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Fertilization: Beginning a new organism

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Textbook: Wolpert L, Beddington R, Jessell T, Lawrence P, Meyerowitz E, Smith J. (2007) Principles of Development. 3th ed. London: Oxford university press.

Gilbert SF. (2003) Development Biology. 7th ed. Sunderland: Sinaure Associates Inc.

Two sex cells (gametes) fuse together to create a new individual with a genome derived from both parents).

First function: transmit genes from parent to offspring Second function: restoration of the diploid number of chromosomes reduced

during meiosis

Fertilization

during meiosis Third function: initiate in the egg cytoplasm those reactions that permit

development to proceed

Generally consists of four major events:

1. Contact and recognition between sperm and egg

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2. Regulation of sperm entry into the egg (only one sperm enter)

3. Fusion of the genetic material of sperm and egg

4. Activation of egg metabolism to star development

Fertilization: Beginning a new organism1. Structure of Gametes

(1)Sperm(2)Egg

2. Recognition of Egg and Sperm(1)Sperm attraction: Action at a distance(2)The acrosome reaction in sea urchins(2)The acrosome reaction in sea urchins(3)Species-specific recognition in sea urchins(4)Gamete binding and recognition in mammals

3. Gamete Fusion and the prevention of Polyspermy(1)Fusion of the egg and sperm cell membranes(2)The prevention of polyspermy

4 The Activation of Egg Metabolism

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4. The Activation of Egg Metabolism(1)Early responses(2)Late responses

5. Fusion of the Genetic Material(1)Fusion of genetic material in sea urchins(2)Fusion of genetic material in mammals

6. Rearrangement of the Egg Cytoplasm

Structure of the Gametes (sperm)

The human infant preformed in the

Fig. 12.21 A human sperm

1. Streamlined2. DNA tightly compressed → size ↓

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sperm, as depicted by Nicolas Hartsoeker (1964)

2. DNA tightly compressed → size ↓3. Haploid4. acrosomal vesicle5. Flagellum 6. Mitochondria enrich

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The maturation of a germ cell from a mammalian sperm

殘餘

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1. Haploid nucleus2. Streamlined3. DNA becomes tightly compressed4. Acrosomal vesicle derived from the Golgi apparatus5. Flagellum formation (Centriole produces )6. Size enlarged 7. Cytoplasm eliminated

Structure of the Gametes (sperm)

Acrosome stained by GFPA construct whereby the GFP gene was combined to the proacrosin

DNA stained blue with DAPIMitochondria stained with green

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was combined to the proacrosin promoter caused GFP accumulate in the acrosome.Tissue specific expression gene.

gFlagellum (tubulin) stained red

proacrosin

promoter

GFP

The motile apparatus of the sperm (flagellum)

Capacitation : 1. Final stages of sperm maturation2.which facilitates fertilization by

removing certain inhibitory factor.

Dynein arm contain ATPase, provide the energy to move. It attached to the microtubules and hydrolyzes ATP and

g y3.Untile the sperm has been inside the

female reproductive tract for a certain period of time

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9+2 arrangement of microtubules

y yconverts the release energy

Capacitation

These are monitor screen images from an instrument which records the movement paths of the sperm p pcells heads (white points) during a certain time span and displays them with a green line.

UPPER PANEL: Before capacitation the majority of the lines are straight.

LOWER PANEL: After capacitation

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palmost all the sperm cells have now gone over to swinging their heads strongly as indicated by the jagged lines.

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The motile apparatus of the sperm (flagellum)

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Dynein arm contain ATPase, provide the energy to move. It attached to the microtubules and hydrolyzes ATP and converts the release energy 10

Microtuble organized around the MTOC and spindle

11Assembly and disassembly cause microtubules → probe →….

