of implantation.
2. Differentiate the phases of gastrulation. Recognize the morphological features of early and late gastrulation.
3. Interpret the essens and mechanisms of extraembryonic organs formation, their structure and function.
4. Interpret mechanisms of formation and the role of the primary strip and the primary node.
5. Interpret the essence, mechanisms, and morphological manifestations of the second phase of gastrulation in
human embryo.
TO ACHIEVE THE AIM IT IS NECESSARY TO CENTRE AROUND THE FOLLOWING POINTS:
1. Implantation. Location. Phases Mechanisms.
2. Changes in trophoblast during invasion.
3. Gastrulation: essence and mechanisms.
4. Early gastrulation. Mechanisms and result.
5. Late gastrulation. Mechanisms and chronology of the processes.
6. Formation of germ layers and notochord.
REFERENCES:
1. The developing human: clinically oriented embryology. Moore K.L., Persaud T.V.N. Saunders Elsvier, 8th
edition, 2008.
2. Conception of Human Anatomy and Physiology. Kent M., Van De Graaff, Stuart Ira Fox. Wm. C. Brown
Publishers, 1995.
WHAT MUST YOU KNOW? (INSTRUCTION FOR YOUR SELF-LEARNING)
Implantation
By 7 day after fertilization the zona pellucida of blastocyst has disappeared, allowing implantation to
begin (fig. 8.1).
Implantation includes 2 phases:
1. Adhesion
2. Invasion.

61
4
3

1
2

Fig. 8.1 Embryo during adhesion (A) and early invasive (B). Histological preparations,. 100.
1 - uterine cavity, 2 - endometrial epithelium, 3 - embrioblast 4 - trophoblast.

In the human, trophoblastic cells over the embryoblast pole begin to penetrate between the epithelial cells
of the uterine mucosa. New studies suggest that L-selectin on trophoblast cells and its carbohydrate receptors
on the uterine epithelium mediate initial attachment of the blastocyst to the uterus. Selectins are carbohydratebinding proteins involved in interactions between leukocytes and endothelial cells that allow leukocyte capture
from flowing blood. A similar mechanism is now proposed for capture of the blastocyst from the uterine
cavity by the uterine epithelium. Following capture by selectins, further attachment and invasion by the trophoblast
involve integrins, expressed by the trophoblast and the extracellular matrix molecules laminin and fibronectin.
Integrin receptors for laminin promote attachment, while those for fibronectin stimulate migration. These molecules
also interact along signal transduction pathways to regulate trophoblast differentiation, so that implantation is the result
of mutual trophoblastic and endometrial action. Hence, by the end of the first week of development, the human zygote
has passed through the morula and blastocyst stages and has begun implantation in the uterine mucosa.
Normally human blastocyst implants in the endometrium along the anterior or posterior wall of the body
of the uterus, where it becomes embedded between the openings of the glands.
After adhesion trophoblast cells produce hydrolytic enzymes, which destroy endometrium, and at the
eighth day of development, the blastocyst is partially embedded in the endometrial stroma. Trophoblast has
differentiated into two layers:
(a) an inner layer of mononucleated cells, the Cytotrophoblast, and
(b) an outer multinucleated zone without distinct cell boundaries, the Syncytiotrophoblast (fig. 8.2).
During the next days the blastocyst is more deeply embedded in the endometrium, and the penetration
defect in the surface epithelium is closed by a fibrin coagulum. The trophoblast shows considerable progress in
development, particularly at the embryonic pole, where vacuoles appear in the syncytium. When these vacuoles
fuse, they form large lacunae, and this phase of trophoblast development is known as the lacunar stage.
By the 11th to 12th day of development, the blastocyst is completely embedded in the endometrial stroma,
and the surface epithelium almost entirely covers the original defect in the uterine wall. Concurrently, cells of
the syncytiotrophoblast penetrate deeper into the stroma and erode the endothelial lining of the maternal
capillaries. These capillaries, which are congested and dilated, are known as sinusoids. The syncytial lacunae
become continuous with the sinusoids, and maternal blood enters the lacunar system. As the trophoblast continues
to erode more and more sinusoids, maternal blood begins to flow through the trophoblastic system, establishing
the uteroplacental circulation.

