Fertilization

Biology 4361 – Developmental Biology

July 1, 2008

Fertilization

Fertilization accomplishes two things:

Major Events:

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.

Lennart Nilsson

Reproduction (initiates reactions in the egg cytoplasm that allow development to proceed)

Sex (combining genes from two genomes)

Fertilization Overview

NOTE ­ Much variation among species ­ models: sea urchin, mouse

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 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

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 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 10 ­4 mm 3

(200 picoliters)

(>200 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

­ cortex layer

actin microvilli – filamentous (f­actin)

Golgi­derived; contain: ­ proteolytic enzymes

­ mucopolysaccharides ­ adhesive glycoproteins

­ hyaline protein

Cortical granules:

cortex – globular (g­actin)

egg jelly

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 multi­step process ­ involves 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) 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 punctulata

eggs produce chemoattractant “resact”

­ 14 aa peptide

­ source – egg jelly

­ species­specific

­ sperm have membrane resact receptors

­ binding: ↑ guanylyl cyclase ­ cGMP activates

Ca 2+ channel

­ ↑Ca 2+ i provides directional cues

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 ­ species­specific stimulatory molecules ­ in S. purpuratus – fucose sulfate

Fucose sulfate binding to sperm receptor activates: ­ Ca 2+ transport channel ­ allows Ca 2+ into sperm head

­ Na + /H + exchanger ­ pumps Na + in/H + out

­ phospholipase ­ produces inositol trisphosphate (IP 3 )

­ elevated Ca 2+ 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 Ca 2+ influx stimulates g­actin polymerization to f­actin

Acrosomal process adheres to vitelline envelope via bindin protein

Bindin – species­specific 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

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 Polyspermy

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

Cell 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

Na K

K

K K

K Na

Na

Na

Na

Na Na

Na

K

Na

K

Sperm binding (or fusion) causes Na+ influx 1­3 sec after sperm binding, membrane potential shifts to ~+20 mV

­ sperm cannot bind to eggs with positive membrane potential

Depolarization

Seconds

transient; membrane re­polarizes

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

Ca 2+ Role in Cortical Granule Reaction Cortical granule reaction mechanism similar to acrosome reaction

­ at fertilization, egg cytoplasmic [Ca 2+ ] rises

­ high Ca 2+ causes cortical granule membranes to fuse with cell membrane

­ internal Ca 2+ released as a self­propagating “wave”

1 2

3 4

­ Ca 2+ causes advancing cortical granule exocytosis

t=0

t=30 sec

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

Ca 2+ released from internal store at fertilization ­ increases concentration from 0.1 – 1.0 μM

Ca 2+ activates metabolic reactions; e.g. ­ NAD + kinase ­ burst of O 2 reduction (to H 2 O 2 )

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 2 nd 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

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

­ sperm­egg 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

Inner­most layer – corona radiata

Gamete Translocation

­ hyperactivated motility in the vicinity of the oocyte or cumulus ­ directional cues from temperature gradients (thermotaxis)

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 transport slows at ampulla (timed­release mechanism?)

­ sperm motility important within the oviduct

sperm motility is not sufficient to move sperm to ampulla

Sperm Capacitation (Mammals)

Capacitation – a series of physiological maturation events that take place in the vaginal tract, uterus, and oviduct

Freshly ejaculated mammalian sperm cannot fertilize the egg ­ fresh sperm “held up” in the cumulus matrix

­ 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

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 species­specific

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 N­acetylglucosamine

Acrosome Reaction ­ Mouse, cont.

Sperm galactosyltransferase crosslinks ZP3 N­acetylglucosamine

­ results in Ca 2+ ­mediated exocytosis of the acrosomal vesicle

­ initiates a cascade that opens membrane Ca 2+ channels

­ crosslinking activates specific G­proteins 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

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

­ replication and transcription allowed

protamine­S­S­protamine

protamine­SH + HS­protamine

GSH

GS

Mammalian oocyte nucleus is arrested in metaphase of 2 nd meiotic division when sperm enters

Sperm entry initiates Ca 2+ oscillations in the oocyte

­ e.g. Ca 2+ inactivates MAP kinase (MEK) – allows DNA synthesis

­ ↑ Ca 2+ 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 down­regulate 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