human fertilization

Maryam Borhani-HaghighiPhD 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..


Cytoplasmic maturation

Cytoplasmicmaturation

Organelle redistribution

MitochondriaEndoplasmic

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 bodyM - mitochondrian - 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.

Maryam Borhani-Haghighi

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

http://en.wikipedia.org/wiki/Glycoprotein

http://en.wikipedia.org/wiki/Plasma_membrane

http://en.wikipedia.org/wiki/Oocyte


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.



Zona pellucida

Inner zone

Outer zone

Birefringence


(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.


(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 ZPMaryam Borhani-Haghighi


Maturation-Ejaculation-CapacitationLeaving 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 capacitationincreases 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 zonapellucida 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 perivitellinespace.

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

SOCStore-operated channels

Progesterone cause Ca2+influx::::::Elevation of Ca2+in the midpiece/proximal 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 ovarythe adrenal glands.In men, is producedin adrenal testicular tissue.



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-freeeggs, 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 bindingIzumo is essential for membrane fusion

On the egg, CD9 is required for fusion and might collaborate with other proteins such as integrins or glycosylphosphatidylinositol (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 itsparticipation in sperm–egg fusion.

• Although both the equatorial regions and posterior head of the spermmembrane 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 polyspermyThe Cortical Reaction

• Calcium not only plays a role in the acrosome reaction, it also mediates the subsequent event of corticalgranuleexocytosis in the egg.

• Ca2+ induces local exocytosisof 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 protamineto histone transition where maternally derived

histones replace protamines resulting in the decondensation of the sperm head


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 cysteinethiols 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

respectsNormal 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


eomesodermin

Cdx2

Trophoblast

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 EmbryoThank you for your attention


human fertilization

Health & Medicine

oocyte cortex

maturation of oocyte

beginning of oocyte

nuclear membrane of

oocyte polarization

cortex cytoplasm

immature oocytes

cell cortex