Fertilization

Biology 4361 – Developmental Biology

June 24, 2009

Fertilization

Fertilization accomplishes two things:

Major Events:

1. Contact and recognition between sperm and eggs.

- must be species-specific

2. Regulation of sperm entry into egg.

3. Fusion of genetic material of sperm and egg.

4. Activation of egg metabolism to start development.

Lennart Nilsson

Reproduction (initiates reactions in the egg

cytoplasm that allow development to

proceed)

Sex (combining genes from two genomes)

Fertilization Overview

NOTE – Tremendous variation among species

- models: sea urchin, mouse, chick

Sperm formation and structure

Egg structure and function

Interactions between sperm and eggs

Chemoattraction

Acrosome reaction

Binding and fusion

Prevention of polyspermy

Egg activation

Pronuclear fusion

Mammalian fertilization

Sperm Formation

Sperm Axoneme

The EggAll materials necessary to begin development are stored in the egg.

Proteins- yolk (made in other organs (liver, fat bodies), transported to egg

Ribosomes and tRNA- burst of protein synthesis after fertilization

Protective chemicals- UV filters- DNA repair enzymes- antibodies- alkaloids (and other protective molecules)

Morphogenic factors- initiate differentiation- e.g. transcription factors,

paracrine factors

mRNA- encode proteins for use in early development- some localized regionally

nanos

mRNA

nucleus

bicoid

mRNA

Egg Maturation at Sperm EntryMost eggs are not fully mature at the time of fertilization;

- sperm entry activates metabolism and relieves meiotic arrest

Egg Structure – Sea Urchin

Volume: 2 x 10-4 mm3 (200 picoliters)

(>200 X sperm volume)

egg jelly

- glycoprotein meshwork

- attract or activate sperm

vitelline envelope

- extracellular (inverts)

- fibrous mat

- sperm-egg recognition

- contains glycoproteins

egg cell membrane

- binds sperm

- fuses with sperm cell membrane

Egg Membrane Structure

- cortex

layer

actinmicrovilli –

filamentous

(f-actin)

- proteolytic enzymes - mucopolysaccharides

- adhesive glycoproteins- hyaline protein

Cortical granules: Golgi-derived

cortex –

globular

(g-actin)

egg jelly

Fertilization Overview

Sperm formation and structure

Egg structure and function

Interactions between sperm and eggs

Chemoattraction

Acrosome reaction

Binding and fusion

Prevention of polyspermy

Egg activation

Pronuclear fusion

Mammalian fertilization

Interactions Between Egg and Sperm

1. Chemoattraction of sperm to egg

- soluble molecules released by egg

4. Passage of sperm through the extracellular envelope

5. Fusion of the egg and sperm cell membranes

2. Exocytosis of the acrosome

- stimulated by binding of egg molecules

3. Binding of sperm to the extracellular envelope

- usually a multi-step process

- binding molecules and receptors located on each gamete

Pronuclear fusion: sperm and egg nuclei (pronuclei) meet, fuse;

development initiated

Sea Urchin Fertilization

Challenges for sea urchins (and others):

1) Bring two very small cells together in a very large space.

2) Ensure that only sperm and eggs of the same species join.

Sperm ChemoattractionChemoattraction: eggs produce chemical attractant for sperm, e.g.

- 14 aa peptide

- source – egg jelly

- species-specific

- A.p. sperm - membrane

resact receptors

- binding: ↑ guanylyl cyclase

- cGMP activates

Ca2+ channel

- ↑Ca2+i provides

directional cues

Resact

Arbacia

punctulata

eggs produce

“resact”

A. 0 sec B. 20 sec

C. 40 sec D. 90 sec

resact

Sea Urchin Acrosome Reaction

Acrosome reaction: fusion of

acrosome and cell membranes

- releases acrosome contents

Acrosome contains enzymes

that digest jelly layer

Exposed sperm membrane

contains proteins that bind

to egg receptors

Sperm acrosomal process

membrane fuses with egg

membrane

Ionic changes stimulate

actin polymerization; forms

acrosomal process

Egg jelly stimulates the sperm

acrosome reaction

Acrosome Reaction – Sea Urchin

AR stimulated by contact with egg jelly

- species-specific stimulatory molecules

- in S. purpuratus – fucose sulfate

Fucose sulfate binding to sperm

receptor activates:

