FERTILIZATION

anD
CLEAVAGE

Lecture - 4
FERTILIZATION
 Competences
The students should be able to explain:
• Sperm and oocyte preparation prior to
fertilization
• Sequences of fertilization
• Why polyspermy has to be prevented and how to
do it
• Oocyte activation
SpermTransport
Needed for internal fertilization:
• Male reproductive tract:
– Seminiferous tubules (rectus tubule-rete testis)
– Vas efferent (vasa efferensia)
– epididymis sperm gain their motility: ATP
production, plasma membrane alteration
– Vas deferent (vasa deferensia)
– urethra
• Female reproductive tract:
– vagina +cervix
– Uterus
– Fallopian tube (place for fertilization)
The number of sperm being deposited into female
reproductive tract

Species S sperm (million) Site of S Sperm reach the

/ejaculate deposition ampulla of Fallopian tube
Mouse 50 uterus <100
Rat 58 uterus 500
Pig 8000 uterus 1000
Rabbit 280 vagina 250-500
Cow 3000 vagina few
Sheep 1000 vagina 600-700
Human 280 vagina 200
Sperm survival in the female reproductive tract

Species Hours
Mouse 6
Rat 14
Guenia pig 21-22
Rabbit 30-32
Sheep 30-48
Cow 28-50
Horse 28-48
Bat 135 days
Human 48
 What are the factors that affect the sperm’s
potential for successfully fertilizing the egg?
Effects of Capacitation on
1. Capacitation - occurs in Sperm
the female’s vagina. • Increased rate of metabolism
 Vaginal secretions cause a • Flagellum beats more rapidly;
molecular change in the Result: Sperm are more motile
sperm plasmalemma • Changes in sperm plasmalemma
(removal of decapacitating proteins allow sperm-egg binding
factor: semen proteins, and occurrence of the acrosome
results in increased reaction
membrane fluidity). • Pro-Acrosin (inactive) is
converted to acrosin (active)
 Takes 4-5 hr in humans, 1 hr
in mice, 6 hr in rabbits. • Sperm become capable of
chemotaxis
What are the factors that affect the sperm’s
potential for successfully fertilizing the egg?

2. Dilution
• Marine invertebrates and oviparous fish – release
the sperm into surrounding water at spawning.
• Mammals - Dilution: 40 - 400 million spermatozoa
in vagina, only a few hundred to a thousand reach
the upper oviduct.
What are the factors that affect the sperm’s
potential for successfully fertilizing the egg?

3. Egg secretions - importance varies among

species - fertilizins - F.R. Lille (1919)
• activating factors, agglutination,
chemoattraction
What are the factors that affect the sperm’s
potential for successfully fertilizing the egg?

4. Structures surrounding the egg – barriers

• Follicle cells (cumulus cells)
• Zona pellucida (vitelline membrane in non-
mammals)
• Oolemma (plasmalemma of egg)
Fertilization:
4 major sequencial events
1. Sperm and egg make contact and must
recognize each other as the same species
2. Regulation of sperm entry into the oocyte
and prevention of polyspermy
3. Fusion of genetic material
4. Activation of egg to begin development
1. Sperm and egg make contact and must
recognize each other as the same species

Sperm and egg recognition

1. Egg of some species produce chemoattractant
2. Exocytosis of achrorosomal content
3. Sperm binding to extra cellular coat of the egg
4. Sperm entry into the egg cytoplasm
5. Fusion of egg and sperm membrane
Chemoattractant
• Attract sperm toward the egg
• Orthopyxis caliculata (cnidaria)
– Released by oocytes completed meiosis II
• Arbacia punctulata (sea urchin)
– Oocytes produce resact (peptide with14 amino acid) –
act as chemoattractant and ‘sperm activating peptide’
– Sperm has receptor which recognize resact
– Species recognizing protein: Bindin protein 30.500 Da
2. Regulation of sperm entry into the oocyte
and prevention of polyspermy
• How does the sperm get through the barriers
surrounding the egg?
• Used to be thought that the acrosome enzymes did it
all.
• More recent data
a.Sperm arrives at zona pellucida with the acrosome still intact. So,
how does it get through the follicle cells that surround the egg?
b.Has to break down the intercellular cement that holds the follicle
cells together - hyaluronic acid
c.Lin and co-investigators (1994) demonstrated that a protein
found in the sperm plasmalemma (PH-20) had hyaluronidase
activity.
Getting through the zona pellucida

