DEVBIO321. LESSON 3 4.

Activation (of the zygote):

FERTILIZATION Cortical reaction (fusion of
cortical vesicles with the egg
FERTILIZATION plasma membrane) and
 The process by which the sperm pronuclear fusion
initiates and participates, with
the egg, in the development of FERTILIZATION PROCESS
the embryo.
 The process of fertilization
involves complex interactions
between egg and sperm. It
includes biochemistry and
binding and activation of
molecular receptors on the
sperm head and egg.

Where does fertilization occur?

 usually occurs in first 1/3 of What are the factors that affect the
uterine tube (oviduct, Fallopian sperm’s potential for successfully
tube) fertilizing the egg?
 can also occur outside uterine
tube associated CAPACITATION
 with Assisted Reproductive  Occurs in the female’s vagina.
Technologies (IVF, GIFT, ZIFT...)  Vaginal secretions cause a
and ectopic pregnancy molecular change in the sperm
o IVF (In-vitro Fertilization) plasmalemma (removal of
decapacitating factor – semen
o GIFT (Gamete Intrafallopian
proteins, results in increased
Transfer)
membrane fluidity).
o ZIFT (Zygote Intrafallopian
 Takes 4-5 hrs. in humans, 1 hr. in
Transfer)
mice, 6 hrs. in rabbits
STAGES OF FERILIZATION
Capacitation is a functional
1. Preparation: Capacitation and
maturation of the spermatozoan.
acrosome reaction. Acrosomal
 The changes take place via the
vesicle fusion is the membrane
sperm cell membrane in which it
fusion event of this stage.
may be that receptors are made
2. Binding: Species-specific
available through the removal of
interaction of gametes.
a glycoprotein layer. The area of
3. Fusion: Merging of sperm and
the acrosomal cap is also so
egg plasma membranes
altered thereby the acrosome
(membrane fusion event)
reaction becomes possible.
 Capacitation is what one calls the 2. Portio / cervix
changes that lead to 3. Cervix canal
hyperactivity of the 4. Isthmus
spermatozoon and which later 5. Ampullary part of the fallopian
allow the spermatozoon to go tube (ampulla)
through the acrosome reaction. 6. Ovary with attached Fimbria
7. Endometrium
EFFECTS OF CAPACITATION ON 8. Myometrium
SPERM 9. Cavum uteri
 Increased rate of metabolism. 10. Meeting place of the sperm
Flagellum beats more rapidly > cells with the oocyte
Sperm are more motile
 Changes in sperm plasmalemma THE CERVICAL CANAL
proteins allow sperm-egg
binding and occurrence of the
acrosome reaction
 Pro-Acrosin (inactive) is
converted to acrosin (active)
 Sperm become capable of
chemotaxis

* Galactosyltransferase protein in the

 Gametes Involved –Sperm are
sperm plasmalemma that MAY act as
small, asymmetrical and motile
the receptor that binds to the zona
cells
pellucida and initiates the acrosome
reaction.

PATH OF THE SPERM CELLS TO

THE OOCYTE- CAPACITATION

1. Tail: contains the flagellar

apparatus, which is composed of
“9 + 2” microtubules and
accessory structures. The sliding
of the microtubule is powered by
the protein dynein.
2. Midpiece: contains a sheath of
1. Rear part of the vaginal cavity
mitochondria, which produce the
 ATP necessary for the beating of
the tail.
3. Head: contains the spermatic
haploid nucleus. Overlaying the
head is a membrane bound
vesicle, the acrosome; Sperm do
not possess any organelles
associated with protein synthesis
(Golgi, RER or lysosomes).

CAPACITATION OF THE SPERM 1. Center of the oocyte

2. Corona radiata (surrounds and
partly covers the oocyte)
3. Head of the spermatozoon

ACROSOMAL REACTED
SPERMATOZOON

7 Membrane residues dropping

behind
The sperm cells meet the oocyte- A Head
THE ACROSOME REACTION B Neck
 Penetrating the cumulus cells C Mid-piece
 Hyaluronidase dissolves the
intercellular matrix between the
cumulus cells, other enzymes
dissolve the pellucid zone that
lies around the oocyte
 Oocytes surrounded by
 cumulus cells hyaluronidase ACROSOME REACTION
dissolves the intercellular matrix  A reaction that occurs in the
between the cumulus cells, other acrosome of the sperm as it
enzymes dissolve the pellucid approaches the egg.
zone that lies around the oocyte  As the sperm approaches the
zona pellucida (necessary for
initiating the acrosome reaction),
the membrane surrounding the
acrosome fuses with the plasma
membrane of the sperm's head,
exposing the contents of the
acrosome allowing fertilization to
occur.
 The contents include surface
antigens necessary for binding to
the egg's cell membrane, and
numerous enzymes which are
responsible for breaking through
the egg's tough coating and
allowing fertilization to occur.

