Fertilization in sea urchin

DUKHIRAM KISKU
18mslsas06
 Fertilization

Fertilization accomplishes two things:

Sex (combining genes from two genomes)
Reproduction (initiates reactions in the egg
cytoplasm that allow development to proceed)

Major Events: Lennart Nilsson

1. Contact and recognition between sperm and eggs.

must be speciesspecific

2. Regulation of sperm entry into egg.

3. Fusion of genetic material of sperm and egg.

4. Activation of egg metabolism to start development.

 Fertilization Overview
1) Sperm formation and structure
2) Egg structure and function
3) Interactions between sperm and eggs

Chemoattraction
Acrosome reaction
Binding and fusion
4) Prevention of polyspermy
5) Egg activation
6) Pronuclear fusion
Sperm Formation
Sperm Axoneme
 The Egg
All 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

mRNA
- encode proteins for use in early development

Morphogenic factors
- Direct the differentiation
- e.g. transcription
factors, paracrine
factors

Protective chemicals
- UV filters
- DNA repair enzymes
- Antibodies
- Distasteful molecules
 Egg Maturation at Sperm Entry
Most eggs are not fully mature at the time of fertilization;
sperm entry activates metabolism and relieves meiotic arrest
Egg maturation stage at fertilization.
Egg Structure – Sea Urchin
Volume: 2 x 104 mm3
(200 picoliters)
(>10,000 X sperm
volume)
egg jelly
- glycoprotein meshwork
- attract or activate sperm

vitelline envelope
- extracellular (inverts)
- fibrous mat
- spermegg recognition
- contains glycoproteins

egg cell membrane

- binds sperm
- fuses with sperm cell
membrane
 Egg Membrane Structure
egg jelly

actin
microvilli –
filamentous
(factin)

cortex –
globular
(gactin)

cortex
layer
Cortical granules: Golgiderived; contain:
- proteolytic enzymes
- mucopolysaccharides
- hyaline protein
- adhesive glycoproteins
 Interactions Between Egg and Sperm
1. Chemoattraction of sperm to egg
 soluble molecules released by egg

2. Exocytosis of the acrosome

 stimulated by binding of egg molecules

3. Binding of sperm to the extracellular envelope

 usually a multistep process
 involves binding molecules and receptors located on each
gamete

4. Passage of sperm through the extracellular

envelope

5. Fusion of the egg and sperm cell membranes

Pronuclear fusion: sperm and egg nuclei (pronuclei) meet, fuse;
development initiated
 Sea Urchin Fertilization
Challenges for sea urchins (and others):
1) How to bring two very small cells together in a very large space.
2) How to ensure that only sperm and eggs of the same species join.
 Sperm Chemoattraction
Chemoattraction: eggs produce chemical attractant for sperm, e.g.

Arbacia A. 0 sec B. 20 sec

punctulata
eggs produce
chemoattractant “resact”

Resact (SAP)
- 14 aa peptide
- source – egg jelly
- speciesspecific resact
- sperm have membrane C. 40 sec D. 90 sec
resact receptors
- binding: ↑ guanylyl cyclase
- cGMP
activates
Ca2+ channel
Fig.(From Ward et al.1985, courtesy of V. D.
- ↑Ca2+i provides
Vacquier.)
directional cues
Sea Urchin Acrosome Reaction

Egg jelly stimulates the sperm

acrosome reaction
Acrosome reaction: fusion of
acrosome and cell membranes
releases acrosome contents
Ionic changes stimulate
actin polymerization; forms
acrosomal process
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
 Acrosome Reaction – Sea Urchin
AR stimulated by contact with egg jelly
- speciesspecific 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
sperm cell membranes

- proteolytic enzymes digest a path

through jelly coat to egg surface
 Acrosome Reaction – Sea Urchin
Ca2+ influx stimulates gactin
polymerization to factin
Bindin
Acrosomal process adheres
to vitelline envelope via
bindin protein Actin
micro
filaments
Bindin – speciesspecific
binding to egg receptor
on vitelline envelope
 Vitelline Membrane Bindin Receptors
Note: regular sperm distribution species specificity
suggests regular bindin
receptor distribution
 Fusion of Sperm and Egg cell Membranes

Acrosome reaction

- acrosomal process adheres

to egg membrane microvilli

- 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
Fig;(Schatten and Mazia 1976, courtesy of G. Schatten;
D courtesy of F. J. Longo.)
 Prevention of Polyspermy

Why?
More than one sperm entering
an egg results in polyploidy;
usually eventual death

Fast block to polyspermy

- Electric potential
- sea urchins, frogs
- not in most mammals

Slow block to polyspermy

- chemical, physical
- most species, including mammals

Fig.J. Holy; Simerly et al. 1999, courtesy

of G. Schatten. Tim Watkins
 Fast Block to Polyspermy
Cell membranes provide a selective ionic barrier: K K Na K
Na Na
seawater: high Na+, low K+ (relatively) Na K
K K Na
cytoplasm: low Na+, high K+ (relatively) K
Na Na
Na Na

This ionic imbalance is maintained by membrane pumps, exchangers

Ionic imbalance creates electrical potential across the membrane;~70 mV
Sperm binding (or fusion) causes Na+ influx
20sec after sperm binding, membrane potential shifts to ~+20 mV

Depolarization transient; membrane repolarizes

sperm cannot bind to

eggs with positive
membrane potential

Seconds
 Slow Block to Polyspermy
Cortical granule reaction
- chemical and mechanical block
- active ~ 1 min after spermegg fusion

R. Bowen

Cortical granules
just beneath plasma membrane
~ 15,000 granules/sea urchin egg
~ 1 μm diameter

Sperm entry initiates fusion of cortical granule membrane with

egg’s cell membrane.