What happens when the sperm gets to the

zona pellucida?
1. Attachment - loose association
2. Binding - strong attachment
3. Acrosome reaction - release of enzymes
4. Penetration of the zona pellucida by the
sperm
Membrane fusion in Sea Urchin
Acrosome reaction in sea urchin
Membrane fusion in mouse
Sperm-egg recognition in mammal

• Zona pelucida analog to vitelline membrane

• Glycoprotein : ZP-1, ZP-2, ZP3
• ZP3 (83 kDa)
– Bind to sperm with intact achrosomal vesicle
– Crosslinking ZP3-receptor on sperm membrane
--- induce acrosomal reaction
• Post achrosomal reaction, the inner acrosomal
membrane bind to ZP2
Acrosome reaction in eutheria
 Prevention of Polyspermy

• Usually done by preventing multiple sperm

from entering the egg
• Sea Urchins have 2 mechanisms:
– Fast Block
• Involves a change in egg cell membrane potential
– Slow Block
• Involves exocytosis of the cortical granules in the
egg
 Fast Block to Polyspermy
(Sea Urchin model)
• The egg has a different ionic concentration from
the seawater in which it exists
• Egg has lower sodium ion concentration; higher
potassium concentration
• This is maintained by sodium/potassium pumps in
the egg cell membrane
• The difference in charge across the egg membrane
can be measured as -70mV and is called the
resting membrane potential
 Fast Block to Polyspermy
(Sea Urchin model)
• 1-3 seconds after first sperm binds, the
membrane potential shifts to +20 mV
• Sperm can no longer fuse to the egg
• Experimental evidence - Polyspermy can
occur if*:
– Eggs are supplied with an electrical current that
keeps charge at -70mV
– Fertilization occurs in water with a low sodium
ion concentration
*Jaffe 1976
 Slow Block to Polyspermy -
Sea urchin model
• Slower, mechanical, permanent block
• Occurs about a minute after sperm-egg fusion
• Upon sperm entry cortical granules fuse with
the cell membrane and release several
molecules
 Cortical Granule Molecues
• Cortical granule serine protease
– Releases vitelline membrane from its anchors to the cell membrane
– Clips off bindin molecules
• Mucopolysaccharides
– Cause osmotic gradient
– Water rushes into space between vitelline envelope
– Vitelline envelope expands (lifts) and becomes the fertilization
envelope
• Peroxidase
– Hardens the fertilization envelope
• Hyaline
– Forms a coating around the egg, protects during early embryonic
development
Formation of fertilization envelope in sea urchin
 Mammalian Cortical Granule Reaction
• Does not form a
fertilization envelope
• Does modify Zona
Pellucida so sperm
cannot bind
• In mice, cortical
granules cleave an
essential portion of
the ZP3 molecule
 Calcium and the Cortical Granule
Reaction
• Upon fertilization, intracellular concentration of
Calcium ion in the egg increases
• This is necessary for the fusion of cortical
granules with the cell membrane
• Calcium comes not from outside the egg, but from
inside the egg itself
• The fusion begins near the site of sperm entry and
continues in a wave across the egg
• A similar wave of calcium ion release can be
observed
Calcium experiments - Ca2+ is directly
responsible for cortical reaction

• A23187 is a calcium ionophore

– Transports Ca2+ across lipid membranes
• Placing sea urchin embryos in sea water
containing A23187 results in cortical
granule reaction & fertilization envelope to
rise (without presence of sperm)
• If Ca2+ chelator is injected into egg, no
cortical reaction occurs
 Why polyspermy has to be blocked ?