62

7 6

Fig. 8.2. Human embryo 7,5 days of development (implantation and the first phase of gastrulation).
Histological specimen, 250.
1 - epithelium of endometrium; 2 - the connective tissue of the mucous membrane of the uterus; 3 - trophoblast; 4 cytotrophoblast; 5 - symplastotrophoblast; 6 - hypoblast; 7 - epiblast; 8 - amnion cavity; 9 - blastocyst cavity.

By the 13th day of development, the surface defect in the endometrium has usually healed. Occasionally,
however, bleeding occurs at the implantation site as a result of increased blood flow into the lacunar spaces.
Because this bleeding occurs near the 28th day of the menstrual cycle, it may be confused with normal menstrual
bleeding and, therefore, may cause inaccuracy in determining the expected delivery date.
The trophoblast is characterized by villous structures. Cells of the cytotrophoblast proliferate locally and
penetrate into the syncytiotrophoblast, forming cellular columns surrounded by syncytium. Cellular columns
with the syncytial covering are known as primary villi.
The syncytiotrophoblast is responsible for hormone production, including human chorionic gonadotropin
(hCG). By the end of the second week, quantities of this hormone are sufficient to be detected by
radioimmunoassays, which serve as the basis for pregnancy testing.
Because 50% of the implanting embryos genome is derived from the father, it is a foreign body that
potentially should be rejected by the maternal system. Recent evidence suggests that a combination of factors
protects the conceptus, including production of immunosuppressive cytokines and proteins and the expression
of an unusual major histocompatibility complex class IB molecule (HLA-G) that blocks recognition of the
conceptus as foreign tissue. If the mother has an autoimmune disease, for example, systemic lupus erythematosus,
antibodies generated during the disease may attack the conceptus and destroy it.
Abnormal implantation sites sometimes occur within the uterus. Normally, the human blastocyst implants
along the anterior or posterior wall of the body of the uterus. Occasionally, the blastocyst implants close to the
internal opening os (opening) of the cervix, so that later in development, the placenta bridges the opening
(placenta previa) and causes severe, even life-threatening bleeding in the second part of pregnancy and during
delivery.
Occasionally, implantation takes place outside the uterus, resulting in extrauterine pregnancy, or ectopic
pregnancy. Ectopic pregnancies may occur at any place in the abdominal cavity, ovary, or uterine tube. However,
95% of ectopic pregnancies occur in the uterine tube, and most of these are in the ampulla. In the abdominal
cavity, the blastocyst most frequently attaches itself to the peritoneal lining of the rectouterine cavity, or Douglas
pouch. The blastocyst may also attach to the peritoneal covering of the intestinal tract or to the omentum.

63
Sometimes, the blastocyst develops in the ovary properly, causing a primary ovarian pregnancy. In most ectopic
pregnancies, the embryo dies about the second month of gestation, causing severe hemorrhaging and abdominal
pain in the mother.
GASTRULATION
Gastrulation is the process of highly coordinated cell and tissue movements during which the cells of
the blastocyst are dramatically rearranged. The blastocyst consists of numerous cells, the positions of which
were established during cleavage. During gastrulation, these cells are given new positions and new neighbors,
and the multilayered body plan of the organism is established.
Gastrulation occurs in the embryo in the course of its implantation and lasts for 7th - 17th days. There
are two stages of implantation:
1) early (7-13 days), which led to formation of extraembryonic organs (amnion, chorion and yolk
sack), and
2 late (14-17 days), resulting in three embryonic layers and notochord formation
Early gastrulation
Cells of the inner cell mass or embryoblast also differentiate into two layers (hyposlast and epiblast)
after delamination (fig. 8.3).
Delamination
of embryoblast

epiblast

hypoblast

B
amnion wall
amnion cavity

blastocyst
cavity

primary
yolk sac

Fig. 8.3. Scheme of early gastrulation (A-D)

.

