Oocyte Maturation and Fertilization Processes

Maryam Borhani-Haghighi
PhD student
of Tehran Medical
University

Maryam Borhani-Haghighi 11
Oocyte maturation
 Nuclear maturation
Cytoplasmic maturation

Oocyte continues meiosis.
Nuclear membrane of oocyte disappears.
First polar body separates, and it enters the perivitelline space.
Second meiotic division takes place and stops in the metaphase II.
This process is known as the maturation of oocyte nucleus.
Nuclear maturation

Nuclear maturation
Maturation Promoting Factor (MPF) activity during oocyte maturation. MPF activity appears just
before germinal vesicle breakdown (GVBD). It falls down at the end of Meiosis I and reappears at the
beginning of Meiosis II (MII). It remains high during MII-arrest of oocytes and decreases following
fertilization..

14
Cytoplasmic maturation
Cytoplasmic
maturation
Organelle
redistribution
Mitochondria
Endoplasmic
reticulum
(ER)
Cortical
granules lipid droplet
Protein
synthesis
Cytoskeleton
dynamics

Mitochondria
 Before the beginning of oocyte maturation. Homogenous distribution of
mitochondria in the cell cortex is observed
 After nuclear maturation, mitochondria are dispersed throughout the cytoplasm
except the very central region of oocytes.
BB - Balbiani body
M - mitochondria
n - nucleus

Endoplasmic reticulum (ER)
 ER :protein and lipid synthesis organelle
 store of Ca2+ oocyte activation in during fertilization.
 In mature oocytes, an accumulation of ER in the oocyte cortex,
specifically in the region with cortical granule exocytosis and sperm-egg fusion

Cortical Granule (CG)
 Exocitosis of cortical granules plays an essential role in the block against
polyspermy.
 In immature oocytes, CGs are found in the whole cytoplasm (the cortex
cytoplasm and the inner cytoplasm)
 In mature oocytes, an asymmetric distribution of CGs in the cortex is observed.

Lipid droplets (LD)
 Energy supply necessary for meiotic, maturation, fertilization and early embryo development
 In GV - stage oocytes, a homogenous distribution has been reported.
 In mature oocytes, Lipid droplets contribute to the oocyte polarization by surrounding the MII
spindle.

20
Cytoskeleton dynamics
 During the GV stage, microfilaments are distributed throughout the cytoplasm,
and during GVBD they migrate toward the oocyte cortex.

The sperm has to pass several barriers to enter
the egg.
1.layer of cumulus cell.
2. zona pellucida

Zona pellucida

The zona pellucida (plural zonae pellucidae, is a glycoprotein membrane
surrounding the plasma membrane of an oocyte.
Zona pellucida

35
ZP3 mediates Sperm-Specific Egg Binding
ZP2 mediates subsequent sperm binding
ZP1 cross-links ZP2 and ZP3 as protein meshwork
Zona pellucida

 As a receptor
 Inducer of Acrosomal Reaction
 Blockade of heterospecific fertilization. Species-specific barrier
to sperm binding and penetration;
 Prevention of polyspermy
 Protection of the embryo before implantation
Functions of the ZP include

• ZP keeps sperm of foreign species out .( Not
100% effective; some cross-species fertilization
can occur (e.g., horse & donkey = mule; some
speciesof monkeys can cross fertilize) )
• Remove the ZP and other species sperm can
fertilize and egg (e.g., Hamster Test for Male
Fertility; human sperm can fertilize the zona-less
hamster egg)

• The mutant mice which lacked ZP3 couldn't bind
the sperm to the zona pellucida and thus did not
get fertilized
• When their missing ZP3 gene was replaced with a
human ZP3 gene, the mice made the human ZP3
and it was incorporated into their zona pellucida.
With the human ZP3 in their zonas the eggs could
be fertilized by mouse sperm revealing that
human and mouse ZP3 can mediate the same
critical events.

34
Zona pellucida
Inner zone
Outer zone
Birefringence

37
(a): Outer surface of the ZP of a human mature oocyte. Many fenestrations
are present in which the filaments form a large meshed network
(b): Outer surface of the ZP of a human atretic oocyte. The filaments form a tight
meshed network.

