Fertilization:

Beginning A New Organism

LEE KUI SOON

The importance of Gametogenesis and Fertilization

Research into how eggs and sperm are created can help us understand
infertility, which affects up 25% of couples in Europe.

In women, infertility can result from premature menopause (premature

ovarian failure), failure to ovulate, polycystic ovarian syndrome (PCOS) or
recurrent miscarriage.

In men, problems at all stages of spermatogenesis contribute to infertility.

Although estimates suggest up to 6,000 genes might be involved in
spermatogenesis, as yet only a few individual gene mutations have been
linked to male infertility.

In pre-menopausal woman and in adult men the sex organs are the major
sites of steroid hormone manufacture.
The importance of Gametogenesis and Fertilization

These steroids - androgens and oestrogens - are not only essential for
formation of eggs and sperm but also have an important impact on the
function of other parts of the reproductive system such as the endometrium
(lining of the womb) in women, and the prostate in men.

In addition the steroids made in the ovaries and testes circulate around the
body and affect the function of the heart, the brain, the skeleton and the
immune system and can contribute to the formation of cancers in tissues
including the breast and prostate.

Health Targets are:

• Infertility
• Premature ovarian failure
• Polycystic ovarian syndrome
• Recurrent miscarriage
• Cancer
The importance of Gametogenesis and Fertilization

Research activities

Investigations at the Human Reproductive Sciences Unit into the formation

of female gametes are aiming to address three different questions:

How do ovarian follicles form and what factors influence the number of
follicles a woman forms during her lifetime?

How is the formation of new blood vessels (angiogenesis) in ovarian

follicles regulated?

How is follicle growth and death regulated?

 Fertilization
Process whereby two sex cells (gametes) fuse together to
create a new individual with genetic potential derived from
both parents

Four major steps:

1. Contact between sperm and egg.
2. Entry of sperm into the egg.
3. Fusion of nuclei of sperm and egg.
4. Activation of egg metabolism.

Accomplishes 4 things:
1. Restoration of diploid genome.
2. Species-specific sperm-egg binding
3. Egg activation
4. Contribution of paternal centrosomal materials by the sperm
 Structure of sperm
Each sperm consists of haploid nucleus, a compulsion
system to move the nucleus and a sac of enzyme that enable
the nucleus to enter the egg

The centriole produces a long flagellum at what will be

the posterior end of the sperm and the Golgi apparatus
forms the acrosomal vesicle at the future anterior end.
The mitochondria (hollow dots) collect around the
flagellum near the base of the haploid nucleus and
become incorparated in to the midpiece of the sperm.
The remaining cytoplasm is jettisoned and the nucleus
condenses. The size of the mature sperm has been
enlarged relative tot eh other stages
Structure of sperm
 Structure of an egg
(A)

(A) structure of the sea urchin egg during fertilization. The drawing also shows the relative sizes of egg
and sperm

(B) Scanning electron micrograph of sea urchin sperm bound to the vitelline envelope of an egg.
Structure of hamster egg
 The egg

All materials necessary for he beginning of growth and

Development must be stored in the mature egg
In contrast to sperm, the developing egg (oocyte) not only
Conserve its materials but is actively accumulating more
The egg cytoplasm behave like a storehouse where the trove
Include
Protein
Ribosomes and tRNA
Messenger RNA
Morphogenesis factors
Protective chemicals
 Recognition of Egg and Sperm

The interaction of sperm and egg generally proceeds

according to five basic steps

• The chemoattraction of the sperm to the egg by soluble molecules

secreted by the egg

• The exocytosis of the acrosomal vesicle to release it enzymes

• The binding of the sperm to the extracellular envelope (vitelline layer

or zona pellucida) of the egg

• The passing of the sperm through this extracellular envelope

• Fusion of egg and sperm cell plasma membrane

Recognition of Egg and Sperm
 Recognition of Egg and Sperm
Meeting between a sperm and an egg is not a simple matter
Eg: Many marine organisms release their gametes into
environment (small as tide pool or large as an ocean).
Moreover, it is shared with other species that may shed their
sex cells at the same time.

Questions:
How can sperms and eggs meet in such a dilute
concentration?
How can sperm be prevented from trying to fertilize eggs of
another species?

* Species-specific attraction of sperm and species specific

sperm activation
 Sperm Attraction: Action at a distance
Species-specific sperm attraction has been documented in
numerous species including cnidarians, molluscs,
echinoderms, and urochordates.

