NEHRU ARTS AND SCIENCE COLLEGE
DEPARTMENT OF COMPUTER SCIENCE
E-LEARNING
CLASS
SUBJECT
: III B Sc Biotechnology
: CORE PAPER: IX ANIMAL BIOTECHNOLOGY
UNIT IV
Embyology: Collection and preservation of embryo, culture of embryos, culture of
embryonic stem cells and its applications. Gametogenesis and fertilization in animals,
Molecular events during fertilization, genetic regulations in embryonic development.
Part – A
1. Embyology
It is a science which is about the development of an embryo from the fertilization of the
ovum to the fetus stage. After cleavage, the dividing cells, or morula, becomes a hollow
ball, or blastula, which develops a hole or pore at one end.
Embryos (and one tadpole) of the wrinkled frog (Rana rugosa)
2. Embryo
An embryo is a multicellular diploid eukaryote in its earliest stage of development, from
the time of first cell division until birth, hatching, or germination. In humans, it is called
an embryo until about eight weeks after fertilization (i.e. ten weeks LMP), and from then
it is instead called a fetus.
3. Embryogenesis
The development of the embryo is called embryogenesis. In organisms that reproduce
sexually, once a sperm fertilizes an egg cell, the result is a cell called the zygote that has
half of the DNA of each of two parents.
4. Gastrulation
It is a phase early in the embryonic development of most animals, during which the
single-layered blastula is reorganized into a trilaminar ("three-layered") structure known
as the gastrula. These three germ layers are known as the ectoderm, mesoderm, and
endoderm.
5. Blastula
The blastula (from Greek βλαστός (blastos), meaning "sprout") is a solid sphere of cells
formed during an early stage of embryonic development in animals. The blastula is
created when the zygote undergoes the cell division process known as cleavage. The
blastula is proceeded by the morula and precedes the gastrula in the developmental
sequence.
6. Collection of embryos from mouse
Collection of embryos from mouse:

Swabbing the abdomen.

Tearing the skin to expose the abdominal wall.

Opening the abdomen.

The uterus in situ.

Removing the uterus.

Removing the membranes.

Collecting the embryos.
7. Embryonic stem cells
Embryonic stem cells (ES cells) are pluripotent stem cells derived from the inner cell
mass of the blastocyst, an early-stage embryo. Human embryos reach the blastocyst stage
4–5 days post fertilization, at which time they consist of 50–150 cells.
8. Properties of Embryonic stem cells
Pluripotency, and ability to replicate indefinitely.
9. Gametogenesis
Gametogenesis is a biological process by which diploid or haploid precursor cells
undergo cell division and differentiation to form mature haploid gametes. Depending on
the biological life cycle of the organism, gametogenesis occurs by meiotic division of
diploid gametocytes into various gametes, or by mitotic division of haploid
gametogenous cells.
10. Fertilization
Fertilisation (also known as conception, fecundation and syngamy) is the fusion of
gametes to produce a new organism. In animals, the process involves the fusion of an
ovum with a sperm, which eventually leads to the development of an embryo.
11. Polyspermy
Polyspermy is the condition when multiple sperm fuse with a single egg. This results in
duplications of genetic material.
12. Activation of the ovum includes the following events:

Cortical reaction to block against other sperm cells

Activation of egg metabolism

Reactivation of meiosis

DNA synthesis
13. In vitro fertilization
In vitro fertilization (IVF) is a process by which egg cells are fertilized by sperm outside
the body, in vitro. IVF is a major treatment in infertility when other methods of assisted
reproductive technology have failed. The process involves hormonally controlling the
ovulatory process, removing ova (eggs) from the woman's ovaries and letting sperm
fertilise them in a fluid medium. The fertilized egg (zygote) is then transferred to the
patient's uterus with the intent to establish a successful pregnancy.
Part – B
1. Collection of embryos from mouse
Collection of embryos from mouse:

Swabbing the abdomen.

Tearing the skin to expose the abdominal wall.

Opening the abdomen.

The uterus in situ.

Removing the uterus.

Removing the membranes.

Collecting the embryos.
2. Preservation of embryo
Preservation of embryo: Cryogenic storage at very low temperatures is presumed to
provide an indefinite, if not near infinite, longevity to cells although the actual “shelf life”
is rather difficult to prove. Cryopreservation is a process where cells or whole tissues
are preserved by cooling to low sub-zero temperatures, such as (typically) 77 K or −196
°C (the boiling point of liquid nitrogen). At these low temperatures, any biological
activity, including the biochemical reactions that would lead to cell death, is effectively
stopped. Embryos that are 2, 4 or 8 cells when frozen. Human Oocyte cryopreservation is
a new technology in which a woman’s eggs (oocytes) are extracted, frozen and stored.
Later, when she is ready to become pregnant, the eggs can be thawed, fertilized, and
transferred to the uterus as embryos. Cryopreservation for embryos are used for embryo
storage, e.g. when in vitro fertilization has resulted in more embryos than is currently
needed.
Pregnancies have been reported from embryos stored for 16 years. Many studies have
evaluated the children born from frozen embryos, or “frosties”. The result has uniformly
been positive with no increase in birth defects or development abnormalities.
3. Gametogenesis
Gametogenesis is a biological process by which diploid or haploid precursor cells
undergo cell division and differentiation to form mature haploid gametes. Depending on
the biological life cycle of the organism, gametogenesis occurs by meiotic division of
diploid gametocytes into various gametes, or by mitotic division of haploid
gametogenous cells.
Scheme showing analogies in the process of maturation of the ovum and the development of the
spermatids, following their individual pathways. The oocytes and spermatocytes are both
gametocytes. Ova and spermatids are complete gametes. In reality, the first polar body typically
dies without dividing.
Animals produce gametes directly through meiosis in organs called gonads. Males and
females of a species that reproduces sexually have different forms of gametogenesis:

spermatogenesis (male)

oogenesis (female)
Stages
However, before turning into gametogonia, the embryonic development of gametes is the
same in males and females.
Common path
Gametogonia are usually seen as the initial stage of gametogenesis. However,
gametogonia are themselves successors of primordial germ cells. During early embryonic
development, primordial germ cells (PGCs) from the dorsal endoderm of the yolk sac
migrate along the hindgut to the gonadal ridge. They multiply by mitosis and once they
have reached the gonadal ridge in the late embryonic stage, they are called gametogonia.
Gametogonia are no longer the same between males and females.
Individual path
From gametogonia, male and female gametes develop differently - males by
spermatogenesis and females by oogenesis. However, by convention, the following
pattern is common for both:
Cell type
ploidy/chromosomes chromatids
gametogonium
diploid/46
Process
2N before replication, gametocytogenesis
4N after it
(mitosis)
primary
diploid/46
gametocyte
secondary
2N before replication, gametidogenesis
4N after it
haploid/23
2N
gametid
haploid/23
1N
gamete
haploid/23
1N
gametocyte
(meiosis 1)
gametidogenesis
(meiosis 2)
In gametangia
Fungi, algae and primitive plants form specialized haploid structures called gametangia
where gametes are produced through mitosis. In some fungi, for example zygomycota,
the gametangia are single cells on the end of hyphae and acting as gametes by fusing into
a zygote. More typically, gametangia are multicellular structures that differentiate into
male and female organs:

antheridium (male)

archegonium (female)
4. Egg activation
Oocyte (or ovum/egg)
activation is a series
of processes that occur
in the oocyte after
fertilisation.
Sperm entry causes
calcium release into
the
oocyte.
mammals,
this
In
has
been proposed to be caused by the introduction of phospholipase C isoform zeta (PLCζ)
from the sperm cytoplasm, although this remains to be established definitively.
Activation of the ovum includes the following events:

Cortical reaction to block against other sperm cells

Activation of egg metabolism

Reactivation of meiosis

DNA synthesis
The sperm may trigger egg activation via the interaction between a sperm protein and an
egg surface receptor. It is possible that a receptor is activated by the sperm binding which
activates a tyrosine kinase which then activates phospholipase C (PLC). The inositol
signaling system has been implicated as the pathway involved with egg activation. IP3
and DAG are produced from the cleavage of PIP2 by phospholipase C. However, another
hypothesis is that a soluble 'sperm factor' diffuses from the sperm into the egg cytosol
upon sperm-oocyte fusion. The results of this interaction could activate a signal
transduction pathway that uses second messengers. A novel PLC isoform, PLCζ, may be
the equivalent of the mammalian sperm factor. A recent paper shows that mammaliam
sperm contain PLC zeta which can start the signaling cascade.
Polyspermy is the condition when multiple sperm fuse with a single egg. This results in
duplications of genetic material. In sea urchins, the block to polyspermy comes from two
mechanisms: the fast block and the slow block. The fast block is an electrical block to
polyspermy. The resting potential of an egg is -70mV. After contact with sperm, an
influx of sodium ions increases the potential up to +20mV. The slow block is through a
biochemical mechanism triggered by a wave of calcium increase. The rise of calcium is
both necessary and sufficient to trigger the slow block. In the cortical reaction, cortical
granules directly beneath the plasma membrane are released into the space between the
plasma membrane and the vitelline membrane (the perivitelline space). An increase in
calcium triggers this release. The contents of the granules contain proteases,
mucopolysaccharides, hyalin, and peroxidases. The proteases cleave the bridges
connecting the plasma membrane and the vitelline membrane and cleave the bindin to
release the sperm. The mucopolysaccharides attract water to raise the vitelline membrane.
The hyalin forms a layer adjacent to the plasma membrane and the peroxidases cross-link
the protein in the vitelline membrane to harden it and make it impenetrable to sperm.
Through these molecules the vitelline membrane is transformed into the fertilization
membrane or fertilization envelope. In mice, the zona reaction is the equivalent to the
cortical reaction in sea urchins. The terminal sugars from ZP3 are cleaved to release the
sperm and prevent new binding.
5. Fate Map
Fate mapping is a technique that is used to show how a cell or tissue moves and what it
will become during normal development. Fate mapping was developed by Walter Vogt as
a means by which to trace the development of specific regions of the early embryo. To do
this, Vogt used agar chips impregnated with vital dyes.
A fate map is a representation of the developmental history of each cell in the body of an
adult organism. Thus, a fate map traces the products of each mitosis from the singlecelled zygote to the multi-celled adult. The process of fate mapping was developed by
Walter Vogt.
The fate map of vulval development in C. elegans has been completely characterized at a
molecular level. In an adult C. elegans, the vulva is the egg-laying organ that consists of
only 22 cells. The differentiation and division of these Px.p cells is dictated by the anchor
cell through a morphogen gradient of LIN-3. Mapping each cell's fate was accomplished
by studying mutants and through tissue grafts.
Part – C
1. Collection and preservation of embryo
Collection of embryos from mouse:

Swabbing the abdomen.