Long-distance movement

microtubule-organizing center is “-”

Structure of the Gametes (egg)

Mature egg = ovum ; the developing egg is called the oocyte before it reaches the stage of meiosis at which it is fertilized

1.More than 10000 times the volume of the sperm2.Haploid3.Remarkable cytoplasmic storehouse

C t l i t iCytoplasmic contain:ProteinRibosomes and tRNA: make it own proteinmRNAMorphogenetic factorProtective chemical: UV filters and DNA repair enzyme

Structure:Cell membrane : regulation the flow of certain ions and fusion with

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Ce e b a e egu a o e o o ce a o s a d us osperm cell membrane

Vitelline envelope: extracellular envelope, that forms a fibrous around the egg and is often involved in sperm-egg recognition. Invertebrate only

In mammalian vitelline envelope is a separate and thick matrix, also called zona pellucida

Jelly layer: glycoprotein, may specialized for receiving sperm

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Egg; store house- all material necessary for the beginning of growth and development

1. Proteins; vit E source and amino acids– York

2. Ribosomal and tRNA

The egg and its

3. mRNA; maternal components4. Morphogenetic factors; segregated

into different cells5. Protective chemicals; Abs, UV filter

and DNA repair enzymeA; cell memb; regulate the flow of ionB; Vitelline envelop; sperm-egg

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The egg and its environment. The laboratory is not where most eggs are found. Egg have evolved remarkable ways to protect themselves in particular environment

B; Vitelline envelop; sperm egg recognition; many glycoproteins, species-specific binding of sperm,

In mammals; zona pellucida, egg is surrounded by cumulus cells

C; Cortex; thin shell of gel-like cytoplam– actin molecules—microfilament

Cumulus

Mammal eggs

Ovum

Cumulus

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_Zona Pellucida

Stage of egg maturation at the time of sperm entry in different animal species

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The egg of invertebrate and mammalian

Cortical granuleshomologous to the acrosomal

Cumulus d

the acrosomal vesicle, it containing proteolytic enzymes

The sea urchin egg cell surface by SEM

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around ovum that were nurturing at the time of release from the ovary.

Hamster eggs immediately before fertilization

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Recognition of egg and sperm1. Contact: Chemoattraction of the sperm to the egg by soluble

molecular secreted by the egg2. Binding of sperm to the extracellular envelope of egg3. Exocytosis of the acrosomal vesicle to release its enzymes4. Passage the sperm through this extracellular envelope5. Fusion of egg and sperm cell membrane

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Chemoattraction of the sperm to the egg by soluble molecular secreted by the egg

Chemotaxis, secrete chemical substance and regulating by time, from eggChemotaxis are differ among species, low concentration, Resact (sperm-activating peptide): a chemotactic molecule, 14 amino acid,

isolated from the egg jelly of sea urchin

1 sec 20 sec 40 sec 90 sec

gg j y

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Sperm chemotaxis in the sea urchin. Add 10nM solution of resact (20ul) into sperm suspension.

Exocytosis of the acrosomal vesicle to release its enzymes

Acrosome reaction (specification)

1.Fusion of the acrosomal vesicle with the sperm cell membrane

2.Extension of the acrosomal process (D)

Sperm contact egg jelly → jelly’s compound bind to specific receptor in sperm head membrane →open calcium channel → Ca2+ influx → exocytosis acrosomal vesicel → digest jelly coat

p ( )

Ca2+ influx → activate RhoB protein (in acrosomal region) → GTP binding protein → polymerizing the actin into actin filaments → protrusion (突出) → fertilization cone

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Sperm touch egg outer

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Acrosome reaction

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Binding of sperm to the extracellular envelope of egg(species-specific recognition)

Bindin (invertebrate)1. Acrosomal protein mediating 2. Non-soluble 305kDa protein, is a acrosomal protein3. Species-specification4. Receptor (350kDa)on the egg, vitilline envelope, or cell membrane

Specific-specific binding of acrosomal process to egg surface in sea

hi

Sperm acrosome

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urchin

Egg microvillus Bindin (sperm) can bind to specific egg outer-membrane

23To identify localization of bind on the acrosomal process

Immunohistochemistry, immunoprecipitation, immunocytochemistryImmunoreactivity, immunofluorescent

Gamete binding and recognition in mammals

1. Invertebrate sperm bindin, in mammal sperm is ZP3 receptor 2. Zona pellucida in mammal analogous to the vitelline envelope in

invertebrate. It contain ZP1, ZP2 and ZP3 (zona protein)3. Binding of sperm to the zona is relatively, but not absolutely, species-

speificspeific.