Sperm are attracted toward eggs of their species by

chemotaxis (following a gradient of a chemical secreted by
the egg)

The mechanism of chemotaxis differ among species

Resact is a chemotactic molecule isolated from egg jelly of

the sea urchin which could attract the sperm migration

Resact also acts as a sperm-activating peptide

Sperm chemotaxis in Sea Urchin
 The acrosomal reaction in sea urchin
Acrosomal reaction has two components:

1. Fusion of the acrosomal vesicle with the sperm plasma

membrane (an exocytosis that results in the relese of the
contents of the acrosomal vesicle)

2. The extension of the acrosomal process

This reaction is initiated by the contact of the sperm with the

egg jelly and this contact causes the exocytosis of the
sperm’s acrosomal vesicle and the release of proteolytic
enzymes that can digest a path through the jelly coat to the
egg surface
The acrosomal reaction in sea urchin
 The acrosomal reaction in hamster sperm

Acrosomal reaction in hamster sperm. (A) Transmission electron micrograph of hamster

sperm undergoing the acrosomal reaction. The acrosomal membrane can be seen to form
vesicles. (B) Interpretive diagram of electron micrographs showing the fusion of the
acrosomal and cell membranes in the sperm head.
 Species Specific recognition in Sea Urchin
Bindin is the acrosomal protein mediating the recognition
between sperm and egg in sea urchin
Biochemical studies showed that bindins of closely related
sea urchin species are indeed different
Species-specific binding of
acrosomal process to egg cell
surface in sea urchins. (A)
Actual contact of a sea urchin
sperm acrosomal process with
an egg microvillus (B) In vitro
model of specirs-specific
binding. The agglutination of
dejellied eggs by bindin was
measured bu adding bin
aggregates to a plastic well
containing a suspension of
eggs. After2-5 minutes of
gentle shaking, the wells were
photographed. Each bindin
bound to and agglutinated only
egs from its own species
 Gamete binding and recognition in mammals

Zona pellucida in mammals is equivalent to viteline envelope

in invertebrate

This matrix is synthesized and secreted by the growing

oocyte and plays two major roles during fertilization:

1. It binds the sperm

2. It initiates the acrosomal reaction after the sperm is bound

 Gamete binding and recognition in mammals

(ZP3): The sperm binding protein of the mouse zona

pellucida

Specific glycoprotein in the mouse zona pellucida to which

the sperm bind

Also initiates the acrosomal reaction after sperm have bound

to it.
 Induction of the mammalian acrosomal
reaction by ZP3

Unlike the sea urchin acrosomal reaction, the acrosomal

reaction in mammals occurs only after the sperm has bound
to the zona pellucida

The acrosomal reaction differences between sea urchin and

mouse may be due to the thickness of the extracellular
envelope surrounding the egg.

Secondary binding of sperm to the zona pellucida

 Gamete Fusion
Fusion of the egg and sperm plasma membrane

Recognition of sperm by the vitelline envelope or zona

pellucida is followed by the lysis of that portion of the
envelope or zona in the region of the sperm head by
acrosomal enzymes

The lysis is followed by the fusion of the sperm plasma

membrane with the plasma membrane of the egg

In sea urchin, all regions of the egg plasma membrane are

capable of fusing with sperm (not apply to every species)

“Fusogenic protein” play important in this process. In sea

urchin, bindin plays a secondary role while in mammal fertilin
are essential for sperm membrane-egg membrane fusion
Sea Urchin fertilizaiton
Golden hamster egg fertilization
 The prevention of polyspermy
As soon as sperm has entered the egg, it is crucial to assure
that ONLY one sperm may fertilize the egg.

Monospermy: One sperm enter the egg

Polyspermy: more than one sperm enter the egg (lead to

disasterous consequences in most organisms)

Species have evolved to prevent the union of more than two

haploid nuclei. Eg: In sea urchin, the egg has two
mechanisms to avoid poluspermy

A fast reaction ( an electric change in the egg)

A slow reaction (exocytosis of the cortical granules)
 The fast block to polyspermy

The fast block to polyspermy is achieved by changing the

electric potential of the egg plasma membrane. However, this
process is transient.

Unfertilized sea urchin eggs have a negative charge inside,

but the charge becomes positive upon fertilization

The membrane provides a selective barrier between the egg

cytoplasm and the outside environment and the ionic
concentration of the egg differs greatly from that of its
surroundings
 The slow block to polyspermy

The egg of sea urchin develop second mechanism to prevent

polyspermy. This is because the fast block is transient and is
not sufficient to prevent polyspermy.

During the fast block mechanism, polyspermy could still

occur because the vetelline envelope are not removed

This removal is accomplised by the cortical granule reaction,

a slower, mechanical block to polyspermy that becomes
active about a minute after the first successful sperm-egg
attachment
Envelop formation & sperm removal
Formation of the fertilization
envelope and removal of
excess sperm. To create
these photographs, sperm
were added to sea urchin
eggs and the suspension
was fixed in formaldehyde
to prevent further reactions.
(A) At10 seconds after
sperm addition, sperm are
seen surrounding the egg.
(B,C) At 25 and 35 seconds
after insemination, a
fertilization envelops is
forming around the egg,
starting at the point of
sperm entry. (D) the
fertilization envelope is
complete, and excess sperm
have been removed
Cortical granule exocytosis
The End

LEE KUI SOON