Delamination means the tangential splitting of blastomeres into two layers:

(a) a layer of small cuboidal cells adjacent to the blastocyst cavity, known as the hypoblast layer; and
b) a layer of high columnar cells adjacent to the amniotic cavity, the epiblast layer.
Together, the layers form a flat disc. At the same time, a small cavity appears within the epiblast. This
cavity enlarges to become the amniotic cavity. Epiblast cells adjacent to the cytotrophoblast are called
amnioblasts and form extraembryonic ectoderm. At the bottom of amnion the rest epiblast is located.
Cells of hypoblast migrate down to blastocoele and form extraembryonic endoderm, which form the
lineng of yolk sack. Next cells from the periphery of epiblst migrate toward walls of amniotic and yolk bubbles
and trophoblast and make extraembrtyonic mesoderm.

64
By the 13 day there 3 extraembryonic organs (fig. 8.4).
1) amnion (extraembryonic Ectoderm and extraembryonic mesoderm) which produces amniotic fluid.
2) chorion (extraembryonic mesoderm and trophoblast) which in future participate in formation of placenta;
3) yolk sack (extraembryonic endoderm and extraembryonic mesoderm), which is the first hematopoietic
organ of embryo.

5
6
7
2
3
4
8
1
10
9

Fig. 8.4. Human embryo at the end of early gastrulation (13th day of development). Scheme.
1 - amniotic vesicle; 2 - yolk sac; 3 - hypoblast; 4 - epiblast; 5 - cytotrophoblast; 6 - symplastotrophoblast; 7 - extraembryonic
mesoderm; 8 - amniotic stem; 9 - the connective tissue of the mucous membrane of the uterus; 10 - lacunae with maternal
blood.

So the main result of early gastrulation is formation of extraembryonic organs which set conditions for
embryo body development.
At the end of the first phase of gastrulation the material of which the embryo is formed - embryonic disc
- is located at the bottom of the amniotic bubbles and formed by epiblast, which is material for formation of all
tissues and organs of the embryo.
The second phase of gastrulation begins at 14-days and finish at 17-days (fig. 8.5, 8.6). At the 14-15-th
day from the material of epiblast primitive streak is formed. This process is identical to that in birds and
mammals and is due to the gradient of proliferation and migration of cells from the brain to the caudal end of the
embryo. The meeting resulted in the lateral flow in the middle of epiblast cells formed thick primitive streak in
central part, which grows to the head end and ends with a thickening - the primary node. Primitive node is the
source of notochord and prenotochordal plate formation. Notochord if important inductor and determine the
axis of body.
The first wave of migrating cells from the front of the primitive streak is directed inwards to hypoblast
to form embryonic endoderm. They penetrate into the hypoblast, moving it outside. The next wave of cells
migration from the primitive streak move inside and in lateral directions, forming an embryonic mesoderm. The
rest of the epiblast form a germinal ectoderm.

65

Fig. 8.5. Formation of primary streak in

embryonic disc at 14-15 days of development.
Dorsal surface of epiblast.

5
1

1 - edge of amnion; 2 - hypoblast; 3 - epiblast;

4 - yolk sac wall; 5 - prenotochordal plate
area; 6 - primary streake.

6
2

Fig. 8.6. Formation of embryonic layers.

Transverse section through the cranial part
of the primary germ streake on 15th day
of development.

3
4

1 - epiblast, 2 - primary node; 3 - the primary

hole, 4 - primary streake, 5 - amnion,
6 - yolk sac wall, 7 - hipoblast; 8 - cell
migration and invagination of the primary
streake.