Polyspermy is disastrous
 Results in triploid
nucleus
 Multiple mitotic
spindles form
Dispermic
Sea Urchin
egg

Dispermic Human Egg -Note 4

centrioles
3. Fusion of genetic material
in mammals
a. The male nucleus enters the egg cytoplasm and
becomes the male pronucleus.
b. As a result of the sperm fusing with the egg
plasmalemma, the oocyte nucleus, which is at
metaphase of the second meiotic division, completes
that division giving rise to another polar body.
c. Following the second meiotic division, what is now the
nucleus of the ovum becomes the female pronucleus.
d. The haploid male and female pronuclei move toward
one and other, meet, and fuse to form the diploid
nucleus of the zygote
3. Fusion of genetic material
in sea urchin

a. The nuclear membrane forms a vesicle

b. Centriole located between the two pronuclei
c. Microtubules integrate the pronuclei to form a
diploid zygote nucleus
4. Activation of egg to begin development

A series of morphological, physiological and

molecular changes that occur in the egg in
response to fusion of the sperm with the egg.
1. Frees egg from constraints that kept it from
developing prior to fertilization.
2. Prepares the egg for development.
Why do “frees the egg” ? Why not “frees the
egg and sperm”?

a. Egg activation does not require the sperm

nucleus
b. In some species, just pricking the egg with a
needle, shocking it, or simply changing the pH
of the solution it’s in will cause activation.
c. In some species eggs that are activated this way
will start to develop. Thus, we have a
mechanism that could allow for selection of
parthenogenesis
The seven events that characterize egg activation
following fusion of the sperm with the egg:

1. Release of Ca++ (calcium) stored in the egg

endoplasmic reticulum - appears to be the critical step
in the process.
2. Cortical reaction - rupture of cortical granules that
occurs concurrently with the Ca++ release.
 Contents of granules are released into perivitelline
space and cause “hardening” of the vitelline membrane
or zona pellucida.
 Causes vitelline/fertilization membrane to rise away
from surface of egg in some species.
The seven events that characterize egg activation
following fusion of the sperm with the egg:

3. In many species, an influx of Na+ (sodium) into the egg

cytoplasm that causes a change in membrane potential -
fast block to polyspermy.
4. In many species a reorganization of the egg cytoplasm.
5. In most cases, completion of meiosis by the egg.
6. An efflux of H+ (hydrogen) ions causing an increase in
cytoplasmic pH - this activates previously inhibited
synthetic pathways.
7. Increase in metabolism - zygote gears up for
development.
Events that occur soon after egg activation:
a. DNA replication as male and female pronuclei
approach each other
b. Male and female pronuclei merge
c. Preparation for first cleavage
CLEAVAGE
 Competences
The students should be able to explain:
• Cleavage proccess
• Cleavage type
• Regulatory factors of cleavage
• Identify morula and blastula
• Blastula formation
 What is cleavage?

Cleavage is a rapid series of mitotic divisions that occur just after fertilization.

There are two critical reasons why cleavage is so important:

1. Generation of a large number of cells that can undergo differentiation and
gastrulation to form organs.
2. Increase in the nucleus / cytoplasmic ratio. Eggs need a lot of cytoplasm
to support embryogenesis. It is difficult or impossible for one nucleus to
support a huge cytoplasm, and oocytes are one of the largest cells that
exist. One small nucleus just cannot transcribe enough RNA to meet the
needs of the huge cytoplasm.
A larger nucleus to cytoplasmic ratio is optimal for cell function. Cell
division occurs rapidly after fertilization to correct this problem.
 Cleavage differs from normal mitoses in 2 respects

1. Blastomeres do not grow in size between successive cell divisions as

they do in most cells. This leads to a rapid increase in the nucleus /
cytoplasmic ratio. Cells undergoing cleavage have mainly S and M
phases of the cell cycle (little or no G1 or G2).
2. Cleavage occurs very rapidly, and mitosis and cytokinesis in each round
of cell division are complete within an hour. Typical somatic cells divide
much more slowly (several hours to days) and even the fastest cancer
cells divide much slower than occurs in a zygote during cleavage.

Cleavage differs in different types of eggs. The presence of large amounts of

yolk alters the cleavage pattern, leading to incomplete cleavage that
characterizes birds and reptiles.