38
(b): The ZP appears completely compact and with a smooth surface.
(d): High magnification of (b) showing the compact and dense structure of the zona.
Human atretic oocyte

This metaphase II oocyte displays an atypical aspect of the
ZP. Projections from the ZP give the oocyte a hairy, brush-
like appearance.
Abnormality of the zona pellucida

Capacitation of sperm

43
Capacitation of sperm
Reduction in membrane cholesterol increase fludity
Hyperactivaty of sperm ;lateral movement of head
Removal of coating factors
Only capacitated sperm can bind to the ZP

Maturation-Ejaculation-Capacitation
Leaving testis-Epididymis-seminal plasma-Oviduct

5 sets of molecular changes are
considered important for capacitation:
1.Sperm membrane sheds cholesterol by albumin
allowing for zona pellucida recognition
2.Protein/carbohydrate loss opens sperm-egg
recognition sites
3.Membrane potential of the cell membrane become
more negative activating cAMP production
4.Protein phosphorylation occurs forming a receptor
5.Acrosomal membrane changes

Reduction In Membrane Cholesterol

Cholesterol efflux during the early phases of capacitation
increases plasma membrane fluidity,
facilitating the entry of bicarbonate (HCO3-) and calcium ions
(Ca2+) into the sperm cytosol through specific membrane
channels.

Model of mammalian sperm
capacitation
soluble
adenylyl
cyclase
(SACY)

which then simultaneously inhibits the activity of tyrosine phosphatases (PTP) and
activates tyrosine kinases (PTK).
cAMP, activate protein kinase A (PKA),
leading to the production of the second messenger cyclic AMP (cAMP).
soluble adenylyl cyclase (SACY) is activated by increases in intracellular bicarbonate, calcium and
pH
increase in protein tyrosine phosphorylation
capacitation

• membrane remodeling events(Cholesterol )
redistribution of membrane rafts to the anterior
region of the sperm head.
• This event may serve to reposition key zona
pellucida receptor molecules
• membrane remodeling events may be
augmented by the action of chaperones that are
themselves activated during capacitation.

EGF
 Epidermal growth factor
(EGF)interacts with its receptor
tyrosine kinase (EGFR)
 stimulates the tyrosine
phosphorylation of several proteins
enhances actin polymerization
during capacitation

Hyperactivaty of sperm
 CatSper, sperm cation channel
that is localized in a principal
piece of the mature sperm tail.
 gene knockouts that are CatSper
have poorly motile sperm that
completely fail to fertilize eggs

Role of actin cytoskeleton in mammalian sperm
capacitation
• In human sperm the regions reported to contain actin include the acrosomal space,
the equatorial and post acrosomal regions, and the tail.
 actin polymerization and depolymerization might be involved in sperm
function.
 actin polymerization occurs during capacitation
 F-actin breakdown should occur in order to achieve the acrosome reaction.
inhibition of actin depolymerization by phalloidin inhibits the reaction.

Sperm penetration to corona radiata

 hyaluronic acid–rich layer of cumulus cells surrounds the ovulated
oocyte.
Sperm penetration to corona radiata

Acrosomal reaction & penetration to zp

The Acrosome Reaction:
• When sperm contacts the egg cell layers, the it
has an intact acrosome.
• stimulation of the sperm by agents from the
corona radiata followed by binding to the
zona pellucida, leads to the acrosome reaction

The Acrosome Reaction:
Galactosyltransferase binding
• Here we will look at only one of the classic
mechanisms of sperm-egg binding that leads
to the acrosome reaction:
Galactosyltransferase binding that occurs at the zona pellucida.

Galactosyltransferase
• Each sperm has galactosyltransferase
(GalTase) enzymes on its head
• Galactosyltransferase is an enzyme that
transfers a sugar group from one molecule to
another

Unlike normal enzyme reactions in which the enzyme binds its
substrate and produces products, this enzyme reaction cannot go
to completion. Since it can’t complete the reaction the enzyme
and the substrate remain attached to each other.

• Each sperm binds N-acetylglucosamine, GlcNAc)
residues in ZP3 via galactosyltransferase (GalTase)
enzymes in the sperm cell membrane. Thus the
GlcNAc is the substrate for the GalTase enzyme.
• so the sperm remain attached via the sugar-enzyme
binding because the enzyme reaction cannot go to
completion .

ZP3 and the Acrosome Reaction

• As more GalTase enzymes bind more GlcNAc substrates the receptors
on the sperm head cluster together
• This clustering alters the sperm cell membrane causing calcium levels
to increase in the sperm cytoplasm
• The increase of intracellular calcium mediates the fusion of the
acrosomal and sperm cell membranes
• This allows the contents of the acrosome to flow out
• The released Acrosomal enzymes now begin to digest a path for the
sperm through the zona pellucida

Calcium & the Acrosome Reaction

In freshly ovulated eggs, ZP3 is in very close physical association with ZP2.
Acrosome-reacted sperm bind to ZP2 via their exposed inner acrosomal membranes, penetrate the
zona, fuse with the oocyte, and fertilize the egg.