6
8

By the 17 th day of development, the last extraembryonic organ allantois - is formed (fig. 8.7). It grows
in the amniotic stalk from the back of the yolk sack. Allantois participate in umbilical cord vessels formation.
Thus, at the 17 th day of embryogenesis formed germ layers: ectoderm, mesoderm and endoderm, the most
important inducer of embryonic characteristic of all vertebrates - notohord, and allantois.
During gastrulation embryos must develop three very important axes that are the foundations of the
body: the anterior-posterior axis, the dorsalventral axis, and the right-left axis. The anterior-posterior (or
anteroposterior) axis is the line extending from head to tail (or mouth to anus in those organisms that lack
heads and tails). The dorsal-ventral (dorsoventral) axis is the line extending from back (dorsum) to belly
(ventrum). The right-left axis is a line between the two lateral sides of the body. Although we may look
symmetrical, recall that in most of us, the heart and liver are in the left half of the body only. Somehow, the
embryo knows that some organs go on one side and other organs go on the other.

66
1
10
7

11

12

10

8
3

2
1
7

13

8
2

6
3
1

2
4

1
3

Fig. 8.7. Development of extraemrionic human organs. Scheme.

1 - amnion; 2 - chorion: 2nd - smooth; 2b - villous; 3 - yolk sac; 4 - allantois; 5 - umbilical cord;
6 - archenteron; 7 - embryonic body; 8 - trunk folds; 9 - embrioblast; 10 - trophoblast;
11 - hypoblast; 12 - epiblast; 13 - extraembryonic mesoderm.

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LESSON 9
THEME: HUMAN EMBRYOLOGY. HISTO- AND ORGANOGENESIS.
Background:
Knowledge of the morphological and functional characteristics and regulatory
mechanisms of interaction in the system of mother-fetus is necessary for the formation of the theoretical
foundations of obstetrics and neonatology. Study of features of morphogenesis of various types of twins will
promote a holistic view about the course of multiple pregnancies and understanding of medical tactics of giving
birth.
AIM OF STUDY: (general): To be able to identify periods of embryogenesis, their patterns, the timing
and mechanisms of the processes of histo- and organogenesis for the interpretation of possible violations,
anomalies and malformations of the embryo in the process of further learning to solve practical problems of
medicine in the prevention and diagnosis of antenatal pathology.
TO ACHIEVE THIS AIM ONE HAS TO (practical skills):
1. Define germ layers and axial complex primordia, to trace their formation and fate.
2. Determine extraembryonic organs: chorion, amnion, yolk sac, allantois, and their development and significance.
3. Interpret the general patterns of histo-, organo-and systemogenesis a human embryo.
4. Interpret the essence of critical periods of human embryogenesis and their importance, principles of
developmental abnormalities, the role of mother-fetus in determining the development of organs and systems.
TO ACHIEVE THE AIM IT IS NECESSARY TO CENTRE AROUND THE FOLLOWING POINTS
1. Axial organs complex formation. Embryonic induction.
2. Neurulation - mechanisms, stages, the effects of developmental disorders.
3. Presomitic and somitic periods, their characteristics.
4. Differentiation of germ layers, histo- and organogenesis.
5. Critical periods of embryogenesis. Factors influencing the development of the embryo.
6. General patterns and chronology of the development agencies.
7. Placentation, the formation of mother-fetus.
8. Features of dizygotic and monozygotic twins.
REFERENCES:
1. The developing human: clinically oriented embryology. Moore K.L., Persaud T.V.N. Saunders Elsvier, 8th
edition, 2008.
2. Conception of Human Anatomy and Physiology. Kent M., Van De Graaff, Stuart Ira Fox. Wm. C. Brown
Publishers, 1995.
WHAT MUST YOU KNOW? (INSTRUCTION FOR YOUR SELF-LEARNING)
The embryonic period, or period of organogenesis, occurs from the third to the eighth weeks of development
and is the time when each of the three germ layers, ectoderm, mesoderm, and endoderm, gives rise to a number
of specific tissues and organs. By the end of the embryonic period the main organ systems have been established,
rendering the major features of the external body form recognizable by the end of the second month.
Derivatives of the Ectodermal Germ Layer
On the third week of development (from 17-18-days) a new stage of embryo development begins
formation of tissues, organs and systems. Firstly the structures of the nervous system are formed by the
process of neurulation (fig. 9.1). It is accompanied by differentiation of germ layers, and the key event is the
formation of the axial complex of organs. They are: notohord, neural tube and paraaxial mesoderm - somites.
The formation of the axial complex organs primordia is based on the principles of embryonic induction.