- Ca2+ transport channel

- allows Ca2+ into sperm head

- Na+/H+ exchanger

- pumps Na+ in/H+ out

- phospholipase - produces

inositol trisphosphate (IP3)

- elevated Ca2+ and basic cytoplasm

triggers fusion of acrosomal and

cell membranes

- proteolytic enzymes digest a path

through jelly coat to egg surface

Acrosome Reaction – Sea Urchin

Ca2+ influx stimulates g-actin

polymerization to f-actin

Acrosomal process adheres

to vitelline envelope via

bindin protein

Bindin – species-specific

binding to egg receptor

on vitelline envelope

Actin

micro-

filaments

Bindin

Vitelline Membrane Bindin Receptors

Note: regular sperm distribution species specificity- suggests regular bindin

receptor distribution

Fusion of Sperm and Egg Membranes

- membranes fuse

(fusogenic protein?)

- causes egg actin polymerization

- fertilization cone formed

- actin from both gametes

form connections

- sperm nucleus and tail pass

through cytoplasmic bridge

- acrosomal process adheres

to egg membrane microvilli

Acrosome reaction

Fertilization Overview

Sperm formation and structure

Egg structure and function

Interactions between sperm and eggs

Chemoattraction

Acrosome reaction

Binding and fusion

Prevention of polyspermy

Egg activation

Pronuclear fusion

Mammalian fertilization

Prevention of Polyspermy

Fast block to polyspermy

- electrical

- sea urchins, frogs

- not in most mammals (why not??)

Slow block to polyspermy

- chemical, physical

- most species, including mammals

More than one sperm entering

an egg results in polyploidy;

usually eventual death

Why?

Tim Watkins

Fast Block to PolyspermyCell membranes provide a selective ionic barrier:

- seawater: high Na+, low K+ (relatively)

- cytoplasm: low Na+, high K+ (relatively)

This ionic imbalance is maintained by membrane pumps, exchangers

resting membrane potential

Seconds

Na

K

K

K

K

KNa

Na

Na

Na

Na

Na

Na

K

Na

K

Ionic imbalance creates electrical potential across the membrane; ~ -70 mV

= -70 mV (inside)

plasma

membrane

Fast Block to Polyspermy

Ionic imbalance creates electrical potential across the membrane; ~ -70 mV

Cell membranes provide a selective ionic barrier:

This ionic imbalance is maintained by membrane pumps, exchangers

Sperm binding (or fusion) causes Na+ influx

Na

K

K

K

K

K

Na

Na

Na

Na

Na

Na

Na

K

Na

KNa

Na

1-3 sec after sperm binding, membrane potential shifts to ~+20 mV

- sperm cannot bind

to eggs with positive

membrane potential

Depolarization

Seconds

- seawater: high Na+, low K+ (relatively)

- cytoplasm: low Na+, high K+ (relatively)

Fast Block to Polyspermy

Ionic imbalance creates electrical potential across the membrane; ~ -70 mV

Cell membranes provide a selective ionic barrier:

This ionic imbalance is maintained by membrane pumps, exchangers

Na

K

K

K

K

KNa

Na

Na

Na

Na

Na

Na

K

Na

K

Sperm binding (or fusion) causes Na+ influx

1-3 sec after sperm binding, membrane potential shifts to ~+20 mV

- sperm cannot bind

to eggs with positive

membrane potential

Depolarization

Seconds

transient; membrane re-polarizes

- seawater: high Na+, low K+ (relatively)

- cytoplasm: low Na+, high K+ (relatively)

Slow Block to Polyspermy

Cortical granule reaction

- chemical and mechanical block

- active ~ 1 min after sperm-egg fusion

Sperm entry initiates fusion of cortical granule membrane with

egg’s cell membrane.