Immediately after fertilization, cortical granules release a ZP2-
specific protease and other enzymes into the perivitelline
space.
This cortical granule protease clips ZP2 and converts it into the
cleaved form (ZP2c) that no longer supports the binding of
acrosome-reacted sperm.
ZP3 dissociates from ZP2c, and undergoes a subtle modification that
converts it into a form (ZP3f) that lacks sperm receptor and acrosome-
inducing activity.

Hyaluronidase
neuraminidase
acrosin
protease
trypsin-like enzyme
Arylsulfatase
PLC
Esterase

 Ca2+-
Calcium is an important modulator for capacitation and AR
and is probably the key messenger in the information
exchange between sperm and egg .
 Progestrone
 Follicular fluid
 Coffein
 K+ and Na+
Acrosomal reaction

SOC
Store-operated
channels
Progesterone
cause Ca2+influx
::::::Elevation of
Ca2+in the
midpiece/proxim
al flagellum
::::::leading to
regulation of
flagellar activity

What is progesterone?
Progesterone is a steroid hormone
made by both men and women.
In women, it is made
the corpus luteum of the ovary
the adrenal glands.
In men, is produced
in adrenal
testicular tissue.Maryam Borhani-Haghighi

Result of Acrosomal Reaction
Acrosomal bulb
Ca / cAMP
Ph (7.1 7.5)
Zp Zp2f

Sperm oolemma binding & fusion

Sperm oolemma binding & fusion
• The observation that acrosome intact sperm can bind to zona-free
eggs, but do not fuse with them, suggests that membrane alterations
occurring during the acrosome reaction are required for fusion.

Sperm Binding to the Zona & the Egg
Cell Membrane
• several proteins that mediate these
membrane interactions, fertilin b. Mouse
mutants that lack fertilin b have a markedly
reduced fertility.

The sperm plasma membrane
• Fertilin α
• Fertilin β
• Cyritestin
• ADAM 4
• ADAM 5
• IZUMO

molecules on the
sperm surface, such
as fertilin and
cyritestin3, involved
in sperm–egg binding
Izumo is essential for
membrane fusion
On the egg, CD9 is
required for fusion
and might
collaborate with
other proteins such
as integrins or
glycosylphosphatidyl
inositol (GPI)-
anchored proteins

• members of a new family of membrane proteins, the ADAM family (a
disintegrin and metalloprotease disintegrin
• cysteine-rich
• EGF-like
Fertilin
• fertilin, a heterodimeric (α and β subunits) sperm membrane protein, is
involved in the fusion process.
• This protein is located on the posterior head of acrosome-reacted sperm.

• Localization of cyritestin in the equatorial region is consistent with its
participation in sperm–egg fusion.
• Although both the equatorial regions and posterior head of the sperm
membrane fuse with the egg membrane,
cyritestin

The Egg
.CD9
.CD81
Glycosylphosphatidyl
inositol (GPI)-anchored

• immunofluorescence microcopy shows that CD9 is localized to the microvillar-
rich region of the egg
CD9

CD81
• CD81, which fairly closely resembles CD9 in structure
and some functions, has a role in gamete fusion.
However, the results of deleting the CD81 gene are
less dramatic than those seen with the deletion of
CD9.

Sperm oolema binding & fusion
The egg plasma membrane can be divided into two major regions.
1.The part of the membrane that directly overlies the metaphase
chromosomes has a smooth surface devoid of microvilli.
2. The remainder of the egg is rich in microvillar protrusions. This is the region
of the egg where sperm both bind and fuse.

Fertilization cone

Block to Polyspermy
• Special blocks to polyspermy exist:
• 1. Fast Block: electrical change in egg
membrane
2. Slow Block: modification of zona pellucida

Slow block to polyspermy
• Elavation of Ca2+
• Cortical granule reaction
Exocytosis Perivitteline space
 Zp2
 β-hexoaminidase
 Tyrosin

Slow block of polyspermy
The Cortical Reaction
• Calcium not only plays a role
in the acrosome reaction, it
also mediates the subsequent
event of corticalgranule
exocytosis in the egg.
• Ca2+ induces local exocytosis
of cortical granules
• Granules release to stimulate
adjacent cortical granules to
undergo exocytosis
• Wave of exocytosis occurs
around egg in 3 dimensions
from original site of sperm
entry Maryam Borhani-Haghighi