Two areas of interest:

1. How does the process of cleavage differ in different organisms?
2. What mechanisms regulate cleavage?
 Eggs are classified by how much yolk is present

1. Isolecithal eggs (iso = equal) have a small amount of yolk that is equally
distributed in the cytoplasm (most mammals have isolecithal eggs).
2. Mesolecithal eggs (meso = middle) have a moderate amount of yolk, and
the yolk is present mainly in the vegetal hemisphere (amphibians have
mesolecithal eggs).
3. Telolecithal eggs (telo = end) have a large amount of yolk that fills the
cytoplasm, except for a small area near the animal pole (fish, reptiles, and
birds).
4. Centrolecithal eggs have a lot of yolk that is concentrated within the
center of the cell (insects and arthropods).
 The pattern of cleavage of the zygote depends upon
the pattern of yolk distribution

1. Holoblastic cleavage: occurs in isolecithal eggs (mammals, sea urchins).

The entire egg is cleaved during each division.
2. Meroblastic cleavage occurs when eggs have a lot of yolk. The egg does
not divide completely at each division. Two types:

a. Discoidal cleavage is limited to a small disc of cytoplasm at the animal

pole. All of the yolk filled cytoplasm fails to cleave (characteristic of
telolecithal eggs such as birds).

b. Superficial cleavage is limited to a thin surface area of cytoplasm that

covers the entire egg. The inside of the egg that is filled with yolk fails
to cleave (centrolecithal eggs such as insects).
 Cleavage Plane
Cleavage plane: this is the plane in which cleavage occurs. It is oriented at
right angles to the metaphase plate. In sea urchins, the first cleavage is
meridional.
Meridional cleavage runs from one pole to another (top to bottom), like the
meridian on a globe.
The second cleavage is also meridional.
Equatorial cleavage encircles the zygote like the equator on the globe. This
creates an animal and vegetal half. The third cleavage in the sea urchin is
equatorial.
Sea urchin
Amphibia
A blastocoel forms within the morula  blastula
Fish
Birds, reptiles, and fishes have telolecithal eggs that completely
support embryogenesis; they undergo meroblastic cleavage
In contrast to holoblastic cleavage, where
the entire zygote divides into blastomeres,
meroblastic cleavage leaves a large
portion of the zygote uncleaved. There are
2 types of meroblastic cleavage, discoidal
and superficial.
Discoidal: In birds and reptiles, the 1st
cleavage is meridional. It starts at the
animal pole but does not progress far. The
2nd and 3rd cleavages are also meridional.
The 4th cleavage is equatorial, and it
creates a layer of small cells on top of the
huge uncleaved area below (yolk).
Blastoderm: when cleavage has
progressed such that there are many
blastomeres in the animal pole, it is a
blastoderm. Chicken eggs have a
blastoderm of about 60,000 cells when the
egg is laid.
Chick’s cleavage
In birds the yolk is so plentiful that it restricts cleavage to the
animal pole: meroblastic cleavage.
 Mammalian eggs have rotational cleavage that is holoblastic

The mammalian egg is a little slow. It begins to cleave in the oviduct and
continues until it implants in the wall of the uterus (1 cleavage / 24 hr).
Asynchronous cleavage: mammalian embryos are unusual in that they have
asynchronous cleavage. Not all blastomeres divide at the same time.
The first cleavage is meridional, and the second cleavage is rotational. The 2
blastomeres divide in different planes (one is equatorial and one is
meridional.
Mammalian cleavage
As the zygote travels down the fallopian tube towards the uterus, it
begins a series of cell divisions:

© 2001 Terese Winslow, U.S. Govt. has certain rights

Timing of cleavage divisions

Normal eukaryotic cells divide slowly, once

every several hours or days. The cell cycle
has G1 and G2 periods. During G1 the cell
synthesizes RNA and other components for
cell growth.

Cleavage consists of very rapid successive

mitoses. Since the egg has stored large
amounts of RNA and other material, it does
not need G1 or G2.

However, as the number of cells increases, the

nucleus / cytoplasmic ratio also increases.
The rate of cell division slows because the cell
now needs to synthesize its own RNA and
grow between divisions. Thus, G1 and G2 are
restored = midblastula transition.