Tearing the skin to expose the abdominal wall.

Opening the abdomen.

The uterus in situ.

Removing the uterus.

Removing the membranes.

Collecting the embryos.
Preservation of embryo: Cryogenic storage at very low temperatures is presumed to
provide an indefinite, if not near infinite, longevity to cells although the actual “shelf life”
is rather difficult to prove. Cryopreservation is a process where cells or whole tissues
are preserved by cooling to low sub-zero temperatures, such as (typically) 77 K or −196
°C (the boiling point of liquid nitrogen). At these low temperatures, any biological
activity, including the biochemical reactions that would lead to cell death, is effectively
stopped. Embryos that are 2, 4 or 8 cells when frozen. Human Oocyte cryopreservation is
a new technology in which a woman’s eggs (oocytes) are extracted, frozen and stored.
Later, when she is ready to become pregnant, the eggs can be thawed, fertilized, and
transferred to the uterus as embryos. Cryopreservation for embryos are used for embryo
storage, e.g. when in vitro fertilization has resulted in more embryos than is currently
needed.
Pregnancies have been reported from embryos stored for 16 years. Many studies have
evaluated the children born from frozen embryos, or “frosties”. The result has uniformly
been positive with no increase in birth defects or development abnormalities.
2.
2. Cleavage and Blastulation
Cleavage is the process after fertilization when early mitotic cell divisions occur that
progressively reduce cell size.

During cleavage, the total embryonic mass, however, remains constant.

In mammals, when the embryo has about 16 cells, its individual cells begin to
adhere to one another and it coalesces to form into a morula.
A blastocyst (blastocyst cavity) forms in the morula when it enters into the uterus.
This cavitation is an important transition from homogeneous cells to differentiated cell
function.
This new structure is called a blastocyst which consists of an outer layer, the trophoblast,
and an inner cluster of cells referred to as the inner cell mass.

Implantation is the process in which the blastocyst attaches to and penetrates into
the uterine wall.
Upon contact with the uterine lining or endometrium during implantation, the trophoblast
cells invade the uterine lining to give the embryo access to the deeper layers of the
uterine wall.
These trophoblast cells differentiate into two new cell types referred to as
syncytiotrophoblasts and cytotrophoblasts.

The syncytiotrophoblasts continue to grow but without cell division and begin to
fuse.

The cytotrophoblasts remain distinct and invade deeper into the uterine wall.

The egg is fertilized in the ampulla of the fallopian tube or first third of the oviduct.

The zygote undergoes a series of cleavages until it forms the blastocyst at the time
of implantation and invades the endometrium.

During the cleavage process there is no increase in cell volume and the zygote
cytoplasm is divided into increasingly smaller cells.
o
This is accomplished by abolishing the growth period between cell divisions.
o
In other words, there is no G1 or G2 phase of the cell cycle in this case.
o
The cells continue dividing without growth at a very rapid rate and this cleavage
ends at the mid-blastula transition at about the time of implantation.
At this point G1 and G2 are again added to the cell cycle and the cells begin to grow and
embryo volume increases.
There are several different types of cleavage patterns which is determined by the
amount and distribution of yolk protein in the cytoplasm and factors influencing the
mitotic spindle.

When one pole of the egg is yolk-free, the cellular divisions occur there at a faster
rate than at the opposite pole.

The pole with the lesser yolk concentration is the animal pole and the pole with the
greater yolk concentration is the vegetal pole.
The zygote nucleus is usually located in the animal pole and the yolk in the vegetal pole
tends to inhibit cleavage.
The influence of yolk is a major factor in the type of cleavage that is seen in different
species.
Two major types of cleavage are seen and referred to as holoblastic (or complete
cleavage) and meroblastic (or incomplete cleavage).

Holoblastic cleavage occurs in mammals and cleavage occurs throughout the entire
egg due to the presence of little yolk.

In organisms such as birds there is a large accumulation of yolk and cleavage
occurs primarily in the animal pole of the blasomere (meroblastic cleavage).

As examples of this, the frog embryo undergoes holoblastic cleavage with divisions
occurring throughout the developing embryo.

However, in zebrafish, meroblastic cleavage occurs and cell cleavage is initially
confined to the animal (or top) half of the embryo.
The symmetry of cleavage is further divided into subtypes such as radial or spiral,
depending on the position of the yolk. < of symmetry types basic some are>
o
The simplest pattern such as occurs in the sea urchin is radial cleavage.
Here successive symmetric cleavages divide the embryo into equal-sized cells.
o
However, in flatworms, the divisions are unequal and the first cleavage of the egg
produces two cells of unequal size.
o
Spiral cleavage is yet another symmetry of cell divisions that occurs in mollusks
and roundworms.
In this case there is also unequal cleavage but the cells arrange in different planes within
the embryo which appears as a spiral formation.
In mammals we see a unique type of cleavage process in the early embryo.

The eggs of mammals are among the smallest in the animal kingdom and cleavage
occurs very slow taking about 12-24 hours.

They also undergo what is referred to a rotational cleavage.
o
In the first division the cells divide in half with the plane from top to bottom.
o
However, in the second cleavage, one of the two blastomeres divides the same as
the first cleavage and the other divides at the equator.
o
This is referred to as rotational cleavage and is unique to mammals.

Also unique to mammalian cleavage is that the cells do not always divide at the
same time producing the 2, 4, or 8 cell stages but sometimes divide at different
times so that odd numbers of cells may be present such as a 5-cell embryo.

One of the most important differences that distinguishes mammalian cell cleavage
from those of other organisms is the process of compaction.
o
Up until the 8-cell stage the blastomeres are loosely arranged and have plenty of
space between them.
o
After the third cleavage, the blastomeres tighten greatly to form a compacted
structure.
o
These changes are the result of changes in cadherin which concentrates at regions of
intracellular contact and now acts for the first time as an adhesion molecule.
o
This process of compaction is where the cells at the 8-cell stage are smooth and
during compaction the cells increase their contact with one another, flatten, and
have more microvilli on their surface.
This increase in microvilli is caused by the contraction of actin filaments drawing the
cortical elements to the surface.

The cells of the compacted embryo divide to produce a 16-cell morula.
These cells are divided into internal and external cells.

The external cells of the morula become the trophoblast cells (trophoderm) that do
not produce any cell of the embryo but are necessary for implantation of the embryo
into the uterine wall.

The trophoblast cells eventually produce the chorion or the embryonic portion of
the placenta which provides oxygen and nourishment from the mother.

The trophoderm also secretes hormones that will regulate the mother's immune
system preventing immune rejection of the new embryo.

The inner cells of the morula eventually form the embryo itself.
The cells of the inner cell mass form a separate group consisting of about 13 cells by
the time the embryo reaches the 64-cell stage (sixth division).

This distinction between the trophoblast and inner cell mass represents the first
differentiation event in mammalian development.
The morula at the compacted stage does not have an internal cavity.

During the process of cavitation the trophoblast cells secrete a fluid into the morula
to produce the blastocoel.

The inner cell mass is positioned on one side of what is now termed the blastocyst
and the trophoblast cells line the cavity.

The position of the cells in the morula either in the internal or external portion of
the cell mass is the major determinant of whether a particular cell will become a
trophoblast or an embryo.
The blastocyst expands within the zona pellucida (which is the extracellular matrix
of the egg) as it travels through the fallopian tubes.

This expansion is caused by a sodium pump in the cell membranes of the
trophoblast cells.
Proteins in the cell membrane pump sodium into the central cavity which draws water in
osmotically.