CG contents released into the space between the cell membrane

and vitelline envelope (perivitelline space)

Cortical granules

- just beneath plasma membrane

~ 15,000 granules/sea urchin egg

~ 1 μm diameter

R. Bowen

Slow Block to Polyspermy

Cortical Granule contents:

1. serine protease

- dissolves protein connections between envelope and membrane

- clips off bindin receptors & connected sperm

2. mucopolysaccharides

- sticky compounds; produce osmotic pressure

- water rushes in, vitelline envelope raises (fertilization envelope)

3. peroxidases – oxidizes and crosslinks tyrosines –

“hardens” fertilization envelope

4. hyaline (protein) forms a coating around the egg: hyaline layer

Cortical Granule Exocytosis

Cortical granule

fusion; release

of CG contents

Elevation of

vitelline

envelope

Cortical Granule Exocytosis

Hyaline layer

Fertilization Envelope

10 sec

Sea urchins -

Time after

sperm addition:

25 sec

35 sec

Ca2+ Role in Cortical Granule ReactionCortical granule reaction mechanism similar to acrosome reaction

- at fertilization, egg cytoplasmic [Ca2+] rises

- high Ca2+ causes cortical granule membranes to fuse with cell membrane

- internal Ca2+ released as a self-propagating “wave”

1 2

3 4

- Ca2+ causes advancing cortical granule exocytosis

t=0

t=30 sec

Fertilization Overview

Sperm formation and structure

Egg structure and function

Interactions between sperm and eggs

Chemoattraction

Acrosome reaction

Binding and fusion

Prevention of polyspermy

Egg activation

Pronuclear fusion

Mammalian fertilization

Activation of Egg Metabolism

Early responses – occur within seconds of cortical reaction

Late responses – start within minutes after fertilization

Fertilization results in:

1. merging of two haploid nuclei

2. initiating the processes that start development

These events happen in the cytoplasm

- occur without nuclear involvement

Sperm fusion activates egg metabolism

- stimulates a preprogrammed set of metabolic events into action

Early Responses

Ca2+ released from internal store at fertilization

- increases concentration from 0.1 – 1.0 μM

Ca2+ activates metabolic reactions; e.g.

- NAD+ kinase

- burst of O2 reduction (to H2O2)

Egg Activation – Early Responses

Late Responses

Egg Activation – Late Responses

Events After Membrane Fusion

Aster microtubules extend throughout the egg; contact female pronucleus

Pronuclei migrate towards one another

Pronuclear fusion forms a diploid zygotic nucleus

- sperm nuclear envelope vesiculates

- sperm DNA decondenses

- transcription and replication can start

After cell membrane fusion, sperm nucleus and centriole separate from

mitochondria and flagellum

- sperm flagellum and mitochondria disintegrate

In sea urchins, fertilization occurs after 2nd meiotic division;

therefore, a haploid female pronucleus is already present at fertilization

The sperm pronucleus rotates 180 - results in sperm centriole

between the sperm and egg pronuclei

- sperm centriole acts as a microtubule organizing center; forms aster

Pronuclear Fusion

♂ ♀

Fertilization Overview

Sperm formation and structure

Egg structure and function

Interactions between sperm and eggs

Chemoattraction

Acrosome reaction

Binding and fusion

Prevention of polyspermy

Egg activation

Pronuclear fusion

Mammalian fertilization

Mammalian Fertilization

Many similarities with sea urchin; some differences:

- translocation of gametes

- sperm capacitation

- chemotaxis, thermotaxis, hyperactivation of motility

- recognition at the zona pellucida (vitelline envelope in urchin eggs)

- gamete adhesion

- sperm-egg binding

- acrosome reaction

- prevention of polyspermy

- fusion of genetic material

- internal fertilization

- heterogeneity of sperm population

- transport of both gametes to the oviduct

- sperm motility

Mammalian Egg

Cumulus – ovarian follicular cells

Inner-most layer – corona radiata

Gamete Translocation

The ovulated egg (surrounded by cumulus cells)

is picked up by the oviduct fimbriae

- ciliary beating and muscle contractions move

oocyte-cumulus complex into oviduct

Sperm are deposited at the cervix

- sperm are transported by the female

reproductive tract via uterine

muscle contractions

- sperm transport slows at ampulla

(timed-release mechanism?)