 How does the sperm get through  Wassermann and co-workers

the barriers surrounding the egg? (1980, 1985, 1987, 1988). Found
o Used to be thought that the that zona pellucida is composed
acrosome enzymes did it all of 3 glycoproteins ZP1, ZP2, ZP3
 Sperm arrives at zona pellucida  Repeating subunits of ZP2 and
with the acrosome still intact. So, ZP3 form filaments that are
how does it get through the bound together by ZP1
follicle cells that surround the  Wassermann et al. found that
egg? when the sperm binds to ZP3, it
o It has to break down the causes a change in Ca+2 and Na+
intercellular cement that flux across the sperm
holds the follicle cells plasmalemma that results in the
together - hyaluronic acid acrosome reaction. (ZP1 and ZP2
 Lin and co-investigators (1994) will not cause this to happen).
demonstrated that a protein  The sperm actually binds to an o-
found in the sperm plasmalemma linked oligosaccharide that is
(PH-20) had hyaluronidase part of ZP3 - (a carbohydrate
activity. component of the glycoprotein)
 The identity of the receptor in
WHAT HAPPENS WHEN THE the sperm plasmalemma that
SPERM GETS TO THE ZONA binds to this oligosaccharide is,
PELLUCIDA? as yet, uncertain. It has been
 Attachment - loose association suggested that a part of PH-20 is
 Binding - strong attachment the receptor; however, more
 Acrosome reaction - release of recent work indicates this may
enzymes not be the case.
 Penetration of the zona pellucida  When the acrosome reaction
by the sperm occurs, a number of proteolytic
enzymes are exposed or released.
HOW IS THIS ACCOMPLISHED?  One or more of these enzymes is
responsible for digesting the hole
through the zona pellucida
 through which the sperm enters spermatozoon has bound
the perivitelline space. itself with the oolemma.
o The "contents" of the sperm
THE DOCKING MECHANISM OF cell are completely taken into
THE SPERMATOZOON ONTO THE the cytoplasma of the oocyte.
OOCYTE (THE KEY-LOCK
PRINCIPLE) THE DOCKING TRIGGERS A
CASCADE OF EVENTS WITH THE
 Acrosomally reacted FOLLOWING GOALS:
Spermatozoon in Perivitelline  Polyspermy block: The
space penetration of further sperm
o Keys and locks are receptor cells should be hindered
Proteins, inserted into the  Hardening of the pellucid zone as
cell membranes of a mechanical protection of the
Spermatozoon and oocyte embryo
and having great mutual  Entry of the spermatozoon into
affinity. the oocyte
o When the acrosomally  Termination of the 2nd meiosis
reacted spermatozoon of the oocyte with expulsion of
encounters the oolemma the 2nd polar body
following the penetration of  Preparation at the molecular
the pellucid zone and touches level of the oocyte for unpacking
it along its length, the the paternal DNA
reciprocal membrane
receptors bind with each THE POLYSPERMY BLOCK
other.  The docking triggers a rapid
wave of depolarization in the
oolemma, leading to changes in
the membrane surface