Purified binding recepto

Sea urchin

24Limited bindin receptor → no sperm contact

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Recognition of egg and sperm1. Contact: Chemoattraction of the sperm to the egg by soluble

molecular secreted by the egg2. Binding of sperm to the extracellular envelope of egg3. Exocytosis of the acrosomal vesicle to release its enzymes4. Passage the sperm through this extracellular envelope5. Fusion of egg and sperm cell membrane

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Bindin bindin receptor

Capacitation

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1. Mouse zona is made of three major glycoprotein, ZP1-32. ZP3 is initially binds the sperm (sperm has ZP3 receptor)3. Induction of the mammalian acrosome reaction

4. Egg (Zona) + Sperm receptor → activated galactosyltransferase-I → G-protein → Ca2+ channel → exocytosis → acrosomal vesicle → protease

Gamete binding and recognition in mammals by ZP3

protein → Ca2 channel → exocytosis → acrosomal vesicle → protease release → lysis zona → hole; fertilization cone → into egg → fusion

27ZP3 immunofluorescence or isotope

Preincubation of different ZP, produced different egg-sperm bindingPre-incubation ZP3 → then sperm binding ↓; ZP3 plays important role of sperm contact with egg jelly

Sperm ZP3-Binding Proteins at the Zona Pellucida;

Sperm ZP3-Binding Proteins in mice; galactosyltransferase-I-binds to ZP-3 –activate G protein—open Ca 2+ channel—acrsomal reactionRelease proteases that lyse the ZP-make a hole in egg

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Gamete fusion

Sperm-egg binding→ → → polymerization of actin →fertilization

cone →cytoplasmic bridge between egg and sperm →DNA entry

SEM of sea urchin egg and sperm

(A)C t t f h d ith i ill th h th l

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(A)Contact of sperm head with egg microvillus through the acrosomal process

(B)Formation of fertilization cone

(C)Internalization of sperm within the egg

(D)Transmission electron micrograph of sperm internalization through the fertilization cone

SEM of golden hamster egg and sperm

Sperm-zona binding Sperm fusion to egg plasma membrane

Entry of sperm into golden hamster egg

Integrin associated CD9 protein

Sperm head passing through zona

Egg membrane

cumulus

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(A)Scanning electron micrograph of sperm fusing with egg

(B)Close-up of sperm-zona binding

(C)Transmission electron micrograph showing the sperm head passing through the zona

(D)Transmission electron micrograph of a hamster sperm fusing parallel to the egg plasma membrane

Egg membrane

Entry of Sperm into Golden Hamster Egg

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Sperm enter → pass egg (contain hyaluronic acid) → sperm head release hyaluronidase → pass Zona, and release galactosyltransferase by exocytosis → release acetylglucosaminidase → break down Zona glycoprotein → enter and fusion

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Prevention of polyspermy

Monospermy: only one sperm enters the egg, a haploid sperm nucleus and a haploid egg nucleus combine to form the diploid nucleus of the fertilized egg (Zygote)

Polyspermy: Multiple sperm entersPolyspermy: Multiple sperm entersPolyspermy induce aberrant development.

Mechanism :1. The fast block (transient), minute; changing the electric potential

of the egg cell membrane by influx of Na+ ions

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2. The slow block, after egg-sperm fusion and fast block; Accomplished by cortical granule reaction

– Protease cleaves bindin receptor, vitelline link– Peroxidase-hardens envelope

Aberrant development is a dispermic sea urchin egg by polyspermy

Fusion of three haploid nuclei, each containing 18 chromosomes →and the division of the two sperm centrioles form four centrosomes → The 54 chromosomes randomly→ The 54 chromosomes randomly assort on the four spindles →anaphase of the first division → duplicated chromosomes

Human dispermic egg at

Two centrioles → four centrioles → 54/4????? 46/4??