- sperm motility important within the oviduct

Sperm motility is not sufficient

to move sperm to ampulla

Fertilization takes place at the ampulla of the fallopian tube

Gamete Translocation

- hyperactivated motility in the vicinity of

the oocyte or cumulus

- directional cues from temperature

gradients (thermotaxis)

Sperm Capacitation (Mammals)

Freshly ejaculated mammalian sperm cannot fertilize the egg

- fresh sperm “held up” in the cumulus matrix

Capacitation – a series of physiological maturation events that take

place in the vaginal tract, uterus, and oviduct

- conditions for capacitation vary among species

- can be accomplished in vitro for many species using:

- oviduct fluid

- culture medium

- albumin (protein)

Capacitation involves changes in: membrane lipid carbohydrates,

proteins, membrane potential (becomes more negative),

protein phosphorylation, internal pH, and enzyme activation

Capacitation is transient; sperm become uncapacitated after a period

WHY?

Sperm Capacitation (Mammals)

WHY?

Timing: nearly all human pregnancies result from sexual intercourse

during a 6-day period ending on the day of ovulation.

- fertilizing sperm may take a long as 6 days to reach the ampulla

Hyperactivation, Thermotaxis, Chemotaxis

Motility patterns change in the oviduct in some species

- hyperactivated motility – higher velocity, greater force

- suited for viscous oviduct fluid

Hyperactivation

Sperm may be able to sense a thermal gradient

- ampulla of oviduct is 2°C warmer than isthmus

- only capacitated sperm can respond thermotactically

Thermotaxis

Oocytes and cumulus cells may secrete chemotactic agents

- follicular fluid shows some chemotactic ability

- only fertilizable follicles had chemotactic activity

- only capacitated sperm respond

Chemotaxis

Recognition at the Zona Pellucida

Mammalian Zona Pellucida

- analogous to vitelline envelope

- sperm binding relatively species-specific

Sequential interactions between sperm

proteins and zona components

1. Weak binding between sperm

and peripheral egg protein

2. Stronger binding between zona

and sperm SED1 protein

3. Sperm protein binds strongly to ZP3

- ZP3 stimulates acrosome reaction

3 glycoproteins: ZP1, ZP2, ZP3

(and some internal accessory proteins)

- ZP matrix is synthesized by oocyte

e.g. mouse zona composed of

Acrosome Reaction - Mouse Sperm

Acrosome reaction induced when ZP3 crosslinks sperm membrane receptors.

[sperm that undergo AR before reaching the zona unable to penetrate]

- sperm galactosyltransferase binds to ZP3 N-acetylglucosamine

Acrosome Reaction - Mouse, cont.

Sperm galactosyltransferase crosslinks ZP3 N-acetylglucosamine

- results in Ca2+-mediated exocytosis of the acrosomal vesicle

- initiates a cascade that opens membrane Ca2+ channels

- crosslinking activates specific G-proteins in sperm membrane

Equatorial

region

Mitochondria

cortical granules

Mammalian Gamete Fusion

Mammalian sperm enter egg tangentially

- contact on the side of the sperm

- membrane fusion at the junction of the

inner acrosomal and cell membrane

= equatorial region

- egg cortical actin polymerizes in the

region of sperm binding

- extends microvilli to sperm

Cortical granules release enzymes that

modify ZP so that it can no longer bind sperm

- N-acetylglucosiminidase cleaves part

of ZP3 carbohydrate chain

- ZP2 is also clipped; loses ability

to bind sperm

Mammalian Pronuclear Fusion

Essentially the same as sea urchin….

- mammalian pronuclear migration takes far longer (12 h v. ~ 1 h)

- glutathione from egg cytoplasm reduces disulfide bonds in sperm

protamines (protamines replace histones in the sperm nucleus)

- allows uncoiling of sperm chromatin

- replication and transcription allowedprotamine-S-S-protamine

protamine-SH + HS-protamine

GSH

GS

Mammalian oocyte nucleus is arrested in metaphase of

2nd meiotic division when sperm enters

Sperm entry initiates Ca2+ oscillations in the oocyte

- e.g. Ca2+ inactivates MAP kinase (MEK) – allows DNA synthesis

- Ca2+i stimulates the cell cycle (i.e. cell division pathways)

Sperm Contribution

Sperm contributes nucleus, centriole, mitochondria, cytoplasm (minor);

Several sperm proteins and mRNAs for transcription and

paracrine factors are brought into the egg

Also, microRNAs imported; may down-regulate receptors

involved in early cell division

- however, mitochondria and mitochondrial DNA are degraded

- therefore, all embryonic mitochondria are derived from the mother

(basis for mtDNA tracing of geneology/phylogenetics)

Egg Activation Pathway

Early responses Late responses