1. Post-acrosomal region
2. Oolemma with microvilli
3. Perivitelline space
4. Pellucid zone

 Docking of the spermatozoon  Pellucid Zone

o The post-acrosomal  Perivitelline Space
membrane region of the  Corical Vesicle
 Oolema
 fuses with the egg, only one male
Blocks to Polyspermy pronucleus survives to fuse with
1. Dilution the female pronucleus. - Does not
2. Fertilizin like secretions cause occur in mammalian eggs.
agglutination of sperm in some Thus, only
invertebrate species a. dilution of the sperm and
3. ‘Hardening” of vitelline b. Hardening of the zona pellucida
membrane or zona pellucida that due to the contents of the cortical
results from cortical granule granules act to prevent
rupture—slow block to polyspermy in most mammals,
polyspermy, takes 1-5 minutes including humans.
4. Depolarization of the oolemma-
membrane potential changes THE ENTRY OF THE
from negative to positive, sperm SPERMATOZOON INTO THE
cannot fuse with positively OOCYTE (IMPREGNATION)
charged oolemma- fast block to  The membrane of the sperm cell
polyspermy, takes 1-2 seconds. head and mid piece coalesces
Caused by influx of sodium ions. with the oolemma and all of the
o Depolarization lasts longer sperm cell contents enter into the
than 5 min, but eventually oocyte. The tail membrane
the potential difference remains as an appendage
across the oolemma returns
to its original value.

o The change in membrane

potential happens in many
species, but does not occur
in mammals 1. Oolema
5. Hydrogen peroxide release by 2. Cell Membrane of the
the eggs of some invertebrate Spermatozoon
species (e.g. sea urchins) - 3. Kinocilium
inactivates sperm - Does not 4. Nucleus (Compact) of the
occur in mammalian eggs. Spermatozoon
6. Unknown mechanisms that cause 5. Centrosome of the Spermatozoon
degeneration of extra sperm
nuclei after they have entered the  The genetic material, lying in the
egg cytoplasm. Seen in nucleus and coming from the
salamanders. As a result, even father, is unpacked and is used
though more than one sperm
 for building the paternal  The three polar bodies can be
pronucleus. recognized: The second polar
 The centrosome plays an body anf the first polar body that
important role in the has divided.
convergence of the two pronuclei
and responsible for building the THE FERTILIZATION IS
first division spindle of the new COMPLETE. THE FORMATION OF
creature. All centrosomes in the THE ZYGOTE.
bodily cells of a human originate (Introduction into the creation
from that of the father. and development of the
 Other sperm components pronuclei.)
transferred to the oocyte
cytoplasm, like the kinocilium, The formation of the paternal
are dissolved. Thus, all pronucleus.
mitochondria in the bodily cells  The unpacked DNA is enclosed in
of an individual normally derive the slowly forming paternal and
from the mother alone. maternal pronuclei.
 During a synthesis phase, the
TERMINATION OF THE SECOND duplication of the DNA occurs in
MEIOSIS OF THE OOCYTE them.
(Secondary oocyte at the moment  After the synthesis phase, which
of impregnation) takes almost a day, the DNA
 The condition in the secondary condenses into chromosomes,
oocyte, which was created These promptly order
shortly before ovulation: themselves on the zygote’s
 The mitotic spindle, arrested in mitotic spindle so this can divide
the metaphase of the second into the two-cell stage.
meiosis, becomes active again
due to the impregnation. From The formation of the maternal
the docking and impregnating pronucleus.
spermatozoon a signal for a  At the same time that the
resumption and termination of paternal pronucleus forms, the
the second oocyte meiosis is maternal pronucleus is also
triggered. One speaks now of an generated.
impregnated oocyte.  The penetration of the
 The 2nd polar body being formed. spermatozoon into the secondary
Beside it the first polar body is oocyte instigated the resumption
visible while dividing the DNA of and termination of its second
the spermatozoon nucleus has meiosis. After the 2nd polar body
begun to unpack itself. is created the maternal
chromosomes that remain in the
 oocyte are enclosed by a nucleic 2. Maternal pronucleus
membrane and decondensed. 3. Paternal centrosome
4. "Inner bodies"
Approach of the pronuclei 5. Maternal astral microtubule

18hrs after impregnation HOW CAN ONE DISTINGUISH THE

 The maternal and the paternal PATERNAL PRONUCLEUS FROM
pronuclei have grown to their THE MATERNAL ONE?
maximal size. They have  The maternal pronucleus is
approached each other closely nearer to the polar bodies.
and the centrosome has divided  The paternal pronucleus is
in preparation for the upcoming slightly larger
cell division.
20hrs after impregnation
 Impregnated oocyte 20 hours
after an assisted fertilization. The
cumulus cells are routinely
peeled away.
 The pronuclei have maximally
converged, but they are not
clearly recognizable
6hrs after impregnation
 An aster has formed out of what
was originally the paternal
centrosome. In the pronuclei the
so-called «inner bodies» can be
seen, which are lined up as can
typically be observed.