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egg at first mitosis. Four centrioles are stained yellow

The fast block

Normal egg, cell membrane inhibit Na+ influx, and prevent K+ out resting membrane potential as -70mV

After binding 1 3 sec

Sperm Resting membrane potential +20mV; for outside Na+

influx

After binding 1-3 sec

When environment Na+ ↓

inhibit

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Fast block for polyspermy

Change in membrane potential → block sperm secondary fertilizationSperm carry a voltage-sensitive component (positively charge)

The prevention of polyspermy; a fast reaction; in frog, not in most mammals

By changing the electric potential of the egg memb;

Environmental; high Na, low K, but low Na and high K in cell

-resting mem poteintial; -70 mV; after binding to sperm; +20mV

-can not fuse the memb at 20 mV

Exp) low Na in lowering Na in

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Exp) low Na in lowering Na in outside; -polyspermy

Table showing the rise of polyspermy with decreasing sodium ion concentration

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490mM 120mM

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Polyspermy in eggs fertilized in similarly high concentrations of sperm in 120mV Na+

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Fig 12.23 Depolarization of the sea urchin egg plasma membrane at fertilization

Before the addition of sperm, the potential difference across the egg plasma membrane is about -70mV

The slow block to polyspermyA minute after the sperm-egg fusion Long term blockMechanical blockCortical granule reaction

Sperm eggCortical granules (down the

egg cell membrane)+ Binding, fusion

Cortical granules serine protease (trypsin-like) release

1. Separated vitelline envelope and cell membrane

Mucopolysacharides release →osmotic gradient → water influx → vitelline envelope and membrane gap increase

exocytosis

enzymes

Peroxidase

Ca2+ dependent, form the storage (endoplasmic reticulum) not environment

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cell membrane2. Clips off the bindin receptors and

sperm attached to them

and membrane gap increase

fertilization envelope

In mammals no fertilization envelope, Cortical granules → release N-acetylglucosaminidase → cleaving N-acetylglucosamine from ZP3 → sperm can bind it

Formation of the fertilization envelope and removal of excess sperm

(A)At 10 seconds after sperm addition, sperm are seen surrounding the eggsurrounding the egg

(B,C) At 25 and 35 seconds after insemination, a fertilization envelope is forming around the egg, starting at the point of sperm entry

(D) The fertilization envelope is l d

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A: sperm 10secB: 25-35 sec, formation of fertilization envelop

complete and excess sperm have been removed

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The slow block to polyspermy;

Cortical granule fuse with egg cell memb;

-release contents;

-protease; clips off the binding receptor and sperm attached

-mucopolysaccharides; osmotic gradient that cause water to rush into the spacemucopolysaccharides; osmotic gradient that cause water to rush into the space

btwn mem and vitelline envelp– envelop expanding and becomes the

fertilization envelop.

-peroxidase; hardens the fertilization envelop by crosslinking Tyr rsidues am

adjacent proteins

-hyalin; forms a coating around the egg; ; hylain layer透明角質

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In mammals; the cortical granule reaction—

Enzymes; modify the ZP sperm receptor; no longer binding

Fig 12.24 The cortical reaction at fertilization in the sea urchin

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Egg → fertilization → cortical granules fuse with plasma membrane → exocytosis some contents → join with vitelline membrane → form fertilization membrane prevent other sperm entry;

Some cortical granule → hyaline layer (surround egg)

Cortical granule exocytosis

C ti l l t i t l th t i li ki th

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Cortical granule → exocytosis →proteases cleave the protein linking the vitelline envelope → mucopolysaccharides release by cortical granule → osmotic gradient → water enter → swell the space between vitelline and membrane;

Other enzyme released from cortical granule →harden fertilization envelope, and release other sperms