Eventually the blastocyst will secrete a protease (strypsin) and lyse the components
of the zona pellucida to make direct contact with the uterus.
The trophoblast cells bind to the uterine cavity and secrete proteases enabling the
blastocyst to bury itself within the uterine wall.
3. Embryo culture
Oocyte Wash Buffer
On the day of egg retrieval (Day 0), this buffer is used for the retrieval of the eggs from
the ovary. Oocyte wash buffer has an ingredient, which prevents a change in pH when the
solution is exposed to air during the retrieval. The eggs are very susceptible to any minute
changes in the pH of their environment. The eggs are washed in this buffer and then
placed into the next medium for culture.
Fertilization Medium
After the wash at retrieval, the eggs are put into the fertilization medium. This medium
contains a variety of salts, sugars, amino acids, protein and other nutrients essential for
the maintenance of the egg (and sperm in IVF) during the process of fertilization (IVF
and ICSI). The fertilization medium and all of the other subsequent culture media, are
buffered with the appropriate components in order to maintain the correct pH of the
solution in the embryo incubator.
Cleavage medium
All of the eggs which undergo normal fertilization are next placed into cleavage medium,
which is formulated specifically to support the growth requirements of the early cleavage
stage embryo. The cleaving (dividing) embryo is cultured in this medium until Day 3. If
the embryo transfer is scheduled for Day 3, the embryos are transferred to the uterus in a
small amount of this medium.
Blastocyst medium
Embryos, that are to be cultured until Day 5 or 6, are placed, later on Day 3, into another
medium referred to as blastocyst medium. The embryos are then maintained in this
medium until embryo transfer on Day 5 or embryo cryopreservation on Day 5 or 6. This
medium has additional components and/or different components required by the embryo
in its transition from a cleavage stage embryo to a blastocyst. If the embryo transfer is
scheduled on Day 5, the embryos are transferred to the uterus in a small amount of this
medium.
Sperm Buffer
The sperm buffer is formulated in order to maintain the correct pH when the solution is
exposed to air. This buffer is used during the preparation of semen samples and solutions
for semen samples, which will be washed and processed outside of the incubator.
Sperm Medium
The sperm medium is similar to the Sperm Buffer except that the buffer is such that the
correct pH of the solution is maintained whilst in the incubator. This medium is important
for the final resuspension of sperm to be used in IVF because the process of fertilization
occurs inside the incubator.
THE EMBRYO CULTURE EQUIPMENT
The Laminar Flow Hood
The preparation of all media and solutions to be used in IVF, ICSI and IUI occurs inside
this specialized hood, which blows air out towards the embryologist. The air is filtered
and the outflow of air prevents any contaminants from blowing in and contaminating the
solutions and embryo dishes being prepared. Preparation of semen samples to be used in
IVF, ICSI and IUI also occurs in this sterile environment.
The Preparation Incubator
All dishes and solutions to be used for an IVF, ICSI or IUI treatment are maintained in
this incubator until use. The incubator is sterile inside, is at 37°C, has a carbon dioxide
concentration of 6.0%, and the environment is fully humidified to prevent any
evapouration. All solutions and dishes to be used for treatment are equilibrated in this
incubator for a minimum of 4 hours before use.
Embryo Culture Incubator
All eggs and embryos are incubated here throughout their time in the VFC laboratory.
The unit is infused with the proper levels of oxygen and carbon dioxide to ensure that the
eggs/embryos are maintained under optimum conditions at all times. The environment in
the incubator is also humidified and kept at 37°C. The temperature and gas levels are
monitored continuously and the incubator is attached to a telephone based alarm system
which will call out to the embryologist during off hours should an unsuitable or
emergency condition arise.
IVF Chamber
Whenever the eggs and embryos need to be outside of the incubator for any reason, they
are handled in our IVF Chamber. The chamber looks like an isolate that you would see in
a special care newborn nursery in the hospital. This chamber however is specially
modified and adapted for the purpose of maintaining eggs and embryos under optimum
conditions even when they are being handled outside of the incubator.
4. Gastrulation
Gastrulation of a diploblast: The
formation of germ layers from a (1)
blastula to a (2) gastrula. Some of the
ectoderm cells (orange) move inward
forming the endoderm (red).
Gastrulation is a phase early in the
development of most animal embryos, during which the morphology of the embryo is
reorganized to form the three germ layers: ectoderm, mesoderm, and endoderm. The
molecular mechanism and timing of gastrulation is different in different organisms.
Gastrulation is followed by organogenesis, when individual organs develop within the
newly formed germ layers.
Development
Gastrulation creates the three embryonic germ layers: the ectoderm, mesoderm, and
endoderm. Each layer gives rise to specific tissues and organs in the developing embryo.


The ectoderm gives rise to:
o
epidermis structures such as the skin, nails, and hair
o
neural crest and neural tissues, which give rise to the nervous system
The mesoderm is found between the ectoderm and the endoderm and gives rise to:
o
somites, which form muscle, the cartilage of the ribs and vertebrae, and
the dermis

o
notochord
o
blood and blood vessels
o
bone and connective tissue
The endoderm gives rise to:
o
epithelium of the digestive system and respiratory system
o
organs associated with the digestive system, such as the liver and pancreas
The embryo must have the correct amount of each germ layer, which must be properly
oriented within the embryo for the organs to develop correctly. Thus, gastrulation must
be tightly regulated for proper embryo development.
Vertebrates
Mice
In mice, gastrulation occurs after implantation of the embryo, on day 6 of mouse
embryogenesis (E6).
Gastrulation occurs in a series of steps:

the embryo becomes asymmetric

the primitive streak forms

cells from the epiblast at the primitive streak undergo a epithelial to mesenchymal
transition and ingress to at the primitive streak to form the germ layers
Loss of Symmetry
In preparation for gastrulation, the embryo must become asymmetric along both the
proximal-distal axis and the anterior-posterior axis. The proximal-distal axis is formed
when the cells of the embryo form the “egg cylinder,” which consists of the
extraembryonic tissues, which give rise to structures like the placenta, at the proximal
end and the epiblast at the distal end. Many signaling pathways contribute to this
reorganization, including BMP, FGF, nodal, and Wnt. Visceral endoderm surrounds the
epiblast. The distal visceral endoderm (DVE) migrates to the anterior portion of the
embryo, forming the “anterior visceral endoderm” (AVE). This breaks anterior-posterior
symmetry and is regulated by nodal signaling.
Epithelial to Mesenchmyal Cell Transition – loss of
cell adhesion leads to constriction and extrusion of
newly mesenchymal cell.
Formation of the Primitive Streak
The primitive streak is formed at the beginning of gastrulation and is found at the
junction between the extraembryonic tissue and the epiblast on the posterior side of the
embryo and the site of ingression.[3] Formation of the primitive streak is reliant upon
nodal signaling[1] within the cells contributing to the primitive streak and BMP4 signaling
from the extraembryonic tissue.[3] Furthermore, Cer1 and Lefty1 restrict the primitive
streak to the appropriate location by antagonizing nodal signaling. The region defined as
the primitive streak continues to grow towards the distal tip.
Epithelial to Mesenchymal Transition and Ingression
In order for the cells to move from the epithelium of the epiblast through the primitive
streak to form a new layer, the cells must undergo an epithelial to mesenchymal transition
(EMT) to lose their epithelial characteristics, such as cell-cell adhesion. FGF signaling is
necessary for proper EMT. FGFR1 is needed for the up regulation of Snai1, which down
regulates E-cadherin, causing a loss of cell adhesion. Following the EMT, the cells