Several proteins released into the space between the cell

membrane and vitelline envelope (perivitelline space)
 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
and formation of the sea urchin
fertilization envelope
Cortical Granule Exocytosis

Elevation of
vitelline envelope

Cortical granule
fusion; release
of CG contents
Cortical Granule Exocytosis

Hyaline layer
 Fertilization Envelope
Sea urchins
Time after
sperm addition:

10 sec 25 sec

35 sec
 Ca2+ Role in Cortical Granule Reaction
Cortical 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 selfpropagating “wave”
- Ca2+ causes advancing cortical granule exocytosis

t=0

t=30 sec
 Activation of Egg Metabolism
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 – occur within seconds of cortical reaction

Late responses – start within minutes after fertilization
 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
In sea urchins, fertilization occurs after 2nd meiotic division;

After cell membrane fusion, sperm nucleus and centriole separate from
mitochondria and flagellum
- sperm flagellum and mitochondria disintegrate
- sperm nuclear envelope vesiculates
- sperm DNA decondenses
transcription and replication can start

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

Aster microtubules extend throughout the egg; contact female pronucleus

Pronuclei migrate towards one another

Pronuclear fusion forms a diploid zygotic nucleus

 Pronuclear Fusion

♂ ♀
 REFERENCES

 Briggs, E. and G. M. Wessel. 2006. In the beginning:

Animal fertilization and sea urchin development.
Dev. Biol. 300: 15–26..
 Glabe, C. G. and V. D. Vacquier. 1978. Egg surface
glycoprotein receptor for sea urchin sperm bindin.
Proc. Natl. Acad. Sci. USA 75: 881–885.
 Developmental Biology 11 edition, Gilbert, Barresi.
 Mammalian Fertilization
Many similarities with sea urchin; some differences:
- internal fertilization
- heterogeneity of sperm population
translocation of gametes
transport of both gametes to the oviduct
sperm motility
sperm capacitation
- chemotaxis, thermotaxis, hyperactivation of motility
- recognition at the zona pellucida (vitelline envelope in urchin eggs)
- gamete adhesion
- spermegg binding
- acrosome reaction
- prevention of polyspermy
- fusion of genetic material
 Mammalian Egg

Cumulus – ovarian follicular cells

Innermost 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
oocytecumulus complex into oviduct

Sperm are deposited at the cervix; but fertilization

takes place at the ampulla of the fallopian tube
- sperm are transported by the
female reproductive tract via
uterine muscle contractions
sperm motility is not sufficient
to move sperm to ampulla
- sperm transport slows at
ampulla (timedrelease
mechanism?)
- sperm motility
important within the
oviduct
- hyperactivated motility in the vicinity of the oocyte or cumulus
 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?
Timing: nearly all human pregnancies result from sexual intercourse
during a 6day period ending on the day of ovulation.
fertilizing sperm may take a long as 6 days to reach the ampulla
Hyperactivation, Thermotaxis, Chemotaxis
Hyperactivation
Motility patterns change in the oviduct in some species
- hyperactivated motility – higher velocity, greater force
- suited for viscous oviduct fluid

Thermotaxis
Sperm may be able to sense a thermal gradient
- ampulla of oviduct is 2°C warmer than isthmus
- only capacitated sperm can respond thermotactically

Chemotaxis
Oocytes and cumulus cells may secrete chemotactic agents
- follicular fluid shows some chemotactic ability
- only fertilizable follicles had chemotactic activity
- only capacitated sperm respond
 Recognition at the Zona Pellucida
Mammalian Zona Pellucida
- analogous to vitelline envelope
- sperm binding relatively speciesspecific

e.g. mouse zona composed of

3 glycoproteins: ZP1, ZP2, ZP3
(and some internal accessory proteins)
ZP matrix is synthesized by oocyte

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
 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 Nacetylglucosamine

 Acrosome Reaction Mouse, cont.
Sperm galactosyltransferase crosslinks ZP3 Nacetylglucosamine
- crosslinking activates specific Gproteins in sperm membrane
- initiates a cascade that opens membrane Ca2+ channels
- results in Ca2+mediated exocytosis of the acrosomal vesicle
 Mammalian Gamete FusionEquatorial
Mammalian sperm enter egg tangentially region
Mitochondria
- contact on the side of the sperm
- membrane fusion at the junction of
the inner acrosomal and cell
membrane
= equatorial region
cortical granules
- egg cortical actin polymerizes in
the region of sperm binding
extends microvilli to sperm

Egg cortical granules release enzymes that

modify ZP so that it can no longer bind sperm
- Nacetylglucosiminidase 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 protamineSSprotamine
GSH
- replication and transcription allowed
GS
protamineSH + HSprotamine

Mammalian oocyte nucleus is arrested in metaphase of

2nd meiotic division when sperm enters
Sperm entry initiates Ca2+ oscillations in the oocyte
-  Ca2+i stimulates the cell cycle (i.e. cell division pathways)
- e.g. Ca2+ inactivates MAP kinase (MEK) – allows DNA synthesis
 Sperm Contribution
Sperm contributes nucleus, centriole, mitochondria, cytoplasm (minor);

- however, mitochondria and mitochondrial DNA are degraded

- therefore, all embryonic mitochondria are derived from the mother
(basis for mtDNA tracing of geneology/phylogenetics)

Several sperm proteins and mRNAs for transcription and

paracrine factors are brought into the egg

Also, microRNAs imported; may downregulate receptors

involved in early cell division
 Egg Activation Pathway

Early responses Late responses