Cortical granule exocytosis

(B,C) Transmission and scanning electron micrographs of the cortex of an unfertilized sea urchin egg

(D E) Transmission and

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(D,E) Transmission and scanning electron micrographs of the same region of a recently fertilized egg

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Egg activation

• Ion flow• Receptor activation• Signal transduction

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The activation of egg metabolism

Early responses: Sperm entry →induced Ca2+ wave in egg

sperm

Every 30 sec

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Endoplasmic reticulum surrounding cortical granules in sea urchin eggs

Ca2+ from the endoplasmic reticulum

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(A)The endoplasmic reticulum has been stained with osmiumreticulum has been stained with osmium--zinc zinc iodide to allow visualization by transmission electron iodide to allow visualization by transmission electron microscopymicroscopy

(B)(B)An entire egg stained with fluorescent antibodies to calciumAn entire egg stained with fluorescent antibodies to calcium--dependent calcium release channelsdependent calcium release channels 48

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Postulated Pathway of Egg Activation in the Sea Urchin

The activation of egg metabolism to the sperm

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The Roles of Inositol Phosphates in Releasing Calcium from theER and the Initiation of Development

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Model of a postulated ( may) pathway of egg activation in the sea urchin

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The possible mechanisms of egg activation

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Possible Mechanisms of Egg Activation

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Possible Mechanisms of Egg Activation

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Fusion of the genetic material

In sea urchin

Egg → 2o meiotic division (haploid) → female pronucleus → Fertilization → sperm and egg fusion → sperm nucleus decondense (male pronucleus) → male pronucleus rotate 180o → centriole between male and female pronucleus → microtubules extend and sperm microtubules degrade

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Sperm, pronucleus

The sperm nucleus, centriole Mito and flagella disintegrate

inside egg;Mitochondrial DNA from egg ,

not from sperm

Sperm nucleus undergo dramatic changes;

--nulcear lamina breakage--pronucleus

--cAMP-dependent protein kinase phosphorylate

the sperm specific Histone --decondensation- sperm

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decondensation sperm specific Histone are replaced by histone from egg

--

calcium oscillation ↓

inactive MAP kinase

Sperm DNA is bound by

protamines, tightly compacted by disulfide bond

Egg 1.contain glutathione reduces disulfide bond2. Egg nucleus attested in metahpase of 20 meiotic di i i

enter

Fusion of the genetic material

In mammalian

Proteolysis cyclin, lead cell cycle to continueProteolysis securin, its holding the metaphase chromosome together

division

DNA synthesis at male and female pronucleus

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Male pronucleus produced asters

Microtubles join two pronuclei

Migrate Sperm mitochondria degrade in egg cytoplasm Nuclear envelopes break down 60

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We need maternal and paternal genetic contributions

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Reorganization of cytoplasm in the newly fertilized frog egg

Blastrulation start

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(A)View looking at the vegetal hemishpere (B)After cytoplasmic rotation, Parallel Microtuble arrays

Fig 12.26 Profile of maturation-promoting factor (MPF) activity in early

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xenopus development

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Hyaluronidase activity on surface of mammalian sperm↓

Sperm penetrate cumulus layer↓

Zona pellucida elicits acrosomal reaction in sperm

Summary: fertilization in mammals

Zona pellucida elicits acrosomal reaction in sperm↓

Enzyme release from acrosome sperm head↓

Sperm plasma membrane fuses with egg plasma membrane↓

Calcium wave↓↓C ti l ti id bl k

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↓↓

Sperm nucleus delivered into cytoplam↓

Egg completes meiosis and development is initiated

Cortical reaction provides block polyspermy

egg activation by fertilization, why?? Cellular mechanism

specific recognition of fertilization

slow block of polyspermy

Stage of egg maturation at the time of sperm entry in different animal specie

Immunohistochemistry, immunoprecipitation, immunocytochemistry

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Immunoreactivity, immunofluorescent

Aberrant development in polyspermy, why?