ingress through the primitive streak and spread out to form a new layer of cells or join
existing layers. FGF8 is implicated in the process of this dispersal from the primitive
streak.
Birds
After cleavage, the blastoderm of chick embryos that sits above the yolk secretes fluid
basally into the space between the yolk and the blastoderm called the subgerminal space.
The region of the blastoderm above the subgerminal space is called the area pellucida.
The region of the blastoderm above the yolk is the area opaca. The region where these
two zones meet is called the marginal zone. At the posterior marginal zone (PMZ), there
is a condensation of cells that is important in gastrulation. Within the PMZ, there is
another thickening of cells called the Koller's sickle. Before gastrulation begins, the
blastoderm forms two layers: the epiblast and the hypoblast. The epiblast gives rise to the
embryo and some of the extraembryonic structures while the hypoblast contributes
entirely to the extraembryonic membranes. The hypoblast comes from the primary
hypoblast which delaminate out of the epiblast. This structure is equivalent to the
organizer in amphibians and the embryonic shield in fish. Cells ingress through the
primitive groove into the blastocoel cavity, migrate anteriorly through Hensen's node and
then laterally through the rest of the groove. Cells that are fated to become the endoderm
migrate to the bottom of the cavity and replace the hypoblast cells. Cells that are fated to
become mesoderm remain in between the future endoderm cells and the epiblast and the
epiblast cells remain to become ectodermal cells. The ectoderm, however, is undergoing
epiboly to surround the yolk mass. The cells at the edge of the area opaca send out long
filopida that attach to fibronectin in the vitelline membrane surrounding the embryo and
yolk mass and pull the ectodermal cells toward the vegetal pole.
As gastrulation proceeds, the primitive streak regresses posteriorly with pharyngeal
endoderm, the head process, and the notochord being laid down as it recedes. This results
in a temporal gradient of development with the anterior forming organs while the
posterior is still going through gastrulation.
Amphibians
During cleavage in amphibians, a higher density of yolk in the vegetal half of the embryo
results in the blastocoel cavity being placed asymmetrically in the animal half of the
embryo. Unlike in sea urchins, the cells surrounding the blastocoel are thicker than a
monolayer. The blastocoel cavity prevents signaling between the animal cap and provides
a space for involuting cells during gastrulation.
There are four kinds of tissue movements that drive gastrulation in Xenopus:
invagination, involution, convergent extension and epiboly. At the vegetal edge of the
dorsal marginal zone, cells change from a columnar shape to become a bottle cell and
drive invagination. At this invagination, cells begin to involute into the embryo. This
initial site of involution is called the dorsal lip. The involuting cells migrate along the
inside of the blastocoel toward the animal cap. This migration is mediated by fibronectin
of the extracellular matrix (ECM) assembled by the blastocoel roof. Eventually, cells
from the lateral and ventral sides begin to involute to form a ring of involuting cells
surrounding the yolk plug. These involuting cells will eventually form the archenteron
which displaces and eventually replaces the blastocoel. Cells from the lateral marginal
zone intercalate with cells closer to the dorsal midline. Directed cell intercalation within
the dorsal mesoderm drives convergent extension. The dorsal cells become the first to
migrate along the roof of the blastocoel cavity and form the anterior/posterior axis of the
embryo. Both prior to and during the involution, the animal cap undergoes epiboly and
spread toward the vegetal pole.
Fish
At the time of mid-blastula transition, the zebrafish embryo is composed of three distinct
cell layers: the enveloping layer (EVL), deep cells, and the yolk syncytial layer (YSL)
formed from the fusion of cells adjacent to the yolk cells.
The first stage of gastrulation begins with the epiboly of the EVL and the deep cells over
the YSL. This epiboly is driven by the migration of nuclei and cytoplasm in the YSL and
attachments between the YSL and the EVL. Intercalation of the deep cells with the EVL
help drive this movement. At about 50% of epiboly, a fate map similar to that of the
Xenopus can be derived. The EVL develops into an extraembryonic membrane and does
not contribute to the embryo.
The second stage of gastrulation occurs when the leading edge of the epibolizing
blastoderm thickens. The dorsal side forms a larger thickening and is known as the
embryonic shield. The deep cells in the embryonic shield form two layers. The epiblast
forms near the surface and will give rise to the ectoderm. The hypoblast forms next to the
YSL and will form a mixture of endoderm and mesoderm. The hypoblast is formed
through involution and/or ingression. The movement of cells in the hypoblast are similar
to the involuting mesoderm of amphibians. The end result of gastrulation is an
asymmetric involution of cells that form the dorsal structures of the embryo.
Invertebrates
Sea urchins
The following description concerns gastrulation in echinoderms, representative of the
triploblasts, or animals with three embryonic germ layers.
Sea urchins deviate from simple cleavage at the fourth cleavage. The four vegetal
blastomeres divide unequally to produce four micromeres at the vegetal pole and four
macromeres in the middle of the embryo. The animal cells divide meridionally and
produce mesomeres.
At the beginning of vertebrate gastrulation, the embryo is a hollow ball of cells known as
the blastula, with an animal pole and a vegetal pole. The vegetal pole begins to flatten to
form the vegetal plate. Some of the cells of the vegetal pole detach and through
ingression become primary mesenchyme cells. The mesenchyme cells divide rapidly and
migrate along the extracellular matrix (basal lamina) to different parts of the blastocoel.
The migration is believed to be dependent upon sulfated proteoglycans on the surface of
the cells and molecules on the basal lamina such as fibronectin. The cells move by
forming filopodia that identify the specific target location. These filopodia then organize
into syncytial cables that deposit the calcium carbonate that makes up the spicules (the
skeleton of the pluteus larva).
During the second phase of gastrulation, the vegetal plate invaginates into the interior,
replacing the blastocoelic cavity and thereby forming a new cavity, the archenteron
(literally: primitive gut), the opening into which is the blastopore. The arechenteron is
elongated by three mechanisms.
First, the initial invagination is caused by a differential expansion of the inner layer made
of fibropellins and outer layer made of hyalin to cause the layers to bend inward.
Second, the archenteron is formed through convergent extension. Convergent extension
results when cells intercalate to narrow the tissue and move it forward.
Third, secondary mesenchyme pull the tip of the archenteron towards the animal pole.
Secondary mesenchyme are formed from cells that ingress from, but remain attached to,
the roof of the archenteron. These cells extend filopodia that use guidance cues to find the
future mouth region. Upon reaching the target site, the cells contract to pull the
archenteron to fuse with the ectoderm. Once the archenteron reaches the animal pole, a
perforation forms, and the archenteron becomes a digestive tract passing all the way
through the embryo.
The three embryonic germ layers have now formed. The endoderm, consisting of the
archenteron, will develop into the digestive tract. The ectoderm, consisting of the cells on
the outside of the gastrula that played little part in gastrulation, will develop into the skin
and the central nervous system. The mesoderm, consisting of the mesenchyme cells that
have proliferated in the blastocoel, will become all the other internal organs.
5. Embryonic Stem Cells
Embryonic stem cells (ES cells) are pluripotent stem cells derived from the inner cell
mass of the blastocyst, an early-stage embryo.[1] Human embryos reach the blastocyst
stage 4–5 days post fertilization, at which time they consist of 50–150 cells. Isolating the
embryoblast or inner cell mass (ICM) results in destruction of the fertilized human
embryo, which raises ethical issues.
Embryonic stem cells are distinguished by two distinctive properties:

their pluripotency, and

their ability to replicate indefinitely
ES cells are pluripotent, that is, they are able to differentiate into all derivatives of the
three primary germ layers: ectoderm, endoderm, and mesoderm. These include each of
the more than 220 cell types in the adult body. Pluripotency distinguishes embryonic
stem cells from adult stem cells found in adults; while embryonic stem cells can generate
all cell types in the body, adult stem cells are multipotent and can only produce a limited
number of cell types.
Additionally, under defined conditions, embryonic stem cells are capable of propagating
themselves indefinitely. This allows embryonic stem cells to be employed as useful tools
for both research and regenerative medicine, because they can produce limitless numbers
of themselves for continued research or clinical use.
Because of their plasticity and potentially unlimited capacity for self-renewal, ES cell
therapies have been proposed for regenerative medicine and tissue replacement after
injury or disease. Diseases that could potentially be treated by pluripotent stem cells
include a number of blood and immune-system related genetic diseases, cancers, and
disorders; juvenile diabetes; Parkinson's; blindness and spinal cord injuries. Besides the
ethical concerns of stem cell therapy (see stem cell controversy), there is a technical
problem of graft-versus-host disease associated with allogeneic stem cell transplantation.
However, these problems associated with histocompatibility may be solved using
autologous donor adult stem cells, therapeutic cloning, stem cell banks or more recently
by reprogramming of somatic cells with defined factors (e.g. induced pluripotent stem
cells). Other potential uses of embryonic stem cells include investigation of early human
development, study of genetic disease and as in vitro systems for toxicology testing.
6. Fertilisation in animals
The mechanics behind fertilisation has been studied extensively in sea urchins and mice.
This research addresses the question of how the sperm and the appropriate egg find each
other and the question of how only one sperm gets into the egg and delivers its contents.
There are three steps to fertilisation that ensure species-specificity:
1. Chemotaxis
2. Sperm activation/acrosomal reaction
3. Sperm/egg adhesion
Internal vs. external
Consideration as to whether an animal (more specifically a vertebrate) uses internal or
external fertilisation is often dependent on the method of birth. Oviparous animals laying
eggs with thick calcium shells, such as chickens, or thick leathery shells generally
reproduce via internal fertilisation so that the sperm fertilise the egg without having to
pass through the thick, protective, tertiary layer of the egg. Ovoviviparous and
euviviparous animals also use internal fertilisation. It is important to note that although
some organisms reproduce via amplexus, they may still use internal fertilisation, as with
some salamanders. Advantages to internal fertilisation include: minimal waste of
gametes; greater chance of individual egg fertilisation, relatively "longer" time period of
egg protection, and selective fertilisation; many females have the ability to store sperm
for extended periods of time and can fertilise their eggs at their own desire.
Oviparous animals producing eggs with thin tertiary membranes or no membranes at all,
on the other hand, use external fertilisation methods. Advantages to external fertilisation
include: minimal contact and transmission of bodily fluids; decreasing the risk of disease
transmission, and greater genetic variation (especially during broadcast spawning
external fertilisation methods).
Sea urchins
Acrosome reaction on a sea urchin cell.
Chemotaxis was discovered as the method by which sperm find the eggs. This
chemotaxis is an example of a ligand/receptor interaction. Resact is a 14 amino acid
peptide purified from the jelly coat of A. punctulata that attracts the migration of sperm.
After finding the egg, the sperm gets through the jelly coat through a process called
sperm activation. In another ligand/receptor interaction, an oligosaccharide component of
the egg binds and activates a receptor on the sperm and causes the acrosomal reaction.
The acrosomal vesicles of the sperm fuse with the plasma membrane and are released. In
this process, molecules bound to the acrosomal vesicle membrane, such as bindin, are
exposed on the surface of the sperm. These contents digest the jelly coat and eventually
the vitelline membrane. In addition to the release of acrosomal vesicles, there is explosive
polymerization of actin to form a thin spike at the head of the sperm called the acrosomal
process.
The sperm binds to the egg through another ligand reaction between receptors on the
vitelline membrane. The sperm surface protein bindin, binds to a receptor on the vitelline
membrane identified as EBR1.
Fusion of the plasma membranes of the sperm and egg are likely mediated by bindin. At
the site of contact, fusion causes the formation of a fertilisation cone.
Mammals
Usually mammals rely on internal fertilisation through copulation. After a male
ejaculates, a large number of sperm cells move to the upper vagina (via contractions from
the vagina) through the cervix and across the length of the uterus toward the ovum. The
capacitated spermatozoon and the oocyte meet and interact in the ampulla of the fallopian
tube. Thermotactic and chemotactic gradients are involved in sperm guiding towards the
egg cell, at least during the final stage of sperm migration. Spermatozoa have been shown
to respond to the temperature gradient of ~2°C between the oviduct and the ampulla, and
chemotactic gradients of Progesterone have been confirmed as the signal emanating from
the cumulus oophorus cells surrounding rabbit and human oocytes. Capacitated and
hyperactivated sperm cells respond to these gradients by changing their behaviour and
moving towards the cumulus-oocyte complex. Other chemotactic signals like formyl
Met-Leu-Phe (fMLF) may also guide spermatozoa.
The zona pellucida of the egg binds with the sperm. In contrast to sea urchins, the sperm
binds to the egg before the acrosomal reaction. The zona pellucida is a thick layer of
extracellular matrix that surrounds the egg and is similar to the role of the vitelline
membrane in sea urchins. A glycoprotein in the zona pellucida, ZP3 was discovered to be
responsible for egg/sperm adhesion in mice. The receptor galactosyltransferase (GalT)
binds to the N-acetylglucosamine residues on the ZP3 and is important for binding with
the sperm and activating the acrosome reaction. ZP3 is sufficient for sperm/egg binding
but not necessary. There are two additional sperm receptors: a 250kD protein that binds
to an oviduct secreted protein and SED1 which binds independently to the zona. After the
acrosome reaction, it is believed that the sperm remains bound to the zona pellucida
through exposed ZP2 receptors. These receptors are unknown in mice but have been
identified in guinea pigs.
In mammals, binding of the spermatozoon to the GalT initiates the acrosome reaction.
This process releases the enzyme hyaluronidase, which digests the matrix of hyaluronic
acid in the vestments surrounding the oocyte. Fusion between the oocyte plasma
membranes and sperm follows, allowing the entry of the sperm nucleus, centriole and
flagellum, but not the mitochondria, into the oocyte. The fusion is likely mediated by the
protein CD9 in mice (the binding homolog). The egg "activates" itself upon fusing with a
single sperm cell, thereby changing its cell membrane to prevent fusion with other sperm.
This process ultimately leads to the formation of a diploid cell called a zygote. The
zygote begins to divide and form a blastocyst and when it reaches the uterus, it performs
implantation in the endometrium. At this point the female's pregnancy has begun. If the
embryo implants in any tissue other than the uterine wall, an ectopic pregnancy results,
which can be fatal to the mother.
In some animals (e.g. rabbits) the act of coitus induces ovulation by stimulating release of
the pituitary hormone gonadotropin. This greatly increases the probability that coitus will
result in pregnancy.
Humans
The term conception commonly refers to fertilisation, the successful fusion of gametes to
form a new organism. 'Conception' is used by some to refer to implantation and is thus a
subject of semantic arguments about the beginning of pregnancy, typically in the context
of the abortion debate. Gastrulation, which occurs around 16 days after fertilisation, is the
point in development when the implanted blastocyst develops three germ layers, the
endoderm, the ectoderm and the mesoderm. It is at this point that the genetic code of the
father becomes fully involved in the development of the embryo. Until this point in
development, twinning is possible. Additionally, interspecies hybrids survive only until
gastrulation, and have no chance of development afterward. However this stance is not
entirely accepted as some human developmental biology literature refers to the
"conceptus" and such medical literature refers to the "products of conception" as the postimplantation embryo and its surrounding membranes. The term "conception" is not
usually used in scientific literature because of its variable definition and connotation.
7. In vitro fertilization
In vitro fertilization (IVF) is a process by which egg cells are fertilized by sperm outside
the body, in vitro. IVF is a major treatment in infertility when other methods of assisted
reproductive technology have failed. The process involves hormonally controlling the
ovulatory process, removing ova (eggs) from the woman's ovaries and letting sperm
fertilise them in a fluid medium. The fertilized egg (zygote) is then transferred to the
patient's uterus with the intent to establish a successful pregnancy. The first successful
birth of a "test tube baby", Louise Brown, occurred in 1978. Robert G. Edwards, the
doctor who developed the treatment, was awarded the Nobel Prize in Physiology or
Medicine in 2010. Before that, there was a transient biochemical pregnancy reported by
Australian Foxton School researchers in 1953 and an ectopic pregnancy reported by
Steptoe and Edwards in 1976. At the same time, Subash Mukhopadyay, a relatively
unknown physician from Kolkata, India was performing experiments on his own with
primitive instruments and a house hold refrigerator and this resulted in a test tube baby,
later named as "Durga" (alias Kanupriya Agarwal) who was born on October 3, 1978.[1]
The term in vitro, from the Latin root meaning in glass, is used, because early biological
experiments involving cultivation of tissues outside the living organism from which they
came, were carried out in glass containers such as beakers, test tubes, or petri dishes.
Today, the term in vitro is used to refer to any biological procedure that is performed
outside the organism it would normally be occurring in, to distinguish it from an in vivo
procedure, where the tissue remains inside the living organism within which it is
normally found. A colloquial term for babies conceived as the result of IVF, "test tube
babies", refers to the tube-shaped containers of glass or plastic resin, called test tubes,
that are commonly used in chemistry labs and biology labs. However, in vitro fertilisation
is usually performed in the shallower containers called Petri dishes. One IVF method,
Autologous Endometrial Coculture, is actually performed on organic material, but is still
considered in vitro.
Theoretically, in vitro fertilization could be performed by aspirating contents from a
woman's fallopian tubes or uterus with a plastic catheter after natural ovulation, mix it
with semen from a man and reinsert into the uterus. However, without additional
techniques, the chances of pregnancy would be extremely small. Such additional
techniques that are routinely used in IVF include ovarian hyperstimulation to retrieve
multiple eggs, ultrasound-guided transvaginal oocyte retrieval directly from the ovaries,
egg and sperm preparation, as well as culture and selection of resultant embryos.
Ovarian hyperstimulation
There are two main protocols for stimulating the ovaries for IVF treatment. The long
protocol involves downregulation (suppression or exhaustion) of the pituitary ovarian
axis by the prolonged use of a GnRH antagonist. Stimulation of the ovaries using a
gonadotrophin starts once the process of downregualtion is complete generally after 10 to
14 days.
The short protocol consist of a regimen of fertility medications to stimulate the
development of multiple follicles of the ovaries. In most patients, injectable
gonadotropins (usually FSH analogues) are used under close monitoring. Such
monitoring frequently checks the estradiol level and, by means of gynecologic
ultrasonography, follicular growth. Typically approximately 10 days of injections will be
necessary. Spontaneous ovulation during the cycle is typically prevented by the use of
GnRH agonists that are started prior or at the time of stimulation or GnRH antagonists
that are used just during the last days of stimulation; both agents block the natural surge
of luteinising hormone (LH) and allow the physician to start the ovulation process by
using medication, usually injectable human chorionic gonadotropins. Ovarian stimulation
carries the risk of excessive or hyperstimulation. This complication is life-threatening and
ovarian stimulation using gonadotrophins must only be carried out under strict medical
supervision
Egg retrieval
When follicular maturation is judged to be adequate, human chorionic gonadotropin
(hCG) is given. Commonly, this is known as the "trigger shot." This agent, which acts as
an analogue of luteinising hormone, makes the follicles perform their final maturation,
and would cause ovulation about 42 hours after injection, but a retrieval procedure takes
place just prior to that, in order to recover the egg cells from the ovary.[3] The eggs are