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1

3
7

II
7

6
9

21

20

20

III
7

22

10

23

21

25

16

24

13
15

12
11

IV

22
14

17

19

18

Fig. 9.1. Formation and differentiation of neural tube and neural crest: A - view from the back; B,
C - cross-sections (B - scheme; - histological specimen). I - formation of neural plate; II formation of neural groove; III - formation of neural tube; IV - differentiation of neural tube and
neural crest.
1 - nervous plate; 2 - notochordal plate; 3 - nervous sulcus; 4 - nervous rollers; 5 - skin ectoderm; 6 - notochord; 7 somites; 8 - neural tube; 9 - nerve crest; 10 - mesenchyme; 11 - neuroepithelial layer; 12 - the mantle layer; 13 - marginal
zone; 14 - ventral root; 15 - dorsal root; 16 - germ of spinal ganglion; 17 - germ of paravertebral autonomic ganglion;18
- aorta; 19 - coelomic epithelium; 20 - nephrogonotom; 21 - anterior neuropore; 22 - posterior neuropore; 23 - the brain;
24 - spinal cord; 25 - spinal canal.

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Notohord induces the development of the neural tube, whereas the neural tube is an inducer of
differentiation and segmentation of the mesoderm.
At the beginning of the third week of development, the ectodermal germ layer has the shape of a disc
that is broader in the cephalic than in the caudal region. Appearance of the notochord and prechordal mesoderm
induces the overlying ectoderm to thicken and form the neural plate. Cells of the plate form the neuroectoderm,
and their induction represents the initial event in the process of Neurulation.
Neurulation - the process of neural tube formation, which determines the early development of the
nervous system and is accompanied by the sequential formation of these structures - neural plate, neural
groove, neural tube and crest.
Once induction has occurred, the elongated, slipper-shaped neural plate gradually expands toward the
primitive streak. By the end of the third week, the lateral edges of the neural plate become more elevated to
form neural folds, and the depressed midregion forms the neural groove. Gradually, the neural folds approach
each other in the midline, where they fuse. Fusion begins in the cervical region (fifth somite) and proceeds
cranially and caudally. As a result, the neural tube is formed. Until fusion is complete, the cephalic and caudal
ends of the neural tube communicate with the amniotic cavity by way of the cranial and caudal neuropores,
respectively. Closure of the cranial neuropore occurs at approximately 25th day (18- to 20-somite stage),
whereas the posterior neuropore closes at 27th day (25-somite stage). Neurulation is then complete, and the
central nervous system is represented by a closed tubular structure with a narrow caudal portion, the spinal
cord, and a much broader cephalic portion characterized by a number of dilations, the brain vesicles.
As the neural folds elevate and fuse, cells at the lateral border or crest of the neuroectoderm begin to
dissociate from their neighbours. This cell population is the neural crest. Crest cells from the trunk region leave
the neural folds after closure of the neural tube and migrate along one of two pathways:
(1) a dorsal pathway through the dermis, where they will enter the ectoderm through holes in the basal
lamina to form melanocytes in the skin and hair follicles, and
(2) a ventral pathway through the anterior half of each somite to become sensory ganglia, sympathetic
and enteric neurons, Schwann cells, and cells of the adrenal medulla.
Neural crest cells also form and migrate from cranial neural folds, leaving the neural tube before closure
in this region. These cells contribute to the craniofacial skeleton, as well as neurons for cranial ganglia, glial
cells, melanocytes, and other cell types.
Induction of neural crest cells requires an interaction between adjacent neural and overlying ectoderm. A
gradient of Bone morphogenetic proteins (BMPs), secreted by nonneural ectoderm, together with FGFs, initiates
the induction process. Thus, the fate of the entire ectoderm is dependent upon BMP concentrations: high levels
result in epidermis formation, lower levels at the border of the neural plate and nonneural ectoderm induce
formation of the neural crest, and BMP inhibition contributes to neural plate induction. Crest cells give rise to a
heterogeneous array of tissues
In general terms, the ectodermal germ layer gives rise to organs and structures that maintain contact
with the outside world:
(a) the central nervous system;
(b) the peripheral nervous system;
(c) the sensory epithelium of the ear, nose, and eye; and
(d) the epidermis, including the hair and nails. In addition, it gives rise to subcutaneous glands, the mammary
glands, the pituitary gland, and enamel of teeth.
At the 17-20-th day the separation of embryo body from extraembryonic material begins by the trunk
folds formation.
Derivatives of the Mesodermal Germ Layer
Formation of the neural tube determines the differentiation of the mesoderm. Initially, cells of the
mesodermal germ layer form a thin sheet of loosely woven tissue on each side of the midline. By approximately
the 17th day, however, cells close to the midline that form the nerve tube proliferate and form a thickened plate