Egg and sperm interactions at fertilization

• Fertilization involves cell-surface interactions between egg and sperm. p

• The sperm has to pass several barriers to enter the egg. • In mammalian eggs, the sperm first passes through a layer of

cumulus cells embedded in hyaluronic acid aided by the hyaluronidase actvity on its surface.

• The 2nd layer is the zona pellucida, a layer of glycoproteins. • The acrosomal reaction (release of enzymes in the sperm head)

is mediated by interaction of the ZP3 species specific receptor

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is mediated by interaction of the ZP3 species-specific receptor and adhesion molecules in the sperm head.

Fertilization requires cell surface interaction

• The acrosome releases acrosin (a protease) and an acetylglucosaminidase which degrades glycoproteins.

• The sperm surface proteins (i.e. fertilin) that can bind the egg's surface are exposed during the acrosomal reactionsurface are exposed during the acrosomal reaction.

• Fertilin binds an intergrin-like receptor of the egg plama membrane to initiate sperm-egg fusion.

• In some invertebrates (i.e. sea urchins), an actin driven acrosomal projection allows the sperm and egg to touch.

• Changes in the egg membrane at fertilization block polyspermy so that only one sperm enters an egg.

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• Enzymes are released that prevent other sperm from binding to the zona pellucida.

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Fertilization and activation of development

• At fertilization a number of events occur to activate development (such as increase in protein synthesis, structural changes [ ti l t ti ])[cortical rotation]).

• Main events are the completion of meiosis, fusion of the nuclei to form a diploid zygotic genome and the start of mitosis.

• Fertilization causes a calcium wave and egg activation. • The sharp increase in calcium initiates the cell cycle by acting

upon proteins that control the cell cycle. • The Xenopus egg is kept in metaphase II by maturation-

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promoting factor (MPF), a complex including cyclin. • The calcium wave activates a kinase that leads to cyclin

degradation, the end of meiosis and nucleus fusion.

Microtuble organized around the MTOC and spindle

70Assembly and disassembly cause microtubules → probe →….

Long-distance movement

microtubule-organizing center is “-”

Heterodimeric tubulin subunits compose the wall of a MT.

Tubulin subunit are formed by and One subunit bind to two GTP, has

GTPase activity.55kDa monomers in all eukaryotes-tubulin are formed by and .

Irreversibly, does not hydrolyze

GTP reversibly

add

In mammals at least 6 alpha and 6 beta isoforms have been identified

The proteins are highly conserved (75% homology between yeast and human)

Most variability is found in the C

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+

Most variability is found in the C-terminal region of the molecules and is likely to affect interactions with accessory proteins

A tubulin homologue, FtsZ, is expressed in prokaryotes

_

Addition of MT fragments demonstrates polarity of tubulin polymerization. (MT assembly and disassembly take place preferentially at the + end)

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Stages in assembly of MTs.

Free tubulin dimers →short protofilaments → unstable and quickly associate more stable curved sheets → wraps around microtubule with 13 protofilaments →composing the microtuble wall → incorporate ( hydrolysis GTP) → bind + end

Slowed 2 fold than +

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MTOCs orient most MTs and determine cell polarity.

MT (doublets) are found in cilia or flagella that are used for cell movement

MT in axon are plus end out, MT in dendrites are plus and minus ends out. These MT are not attached to any nucleating structure.

Cells have stable (half life >1 hr) and unstable/dynamic (half-life = 5-10 min) microtubules. These subsets differ in post-translational modification of tubulin.

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Aberrant development in a dispermic sea urchin egg(A) Fusion of three haploid nuclei, each containing 18 chromosomes and the division of the two sperm centrioles to form four centrosomes

The prevention of polyspermy

form four centrosomes

(B,C) The 54 chromosomes randomly assort on the four spindles

(D) At anaphase of the first division the duplicated chromosomes are pulled to the

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four poles

(E) Four cells containing different numbers and types of chromosomes are formed, thereby causing

(F) The early death of the embryo