retrieved from the patient using a transvaginal technique (transvaginal oocyte retrieval)
involving an ultrasound-guided needle piercing the vaginal wall to reach the ovaries.
Through this needle follicles can be aspirated, and the follicular fluid is handed to the
IVF laboratory to identify ova. It is common to remove between ten and thirty eggs. The
retrieval procedure takes about 20 minutes and is usually done under conscious sedation
or general anaesthesia.
Egg and sperm preparation
In the laboratory, the identified eggs are stripped of surrounding cells and prepared for
fertilisation. An oocyte selection may be performed prior to fertilisation to select eggs
with optimial chances of successful pregnancy. In the meantime, semen is prepared for
fertilisation by removing inactive cells and seminal fluid in a process called sperm
washing. If semen is being provided by a sperm donor, it will usually have been prepared
for treatment before being frozen and quarantined, and it will be thawed ready for use.
Fertilization
The sperm and the egg are incubated together at a ratio of about 75,000:1 in the culture
media for about 18 hours. In most cases, the egg will be fertilised by that time and the
fertilised egg will show two pronuclei. In certain situations, such as low sperm count or
motility, a single sperm may be injected directly into the egg using intracytoplasmic
sperm injection (ICSI). The fertilised egg is passed to a special growth medium and left
for about 48 hours until the egg consists of six to eight cells.
In gamete intrafallopian transfer, eggs are removed from the woman and placed in one of
the fallopian tubes, along with the man's sperm. This allows fertilisation to take place
inside the woman's body. Therefore, this variation is actually an in vivo fertilisation, not
an in vitro fertilisation.
Embryo culture
Typically, embryos are cultured until having reached the 6–8 cell stage three days after
retrieval. In many Canadian, American and Australian programmes, however, embryos
are placed into an extended culture system with a transfer done at the blastocyst stage at
around five days after retrieval, especially if many good-quality embryos are still
available on day 3. Blastocyst stage transfers have been shown to result in higher
pregnancy rates. In Europe, transfers after 2 days are common.
Culture of embryos can either be performed in an artificial culture medium or in an
autologous endometrial coculture (on top of a layer of cells from the woman's own
uterine lining). With artificial culture medium, there can either be the same culture
medium throughout the period, or a sequential system can be used, in which the embryo
is sequentially placed in different media. For example, when culturing to the blastocyst
stage, one medium may be used for culture to day 3, and a second medium is used for
culture thereafter. Single or sequential medium are equally effective for the culture of
human embryos to the blastocyst stage. Artificial embryo culture media basically contain
glucose, pyruvate, and energy-providing components, but addition of amino acids,
nucleotides, vitamins, and cholesterol improve the performance of embryonic growth and
development.
Embryo selection
Laboratories have developed grading methods to judge oocyte and embryo quality. In
order to optimise pregnancy rates, there is significant evidence that a morphological
scoring system is the best strategy for the selection of embryos. However, presence of
soluble HLA-G might be considered as a second parameter if a choice has to be made
between embryos of morphologically equal quality. Also, two-pronuclear zygotes (2PN)
transitioning through 1PN or 3PN states tend to develop into poorer-quality embryos than
those who constantly remain 2PN. In addition to tests that optimise pregnancy chances,
Preimplantation genetic diagnosis (PGD) or screening may be performed prior to transfer
in order to avoid inheritable diseases. Methods are emerging in making comprehensive
analyses of transcriptomes of embryos in order to assess embryo quality.
Embryo transfer
Embryos are graded by the embryologist based on the number of cells, evenness of
growth and degree of fragmentation. The number to be transferred depends on the
number available, the age of the woman and other health and diagnostic factors. In
countries such as Canada, the UK, Australia and New Zealand, a maximum of two
embryos are transferred except in unusual circumstances. In the UK and according to
HFEA regulations, a woman over 40 may have up to three embryos transferred, whereas
in the USA, younger women may have many embryos transferred based on individual
fertility diagnosis. Most clinics and country regulatory bodies seek to minimise the risk of
pregnancies carrying multiples. As it is not uncommon for more implantations to take
than desired, the next step faced by the expectant mother is that of selective abortion. The
embryos judged to be the "best" are transferred to the patient's uterus through a thin,
plastic catheter, which goes through her vagina and cervix. Several embryos may be
passed into the uterus to improve chances of implantation and pregnancy.
8. Embryonic stem cell culture
Techniques and Conditions for Embryonic Stem Cell Derivation and Culture
Embryonic stem cells are derived from the inner cell mass of the early embryo, which are
harvested from the donor mother animal. Martin Evans and Matthew Kaufman reported a
technique that delays embryo implantation, allowing the inner cell mass to increase. This
process includes removing the donor mother’s ovaries and dosing her with progesterone,
changing the hormone environment, which causes the embryos to remain free in the
uterus. After 4–6 days of this intrauterine culture, the embryos are harvested and grown
in in vitro culture until the inner cell mass forms “egg cylinder-like structures,” which are
dissociated into single cells, and plated on fibroblasts treated with mitomycin-c (to
prevent fibroblast mitosis). Clonal cell lines are created by growing up a single cell.
Evans and Kaufman showed that the cells grown out from these cultures could form
teratomas and embryoid bodies, and differentiate in vitro, which all indicate the cells are
pluripotent.
Gail Martin derived and cultured her ES cells differently. She removed the embryos from
the donor mother at approximately 76 hours after copulation and cultured them overnight
in media containing serum. The following day, she removed the inner cell mass from the
late blastocyst using microsurgery. The extracted inner cell mass was cultured on
fibroblasts treated with mitomycin-c in media that containing serum and was conditioned
by EC cells. After approximately one week, colonies of cells grew out. These cells grew
in culture and demonstrated pluripotent characteristics, as demonstrated by the ability to
form teratomas, differentiate in vitro, and form embryoid bodies. Martin referred to these
cells as ES cells.
It is now known that the feeder cells provide leukemic inhibitory factor (LIF) and serum
provides bone morphogenetic proteins (BMPs) that are necessary to prevent ES cells
from differentiating. These factors are extremely important for the efficiency of deriving
ES cells. Furthermore, it has been demonstrated that different mouse strains have
different efficiencies for isolating ES cells. Current uses for mouse ES cells include the
generation of transgenic mice, including knockout mice. For human treatment, there is a
need for patient specific pluripotent cells. Generation of human ES cells is more difficult
and faces ethical issues. So, in addition to human ES cell research, many groups are
focused on the generation of induced pluripotent stem cells (iPS cells).
GAMETOGENESIS
Spermatogenesis is the process by which male spermatogonia develop into mature
spermatozoa. Spermatozoa are the mature male gametes in many sexually reproducing
organisms. Thus, spermatogenesis is the male version of gametogenesis. In mammals it
occurs in the male testes and epididymis in a stepwise fashion, and for humans takes
approximately 64 days.[1] Spermatogenesis is highly dependent upon optimal conditions
for the process to occur correctly, and is essential for sexual reproduction. It starts at
puberty and usually continues uninterrupted until death, although a slight decrease can be
discerned in the quantity of produced sperm with increase in age. The entire process can
be broken up into several distinct stages, each corresponding to a particular type of cell:
A mature human Spermatozoon
Purpose
Spermatogenesis produces mature male gametes, commonly called sperm but specifically
known as spermatozoa, which are able to fertilize the counterpart female gamete, the
oocyte, during conception to produce a single-celled individual known as a zygote. This
is the cornerstone of sexual reproduction and involves the two gametes both contributing
half the normal set of chromosomes (haploid) to result in a chromosomally normal
(diploid) zygote.
To preserve the number of chromosomes in the offspring, which differs between species,
each gamete must have half the usual number of chromosomes present in other body
cells. Otherwise, the offspring will have twice the normal number of chromosomes, and
serious abnormalities may result. In humans, chromosomal abnormalities arising from
incorrect spermatogenesis can result in Down Syndrome, Klinefelter's Syndrome, and
spontaneous abortion. Most chromosomally abnormal zygotes will not survive for long
after conception; however, plant reproduction is a little more robust, and viable new
species may arise from cases of polyploidy.
Location
Spermatogenesis takes place within several structures of the male reproductive system.
The initial stages occur within the testes and progress to the epididymis where the
developing gametes mature and are stored until ejaculation. The seminiferous tubules of
the testes are the starting point for the process, where stem cells adjacent to the inner
tubule wall divide in a centripetal direction—beginning at the walls and proceeding into
the innermost part, or lumen—to produce immature sperm. Maturation occurs in the
epididymis and involves the acquisition of a tail and hence motility.
Stages
Spermatocytogenesis
Spermatocytogenesis is the male form of gametocytogenesis and results in the formation
of spermatocytes possessing half the normal complement of genetic material. In
spermatocytogenesis, a diploid spermatogonium which resides in the basal compartment
of seminiferous tubules, divides mitotically to produce two diploid intermediate cell
called a primary spermatocyte. Each primary spermatocyte then moves into the adluminal
compartment of the seminiferous tubules and duplicates its DNA and subsequently
undergoes meiosis I to produce two haploid secondary spermatocytes. This division
implicates sources of genetic variation, such as random inclusion of either parental
chromosomes, and chromosomal crossover, to increase the genetic variability of the
gamete.
Each cell division from a spermatogonium to a spermatid is incomplete; the cells remain
connected to one another by bridges of cytoplasm to allow synchronous development. It
should also be noted that not all spermatogonia divide to produce spermatocytes,
otherwise the supply would run out. Instead, certain types of spermatogonia divide to
produce copies of themselves, thereby ensuring a constant supply of gametogonia to fuel
spermatogenesis.
Spermatidogenesis
Spermatidogenesis is the creation of spermatids from secondary spermatocytes.
Secondary spermatocytes produced earlier rapidly enter meiosis II and divide to produce
haploid spermatids. The brevity of this stage means that secondary spermatocytes are
rarely seen in histological preparations.
Spermiogenesis
During spermiogenesis, the spermatids begin to grow a tail, and develop a thickened midpiece, where the mitochondria gather and form an axoneme. Spermatid DNA also
undergoes packaging, becoming highly condensed. The DNA is packaged firstly with
specific nuclear basic proteins, which are subsequently replaced with protamines during
spermatid elongation. The resultant tightly packed chromatin is transcriptionally inactive.
The Golgi apparatus surrounds the now condensed nucleus, becoming the acrosome. One
of the centrioles of the cell elongates to become the tail of the sperm.
Maturation then takes place under the influence of testosterone, which removes the
remaining unnecessary cytoplasm and organelles. The excess cytoplasm, known as
residual bodies, is phagocytosed by surrounding Sertoli cells in the testes. The resulting
spermatozoa are now mature but lack motility, rendering them sterile. The mature
spermatozoa are released from the protective Sertoli cells into the lumen of the
seminiferous tubule in a process called spermiation.
The non-motile spermatozoa are transported to the epididymis in testicular fluid secreted
by the Sertoli cells with the aid of peristaltic contraction. Whilst in the epididymis they
acquire motility and become capable of fertilisation. However, transport of the mature
spermatozoa through the remainder of the male reproductive system is achieved via
muscle contraction rather than the spermatozoon's recently acquired motility.
Role of Sertoli cells
Labelled diagram of the organisation of Sertoli cells (red) and spermatocytes (blue) in the
testis. Spermatids which have not yet undergone spermination are attached to the lumenal
apex of the cell
At all stages of differentiation, the spermatogenic cells are in close contact with Sertoli
cells which are thought to provide structural and metabolic support to the developing
sperm cells. A single Sertoli cell extends from the basement membrane to the lumen of
the seminiferous tubule, although the cytoplasmic processes are difficult to distinguish at
the light microscopic level.
Sertoli cells serve a number of functions during spermatogenesis, they support the
developing gametes in the following ways:

Maintain the environment necessary for development and maturation via the
blood-testis barrier

Secrete substances initiating meiosis

Secrete supporting testicular fluid

Secrete androgen-binding protein, which concentrates testosterone in close
proximity to the developing gametes
o
Testosterone is needed in very high quantities for maintenance of the
reproductive tract, and ABP allows a much higher level of fertility

Secrete hormones effecting pituitary gland control of spermatogenesis,
particularly the polypeptide hormone, inhibin

Phagocytose residual cytoplasm left over from spermiogenesis

They release Antimullerian hormone which prevents formation of the Mullerian
Duct / Oviduct.
OOGENESIS
In mammals, the first part of OOGENESIS occurs in the ovarian follicle that is the
functional unit of the ovary.
It is interesting to note that such an important process in animal life cycles is done
completely without the aid of spindle-coordinating centrosomes.
It consists of several processes: oocytogenesis, ootidogenesis and the final maturity to
form an ovum. Folliculogenesis is a separate process during ootidogenesis.
Oogonium --(Oocytogenesis)--> Primary Oocyte --(Meiosis I)-->First Polar Body
(Discarded afterward) + Secondary oocyte --(Meiosis II)--> Secondary Polar
Body(Discarded afterward) + Ovum
Creation of oogonia
The creation of oogonia traditionally doesn't belong to oogenesis, but to the common path
of gametogenesis together with spermatogenesis.
Oocytogenesis
Oogenesis
starts
with
oogonial
transformation
into
primary oocytes,
called
oocytogenesis[1]. Oocytogenesis is completed either before or shortly after birth.
Number of primary oocytes
It is commonly said that when oocytogenesis is completed, no additional primary oocytes
are created, in contrast to the male spermatogenesis, where gametocytes are continuously
created. In other words, oocytes reaches their maximum at ~20[2] weeks of gestational
age, when there are approximately seven million of them.
Recently, however, two publications have challenged the ovarian biology dogma that a
finite number of oocytes are set around the time of birth.[3][4] Renewal of ovarian follicles
from germline stem cells (originating from bone marrow and peripheral blood) was
reported in the postnatal mouse
vary. Due to the revolutionary nature of these claims, further experiments are required to
examine the dynamics of small follicle formation.
Ootidogenesis
The succeeding ootidogenesis is the step in which the primary oocyte turns into an ootid.
It is achieved by meiosis. The primary oocyte is even defined from its role to undergo
meiosis[5].
However, although this process begins at prenatal age, it stops at prophase I. In late fetal
life, all oocytes, still primary oocytes, have taken this halt in development, called
dictyate. First after menarche they continue to develop, although only a few does so every
menstrual cycle.
Meiosis I
Meiosis I of ootidogenesis starts at embryonic age, but halts in diplotene of prophase I
until puberty. For those primary oocytes continuing to develop in each menstrual cycle,
however, synapsis occurs and tetrads form, enabling and crossing over. As a result of
meiosis I, the primary oocyte becomes the secondary oocyte and the first polar body.
Meiosis II
Immediately after meiosis I, the haploid secondary oocyte initiates meiosis II. However,
this, too is halted in metaphase II. However, this only lasts until fertilization, if such
occurs. When meiosis II is completed, an ootid and another polar body is created.
Folliculogenesis
Synchronously as ootidogenesis, the ovarian follicle surrounding it develops from a
primordial follicle to a preovulatory one.
Maturation into ovum
Both polar bodies at the end of Meiosis II disintegrate leaving only the ootid which
undergoes maturation and eventually matures into an ovum.
The function of forming polar bodies is to discard the extra haploid set of chromosome(n)
Oogenesis in non-mammals
Many protists produce egg cells in structures termed archegonia. Some algae and the
oomycetes produce eggs in oogonia. In the brown alga Fucus, all four egg cells survive
oogenesis, which is an exception to the rule that generally only one product of female
meiosis survives to maturity.
In plants, oogenesis occurs inside the female gametophyte via mitosis. In many plants
such as bryophytes, ferns, and gymnosperms, egg cells are formed in archegonia. In
flowering plants, the female gametophyte has been reduced to an eight-celled embryo sac
within the ovule inside the ovary of the flower. Oogenesis occurs within the embryo sac
and leads to the formation of a single egg cell per ovule.
In ascaris, the oocyte does not even begin meiosis until the sperm touches it, in contrast
to mammals, where meiosis is completed in the menstrual cycle.
FERTILIZATION is more a chain of events than a single, isolated phenomenon.
Indeed, interruption of any step in the chain will almost certainly cause fertilization
failure. The chain begins with a group of changes affecting the sperm, which prepares
them for the task ahead.
Successful fertilization requires not only that a sperm and egg fuse, but that not more than
one sperm fuses with the egg. Fertilization by more than one sperm - polyspermy - almost
inevitably leads to early embryonic death. At the end of the chain are links that have
evolved to efficiently
prevent polyspermy.
In overview, fertilization can be described as the following steps:
Sperm Capacitation
Freshly ejaculated sperm are unable or poorly able to fertilize. Rather, they must first
undergo a series of changes known collectively as capacitation. Capacitation is associated
with removal of adherent seminal plasma proteins, reorganization of plasma membrane
lipids and proteins. It also seems to involve an influx of extracellular calcium, increase in
cyclic AMP, and decrease in intracellular pH. The molecular details of capacitation
appear to vary somewhat among species.
Capacitation occurs while sperm reside in the female reproductive tract for a period of
time, as they normally do during gamete transport. The length of time required varies
with species, but usually requires several hours. The sperm of many mammals, including
humans, can also be capacitated by incubation in certain fertilization media.
Sperm that have undergone capacitation are said to become hyperactiviated, and among
other things, display hyperactivated motility. Most importantly however, capacitation
appears to destabilize the sperm's membrane to prepare it for the acrosome reaction, as
described below.
Sperm-Zona Pellucida Binding
Binding of sperm to the zona pellucida is a receptor-ligand interaction with a high degree
of species specificity. The carbohydrate groups on the zona pellucida glycoproteins
function as sperm receptors. The sperm molecule that binds this receptor is not known
with certainty, and indeed, there may be several proteins that can serve this function.
The Acrosome Reaction
Binding of sperm to the zona pellucida is the easy part of fertilization. The sperm then
faces the daunting task of penetrating the zona pellucida to get to the oocyte. Evolution's
response to this challenge is the acrosome - a huge modified lysosome that is packed with
zona-digesting enzymes and located around the anterior part of the sperm's head - just
where
it
is
needed.
The acrosome reaction provides the sperm with an enzymatic drill to get throught the
zona pellucida. The same zona pellucida protein that serves as a sperm receptor also
stimulates a series of events that lead to many areas of fusion between the plasma
membrane and outer acrosomal membrane. Membrane fusion (actually an exocytosis)
and vesiculation expose the acrosomal contents, leading to leakage of acrosomal enzymes
from the sperm's head.
As the acrosome reaction progresses and the sperm passes through the zona pellucida,
more and more of the plasma membrane and acrosomal contents are lost. By the time the
sperm traverses the zona pellucida, the entire anterior surface of its head, down to the
inner acrosomal membrane, is denuded. The animation to the right depicts the acrosome
reaction, with acrosomal enzymes colored red.
Sperm that lose their acrosomes before encountering the oocyte are unable to bind to the
zona pellucida and thereby unable to fertilize. Assessment of acrosomal integrity of
ejaculated sperm is commonly used in semen analysis.
Penetration of the Zona Pellucida
The constant propulsive force from the sperm's flagellating tail, in combination with
acrosomal enzymes, allow the sperm to create a tract through the zona pellucida. These
two factors - motility and zona-digesting enzymes- allow the sperm to traverse the zona
pellucida. Some investigators believe that sperm motility is of overriding importance to
zona penetration, allowing the knife-shaped mammalian sperm to basically cut its way
through the zona pellucida.
Sperm-Oocyte Binding
Once a sperm penetrates the zona pellucida, it binds to and fuses with the plasma
membrane of the oocyte. Binding occurs at the posterior (post-acrosomal) region of the
sperm head.
The molecular nature of sperm-oocyte binding is not completely resolved. A leading
candidate in some species is a dimeric sperm glycoprotein called fertilin, which binds to
a protein in the oocyte plasma membrane and may also induce fusion. Interestingly,
humans and apes have inactivating mutations in the gene encoding one of the subunits of
fertilin, suggesting that they use a different molecule to bind oocytes.
Egg Activation and the Cortical Reaction
Prior to fertilization, the egg is in a quiescent state, arrested in metaphase of the second
meiotic division. Upon binding of a sperm, the egg rapidly undergoes a number of
metabolic and physical changes that collectively are called egg activation. Prominent
effects include a rise in the intracellular concentration of calcium, completion of the
second meiotic division and the so-called cortical reaction.
The cortical reaction refers to a massive exocytosis of cortical granules seen shortly after
sperm-oocyte fusion. Cortical granules contain a mixture of enzymes, including several
proteases, which diffuse into the zona pellucida following exocytosis from the egg. These
proteases alter the structure of the zona pellucida, inducing what is known as the zona
reaction. Components of cortical granules may also interact with the oocyte plasma
membrane.
The Zona Reaction
The zona reaction refers to an alteration in the structure of the zona pellucida catalyzed
by proteases from cortical granules. The critical importance of the zona reaction is that it
represents the major block to polyspermy in most mammals. This effect is the result of
two measurable changes induced in the zona pellucida:
1. The zona pellucida hardens. Crudely put, this is analogous to the setting of
concrete. Runner-up sperm that have not finished traversing the zona pellucida by
the time the hardening occurs are stopped in their tracks.
2. Sperm receptors in the zona pellucida are destroyed. Therefore, any sperm that
have not yet bound to the zona pellucida will no longer be able to bind, let alone
fertilize the egg.
The loss of sperm receptors can be demonstrated by mixing sperm with both unfertilized
oocytes (which have not yet undergone the zona reaction) and two-cell embryos (which
have previously undergone cortical and zona reactions). In this experiment, sperm attach
avidly to the zona pellucida of oocytes, but fail to bind to the two-cell embryos.
Post-fertilization Events
Following fusion of the fertilizing sperm with the oocyte, the sperm head is incorporated
into the egg cytoplasm. The nuclear envelope of the sperm disperses, and the chromatin
rapidly loosens from its tightly packed state in a process called decondensation. In
vertebrates, other sperm components, including mitochondria, are degraded rather than
incorporated into the embryo.
Chromatin from both the sperm and egg are soon encapsulated in a nuclear membrane,
forming pronuclei. The image to the right shows a one-cell rabbit embryo shortly after
fertilization - this embryo was fertilized by two sperm, leading to formation of three
pronuclei, and would likely die within a few days. Pass your mouse cursor over the
image to identify pronuclei.
Each pronucleus contains a haploid genome. They migrate together, their membranes
break down, and the two genomes condense into chromosomes, thereby reconstituting a
diploid organism.
BLASTUALTION AND GASTRULATION
In this early stage in animal development, the first cavity (blastocoel) is formed, when the
embryonic cells form a hollow ball called a blastula.
Cleavage is a process that the fertilized egg undergoes to form blastomeres, which make
up a blastula. A blastula is simply another form of the fertilized egg. Inside the blastula,
blastomeres divide to form an embryo. A gastrula is formed from the blastula when a
small pocket begins to form on the blastula. Blastulation is the process that creates a
blastula, and gastrulation is the process that forms a gastrula.
Gastrulation
In this stage of animal embryonic development, an external layer of cells fold inward to
the interior to form the beginning of the digestive tract. This is the earliest formation of
the gut in an organism.
The sexing of embryos is also done permitting more female progeny if required. Another technique
involves splitting of embryos into segments, each segment developing into an offspring using the
above technology, so that 20-30 calves per year can be obtained from one valuable donor.