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of tissue known as Paraxial mesoderm. These areas of mesoderm fall into segmentation separated into
metamerically located areas - primary segments, or somites (fig. 9.2).
5
4

B
2
3

6
5
4
7

D
6
Fig. 9.2. Stages of somites development (A - D).

1 - nervous sulcus; 2 - ventral part of somite; 3 - notochord; 4 - sclerotome; 5 - neural tube; 6 - dorsal aorta; 7 - dermatome;
8 - myotome.

By the number of somites the time of embryo development can be determined. Segmentation of the
mesoderm is gradual and begins at the cranial end of the body, spreading in the caudal direction. On average,
some 2 or 3 somites are formed every day. Somites appear on the 20 th day of embryogenesis, beginning with
the third pair. 22-23rd day embryo already have 7 pairs of somites, the 25-day embryo - 17-18 pairs, by the 30th day, there are 30 pairs of them, and by the 35-th day - 43-44. The second, and then the first pair of somites
develops with a delay, their material goes to the development of the eye muscles.
Three zones can be distinguished in somites. Ventromedial region of somite is the sclerotome. It is close
to notohord and in the future sclerotome forms axial skeleton - cartilage and bone tissue. From dorsolateral part
of somite lying close to the skin ectoderm dermatome developes, which forms connective tissue of the skin.
Part of the somite that lies between dermatomes and sclerotome goes to the development of myotomes - the
germ of skeletal muscle. Myotomes cells rapidly divide, stretch in length and convert into myoblasts that fuse to
form muscle fibers of striated skeletal muscle tissue. To the lateral side of somites there are partially segmented
regions of intermediate mesoderm - nefrogonotomes, which form the structure of kidneys, gonads and sperm
paths.
Lateral parts of mesoderm remain non-segmented and give rise to splanhnotomes. Splanhnotome is
stratified into 2 sheets - parietal (somatopleure), which is adjacent to the cutaneous ectoderm and the visceral