Formation of Zygote
 22 hours after the fertilization a
mitotic spindle has formed.
 The nucleic membranes of the
two pronuclei dissolve. This is
the visible sign that the zygote
has been created.

1. Paternal pronucleus
 EGG ACTIVATION
 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
 The mitotic spindle divides the
development.
chromosomes that have just been
brought together into the two
Why “frees the egg”? Why not
first cells of the embryo. This
“frees the egg and sperm”?
proceeding towards the two-cell
1. Egg activation does not require
stage occurs on average between
the sperm nucleus
22 and 26 hours after
2. In some species, just pricking the
fertilization.
egg with a needle, shocking it, or
simply changing the pH of the
FOLLOWING FUSION OF THE
solution it’s in will cause
SPERM WITH THE EGG
activation.
3. In some species eggs that are
1. The male nucleus enters the egg
activated this way will start to
cytoplasm and becomes the male
develop. Thus, we have a
pronucleus.
mechanisms that could allow for
2. As a result of the sperm fusing
selection of parthenogenesis.
with the egg plasmalemma, the
oocyte nucleus, which is at
Seven events that characterize egg
metaphase of the second meiotic
activation following fusion of the
division, completes that division
sperm with the egg:
giving rise to another polar body.
3. Following the second meiotic
1. Release of Ca++ (calcium) stored
division, what is now the nucleus
in the egg endoplasmic reticulum.
of the ovum becomes the female
2. Cortical reaction - rupture of
pronucleus.
cortical granules that occurs
4. The haploid male and female
concurrently with the Ca++
pronuclei move toward one and
release.
other, meet, and fuse to form the
o Contents of granules are
diploid nucleus of the zygote.
released into perivitelline
5. The zygote will now proceed to
space and cause “hardening”
undergo cleavage.
 of the vitelline membrane or 3. The genetic sex of the future
zona pellucida. embryo is determined by the
o Causes vitelline/ fertilization chromosomal complement of the
membrane to rise away from spermatozoon. (If the sperm
surface of egg in some contains 22 autosomes and an X
species. chromosome, the embryo is a
3. In many species, an influx of Na+ genetic female, and if it contains
(sodium) into the egg cytoplasm 22 autosomes and a Y
that causes a change in chromosome, the embryo is a
membrane potential- fast block male.
to polyspermy. 4. Through the mingling of
4. In many species a reorganization maternal and paternal
of the egg cytoplasm. chromosomes, the zygote is a
5. In most cases, completion of genetically unique product of
meiosis by the egg. chromosomal reassortment,
6. An efflux of H+ (hydrogen) ions which is important for the
causing an increase in viability of any species.
cytoplasmic pH - this activates 5. The process of fertilization
previously inhibited synthetic causes metabolic activation of the
pathways. egg, which is necessary for
7. Increase in metabolism - zygote cleavage and subsequent
gears up for development. embryonic development to occur.
SUMMARY:
EVENTS THAT OCCUR SOON AFTER  The zygote - by definition the
EGG ACTIVATION: first cell of the embryo –
1. DNA replication as male and undergoes only an incomplete
female pronuclei approach each cell cycle.
other  With the penetration of the
2. Male and female pronuclei merge sperm cell, the genetic
3. Preparation for first cleavage information of the two parents
are not instantly brought
WHAT IS ACCOMPLISHED BY together in order that they are
FERTILIZATION? then duplicated together and
The process of fertilization ties distributed in the subsequent
together many biological loose ends, mitosis. In reality, the paternal
as follows: and also the maternal genetic
1. It stimulates the egg to complete information are first duplicated
the second meiotic division. in two independent pronuclei
2. It restores to the zygote the and also there condensed again
normal diploid number of into chromosomes so they can
chromosomes (46 in humans).
 align themselves along the
equator of the mitotic spindle.
 The goal of the fertilization
cascade is thus achieved:
o The fabrication of a diploid
set of chromosomes
o The determination of the
chromosomal gender of the
new individual
o The induction of normal
"cleavage division" for
embryogenesis.