Calcium signals at fertilization
• A “Ca2+ wave” that starts from the site of sperm–egg fusion
and propagates across the egg cytoplasm has been
extensively analyzed

Fast block of polyspermy
• is the electrical charge across the surface of the egg, which
is caused by the fusion of the first sperm with the egg.
 Elevation of intracellular PH
 Change the membrane potential
 Resting potential: -75 mV
 Fertilization potential: +20 mV

a) Glutathione
b) Heparin
c) Zn
Decondensation of sperm nucleus

From the following cell is seen the protamine
to histone transition where maternally derived
histones replace protamines resulting in the
decondensation of the sperm headMaryam Borhani-Haghighi

Human sperm nuclear decondensation in vivo involves
protamine disulfide bond reduction by glutathione
(GSH) and protamine/histone exchange, presumably
with heparan sulfate (HS) as the protamine acceptor

A significant fraction of the zinc is lost
from sperm chromatin when the cysteine
thiols in protamine are oxidized into
disulfide bonds.
Extraction of zinc from the freshly
ejaculated spermatozoon allows
immediate decondensation

• Male PN : 22.1 µm
• Female PN : 24.4 µm
• Male PN has more Nucleoli
• Female PN is close to polar body
The male pronucleus is colorized in blue;
female in pink.

Role of tail
sperm-aster
 A centrosome with astral rays in the cytoplasm of
an inseminated ovum;
 it is brought in by the penetrating spermatozoon
and gives rise to the mitotic spindle of the first-
cleavage division.
Proximal centriol

syngamy

Syngamy
Union of gametes resulting
in formation of a zygote
syngamy

Singamy

At gamete fusion the sperm tail is incorporated into the ooplasm,
the centriolar region forms the sperm aster while the sperm head is
decondensing
; this aster acts to guide the female pronucleus towards the male pronucleus.
The centriole duplicates during the pronuclear stage,
and at syngamy centrioles are found at opposite poles of the first cleavage.
The centrosome has several implications for human infertility.
It is possible that immotile or nonprogressively motile spermatozoa may
possess centriolar abnormalities or an absence of centrioles.
Similarly, antisperm antibodies against centrioles may be responsible for
mitotic arrest.
One way of solving this problem would be the use of donor centrosomesMaryam Borhani-Haghighi

Pronuclear scoring system
• 16-18 hours after fertilization
• Grading is based on:
– Pronuclear size, symmetry & position
– Size, number, equality & distribution of nucleoli
– Appearance of cytoplasm
Van Blerkom (1990)

Pronuclear size & symmetry
 Embryos containing tree or More PNs are polyploid:
 injection of more than one sperm
 sperm chromatin disperse
 second polar body extrusion failure

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.

Cleavage differs from normal mitoses in 2
respects
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

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

Cleaved embryo assessment
24-28 hours after insemination
2 cells
4 cells
8 cells

•Morula stage in mammals begins when the embryo
consist of 16 blastomers , occurs 3 to 4 days after
fertilization, when embryo passes from oviduct into
the uterus
MORULA

Compaction
During compaction each of the eight blastomeres undergo a polarization.
Polarization and the formation of tight junction allow the blastomers to
create an inner embryo environment that is different from the outside
environment
Tight junctions develop beneath ext. surfaces and gap junction form
between the internal surfaces

Blastocyst Morphology
• Thin zona pellucida.
• Smooth trophoectoderm.
• Equality & close adhesion of blastomeres.
• Clearly visible blastocyst cavity.
• Well developed inner cell mass.
Blastocyst scoring is based on:
blastocyst, Inner cell mass, trophectoderm

eomesoder
min
Cdx2
Trophobla
st
Oct4
Nanog
ICM

The act of hatching involves the local digestion of the zona by an enzyme produced in
patch of trophoblast cells situated opposite the inner cell mass (this location
minimizes the risk of collateral enzymatic damage to the embryo
Having escape from the zona ,the blastocyst begins the process of implantation
Hatching

Metabolic Shift
• Maturing oocytes use more pyruvate than immature and non-transitional oocytes.
• In the early stages of early development the embryo uses pyruvate switching to
glucose.
• The use of pyruvate, glucose and lactate production indicating embryo health.

• Blastocyst with a glucose uptake >5µg/h develop
better in culture and give rise to more pregnancies.
• Morphologically normal blastocyst use more
glucose than degenerating blastocyst.

Thank you for your
attention
Thank you for your attentionMaryam Borhani-Haghighi

Thank you for your
attention

Developing Embryo
Thank you for your attention

Oocyte Maturation and Fertilization Processes

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