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(splanhnopleure), which is bordered by the endoderm. Cavity located between them is a secondary body cavity
- coelom. Thereafter, it is subdivided into pleural, pericardial and abdominal cavities.
The parietal splanhnotome sheet (somatopleure) differentiates into the serous membranes, which cover
the chest, abdominal and pericardial cavities - the parietal pleura, peritoneum, pericardium. Visceral sheet
(splanhnopleure) is a part of the internal organs such as visceral pleura, visceral peritoneum, epicardium.
Splanhnotome develops into coelomic epithelium and mesenchyme. Coelomic epithelium is involved in the
formation of the mesothelium, which covers the serous membranes of adrenal cortex, supporting cells of the
testis and follicular epithelium of the ovary. Splanhnotome mesenchyme is the source of connective tissue,
smooth muscle, myeloid and lymphoid tissues, blood vessels and microglia. Splanhnopleure is also a source of
myoepicardial plate from which striated cardiac muscle tissue is formed.
The separation of the body of the embryo from extraembryonic material develops, as mentioned earlier,
with the help of the trunk folds, which grow under the body of the embryo toward each other from the 20-th day
of development, turning the body into the tube.
Derivatives of Endoderm
With the help of the trunk folds embryonic entoderm plunges into the body of the embryo, forming the
primitive gut. Its cranial part is formed by prenotochordial plate, which is formed from the material of the
primary node. Prenotochordial plate participates in the formation of the coating and glandular epithelia of the
anterior gut tube (oral cavity, pharynx, esophagus, salivary glands) and respiratory system (from trachea to
pulmonary alveoli). In the mouth the boundary between the ectoderm derivates and prenotochordial plate ends
approximately at the line of teeth. Endoderm and adjacent to it splanhnopleure form the gut tube, which is a
source of digestive organs. Endoderm differentiates into covering and glandular epithelium of the stomach,
intestines, liver and pancreas.
Separation of the embryo with the trunk folds leads to the formation of the umbilical cord (fig. 9.3).
2

7
6

9
11

6
10
2

7
8

12
6

Fig. 9.3. Scheme of structure of umbilical cord of human embryo: A - primary structure of the embryo umbilical
ring 5 weeks of embryonic development; B - primary umbilical cord 10 weeks of embryonic development; C cross-section of the umbilical cord structures; D - cross-section of primary umbilical cord loops with bowel.
1 - amnion; 2 - chorion; 3 - horion plate; 4 - yolk sac vessels; 5 - yolk sac; 6 - connecting stalk; 7 - intestine loop; 8 amnion cavity; 9 - allantois; 10 - abdominal wall of the embryo; 11 - primary umbilical ring; 12 - umbilical vessels.

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Apart from allantois it includes and reduced yolk sac, which on the surface is covered with amniotic
membrane. Further, in umbilical cord umbilical arteries and vein pass. Umbilical arteries are branches of fetal
descending aorta and carry venous blood to the chorion. Umbilical vein carries arterial blood from placenta to
fetus body.
Development of congenital anomalies may not only be the result or manifestation of genetic defects and
diseases, but also the result of the influence of harmful factors. The nature of the violation of bodies depends on
the timing of the action of harmful factors and the leading mechanism of morphogenesis of the body. For
example, one embodiment of developmental abnormalities of organs (kidney, liver, heart, etc.) is agenesis or
hypoplasia which develops due to violation of the inductive effects of germs. Breach of the merger process
leads to disrafii (anomalies of the face, partition walls of the heart), the violation of the reduction - to preserve
the extra structure and formation of fistulas or conservation bridges between the fingers.

CONTENTS

1. Methods of histological investigation...............................................................................1

2. Lesson 1. General organization of cell. Plasma membrane............................................... 2
3. Lesson 2.Cell. Cytoplasm. Hyaloplasm, organelles.........................................................10
4. Lesson 3. Cell inclusions................................................................................................20
5. Lesson 4. Cell nucleus...................................................................................................24
6. Lesson 5. Cell cycle and replication. Cell differentiation. Cell death.................................31
7. Lesson 6. Embryology of birds and mammals.................................................................42
8. Lesson 7. Human embryology. Fertilisation. Cleavage.....................................................51
9. Lesson 8. Human embryology. Implantation. Gastrulation................................................60
10. Lesson 9. Human embryology. Histo- and organogenesis................................................67