It is also possible to replace the nucleus of an egg of a genetically less desirable female
animal with that of a somatic cell of a superior female so that superior progeny may be
obtained. This will overcome the problem of non availability of superior eggs in large
number.
Methods are also being developed to transform the egg or embryo before embryo transfer
is affected. Transgenic animals in this manner have been produced in mice, cows, sheep,
goats, chicken, pigs, etc.
In farm animals, more efficient methods are being developed for superovulation, in vitro
fertilization, embryo sexing, embryo culture, transformation of embryo, and embryo
transfer.. The nutritional requirements for culturing embryos of farm animals have
generally been extrapolated from work with laboratory animals like mice and rabbits.
However, embryos of most mammals can be maintained outside the bodies of the animals
for half a day or more with no irreversible damages. If transgenic animals produced by
the above methods are genetically superior and can be multiplied using the methods
outlined above, the expense involved will be rewarded by producing many superior
animals through super ovulation, and embryo transfer in surrogate mothers.
The genetic potential of an embryo can be multiplied by dividing the embryo into groups
of cells to obtain identical twins, triplets or quadruplets. The technology for freezing
semen and embryos has also been developed, and will provide enormous flexibility in
using these techniques for genetic improvement of farm animals.
1. What is spermiogenesis? Explain the three phases which are involved in it.
2. Write short note on in vitro fertilization
3. What are the different types of animal cell culture? Explain.
4. Explain primary explant culture.
5. Explain gamatogenesis in detail with a neat labeled diagram.
6. Differentiate spermatogenesis and oogenesis.
7. Explain fertilization in animals in detail.
8. Write short notes on Collection, preservation and culturing of embryos.
9. What is blastulation?
10. what is gastrulation?
UNIT-1
ANIMAL TISSUE CULTURE MEDIA
Animal tissue culture media which are used for the preparation of animal cell cultures,
Without media we cannot culture plant cell and animal cell.
Objectives
1.
2.
3.
Introduction
Culture Media Containing Naturally Occurring Ingredients
Inspite of vast use of chemically defined media in tissue culture, it is still necessary
inmost undertakings to depend on naturally occurring substances derived from the
organism.
The various kinds of such media, which are used, may be:
(i) Blood plasma, (ii) blood serum, (iii) tissue extract and (iv)
complex natural media.
Blood Plasma
The first tissue culture was done by Harrison (1907) in clotted frog lymph. Burrows
(1910) substituted a coagulum prepared from chicken plasma. After many years it was
found that plasma provided a complete nutrient in which cells could survive and multiply
slowly for extended periods under conditions that resembled in many respect those found
in body. Plasma is still being used to advantage for the following purposes:
1. To provide a nutritive substrate and a supporting structure for many types of cultures,
Just
as it also provides a matrix for new cells during’ the repair of injury in the body.
2. To provide a means of conditioning the surface t>f glass for
better attachment of cells.
3. To provide a means of protecting cells and tissues from excessive traumatic damage
during sub culture. To provide some degree of protection from sudden changes in the
environment at times fluid change.
4. To provide localized pockets of conditioned medium around cells. For culture work,
plasma from the adult chicken is preferred to mammalian plasma because it forms a clear,
solid coagulum even when diluted several times. Mammalian plasma is either too opaque
for good optical work or else it fails to produce solid clots. The plasma is obtained by
centrifugation of whole blood before coagulation ‘takes place. The tissue is then placed in
a small quantity of the plasma and coagulation encouraged by addition of a small amount
of tissue extract or thrombin. This is done because the cells in culture require a solid
support for continued growth and activity. In case of fowl the blood is obtained from the
wing, heart or carotid artery. In case of mammals it is obtained from carotid artery and
heart.
Blood Serum
Blood serum (plasma minus fibrinogeri) with or without other nutritive substances may
be used either as the entire culture medium or as the fluid phase of a medium consisting
partly of a plasma coagulum. For many years it was assumed that whole serum was toxic,
that plasma was useful only as a supportive structure and that the nutritive requirements
of the cells were supplied by the embryo extract that was usually added to the medium.
Eventually, however, it was found possible to cultivate tissues in serum alone without
plasma or tissue extract. In 1928, des Ligneris reported the successful cultivation of many
mammalian tissues in diluted serum and later Parker (1933, 1936) cultivated chick tissues
in serum. Simms (1936), and Simms and Sanders (1942) introduced an ultrafiltrate of
serum that was used as a basal medium for many purposes including the propagation of
viruses. Fischer and coworkers (1948) stressed the importance of the low molecular
weight growth factors provided by serum. As some of the more elaborate chemically.
Defined solutions were developed, it was found that they had to be supplemented with 10
to 20 per cent serum to provide completely adequate medium for the continuous
propagation of established cell lines and freshly explanted tissues for extended periods.
Harris (1959) concluded that medium 199 and NCTC-109, as well as the simpler basal
medium of Eagle (1955) are all deficient in one or more factors that occur in serum
dialysate and are essential for the growth and maintenance of chick skeletal muscle
fibroblasts. Thus serum does provide some of the growth factors or some of the physical
conditions, or both, that are presently lacking insynthetic media.
Preparation of Chicken Serum
The fluid plasma from which the serum is prepared should be completely coagulated. The
plasma is coagulated deliberately by adding to each tube a drop or two of embryo tissue
extract or an equivalent amount of thrombin and leaving the tubes to
incubate for several hours at 37° C. The coagulated plasma is broken up into fragments
and it is ground in a mortor with sterile quartz sand. After grinding the serum is separated
by
centrifugation.
Preparation of Mammalian Serum
The mammalian blood is left at room temperature for an hour. The clot is removed by a
glass rod and then centrifuged for 30 minutes at 3000 rpm and the serum is separated.
Serum Free Media
The use of serum in culture media is not so common because it has following
disadvantages:
1. The quality of serum varies from batch to batch and
deteriorates within one year. Therefore every batch of serum
needs fresh testing.
2. If more than one cell types are used, each may require
different serum batch, therefore, many batches are to be maintained and co-culturing may
be difficult.
3. The demand of serum usually exceeds the supply for a variety of reasons.
4. When cell culture is used for downstream processing to recover cell products, the
presence of serum is an obstacle to purification; Serum increases the cost of the medium.
5. Serum may stimulate undesirable growth and may even inhibit growth in some cases.
Advantage of Serum Free Media
1. It has the ability to make a medium selective for a particular cell type, since each cell
type appears to require a different recipe.
2. It has high degree of purity of reagents and water.
3. It needs high degree of cleanliness of all apparatus.
How to Develop Serum Free Media?
1. A known recipe for a related cell type may be used.
2. Individual constituents of the serum may be altered, until the medium is optimized.
3. The development and assessment constituent is a time-consuming^ process.
Sometimes it takes about three years for development of a new medium.4. In the second
approach, an existing medium like RPMI 1640 or Ham’s F12 or DMEM (Dulbecco’s
modified minimal essential medium) is taken and a shorter list of constituents like
selenium, transferrin, albumin, insulin, androgen, hydrocortisone, estrogen, etc. is use!
for manipulation.
Tissue Extracts
Carrel (1912) discovered that embryo tissue extract had remarkable powers of promoting
eel growth and multiplication in cultures of connective tissue cells from chick embryo
heart. Since Carrel’s experiments, there have been many attempts to determine the
chemical nature of the substances responsible for the stimulating effect of embryo
extract. Baker and Carrel (1926) obtained active fractions of the extract by precipitation
with carbon dioxide and found that the activity was concentrated largely in the protein
portion containing nucleoproteins and glycoproteins. This was further observed(Carrel
and Baker, 1926) that proteoses and higher molecular weight protein degradation
products also had very potent
growth promoting properties. Growth promoting activity appeared to be associated
particularly with fractions containing predominantly nucleoproteins of the ribonucleic
acid type. Fractions relatively high in deoxyribonucleic acid appeared much less active.
Active nucleoprotein fractions from adult chicken heart, brain, liver and spleen have been
used but no indications of organ specificity were observed. The activity of the fraction
also did not depend on their total nucleic acid content or on the age of the individuals
from which they were prepared.
Preparation of Embryo Extract
Chick embryo extract is made from 10 to 11 days old embryos (before the calcifying
mechanisms have become too active). The embryos are removed from the egg, then
homogenized in a motordriven homogenizer. Six to eight embryos and a
measured quantity of balanced salt solutions (e.g., 2.0 ml per embryo) may be processed
at one time. After homogenization, it is centrifuged and further diluted 10 to 20 times.
Embryo
extract may be stored indefinitely after it has been dried from the frozen state.
Complex Natural Media
Some of the complex natural media are as follows: Supplemented Hanks-Simms medium
Weller and co-workers (1952) in their earlier work with polioviruses made excellent use
of a combination of 3 parts Hanks’s balanced salt and 1 part Simm’s ox serum
ultrafiltrate. For
rollertube cultures of various human and animal tissues (embryonic, infant and adult), the
complete medium consisted of Hanks-Simms solution (85%), beef embryo extract (10%),
horse serum inactivated at 56° C for 30 minutes (5 to 20%), penicillin (50 jig/ml) and
streptomycin (50 Hg/ml).Supplemented bovine amniotic fluid medium Milovanic and coworkers (1957, 1958) used the following medium:
Bovine amniotic fluid (37.5%), horse serum inactivated at 56° C for 30 minutes (20%),
bovine embryo extract (5%), Hanks balanced salt solution (37.5%), streptomycin (lOOu
/ml), penicillin (lOOu/ml) and mycostatin (lOOu/ml).
ROLE OF CARBON DI OXIDE
1. Carbon di oxide influences the pH of the media.
2. Buffering action of bicarbonate helps maintain PH.
3. HEPES containing media well regulates ph .
For more details on this topic, see Carbon dioxide (data page).
Carbon dioxide is a colorless, odorless gas. When inhaled at concentrations much
higher than usual atmospheric levels, it can produce a sour taste in the mouth and
a stinging sensation in the nose and throat. These effects result from the gas
dissolving in the mucous membranes and saliva, forming a weak solution of
carbonic acid. This sensation can also occur during an attempt to stifle a burp
after drinking a carbonated beverage. Amounts above 5,000 ppm are considered
very unhealthy, and those above about 50,000 ppm (equal to 5% by volume) are
considered dangerous to animal life.[4]
At standard temperature and pressure, the density of carbon dioxide is around 1.98 kg/m³,
about 1.5 times that of air. The carbon dioxide molecule (O=C=O) contains two double bonds
and has a linear shape. It has no electrical dipole, and as it is fully oxidized, it is moderately
reactive and is non-flammable, but will support the combustion of metals such as
magnesium.
QUESTION BANK
SECTION B & C
1. Write short note on culture media.
2. Explain in the importance of growth factors in the culture medium.
3. Give a detailed account on the serum and Protein Free Media. Also differentiate
between serum containing and serum free media and give their applications.
4. Write the relationship between bicarbonate, carbon dioxide and HEPES in the
Culture media.
5. Write short notes on natural media.
6. Discuss the role of hormones in culture medium
7. Explain the role of glutathione in media?
8. Explain the role of glutamine in media?
9. Differentiate between Natural Media and synthetic media.
10. What is serum? Explain the composition of serum.
11. Explain the role of glutamine in the media.
12. What is BSS? Explain its importance.
13. Explain in detail about the physical, chemical and metabolic functions
of different constituents of culture media.
14. Differentiate natural and synthetic media in detail
Section -A
I. Define the following:
1. Media
2. Serum.
3. Balanced salt solution.
4. Glutamine
5. Osmolality to be maintained in the media for animal tissue culture is ……..
6. …….. is a growth hormone which has a dual role of stimulation and inhibition of
growth of the cells.
7. HEPES
8. What are hormones?
9. What are growth regulators?
10. Define media.