Fertilization

Biology 4361 – Developmental Biology

October 18, 2007

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

Sperm formation and structure

Egg structure and function

Many variations among species ­ models: sea urchin, mouse

Interactions between sperm and eggs

Chemoattraction

Acrosome reaction

Binding and fusion

Prevention of polyspermy

Egg activation

Pronuclear fusion

Sperm Formation

Sperm Axoneme

The Egg All material necessary to begin development and growth is stored in the egg.

Eggs actively accumulate material as they develop:

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)

mRNA ­ encode proteins for use in early development

bicoid

nanos

nucleus

Morphogenic factors ­ direct differentiation of cells into certain types ­ transcription factors, paracrine factors ­ some localized regionally; segregated into different cells during cleavage

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 at Sperm Entry Most eggs are not fully mature at the time of fertilization; sperm entry activates metabolism and relieves meiotic arrest

Egg Structure – e.g. Sea Urchin

cell membrane functions: ­ fusion with sperm

cell membrane ­ regulates ion flow

egg jelly ­ glycoprotein meshwork ­ attract or activate sperm

Volume: 2 x 10 ­4 mm 3 (200 picoliters)

>200 X sperm volume

extracellular envelope ­ inverts ­ vitelline envelope ­ fibrous mat ­ sperm­egg recognition ­ contains glycoproteins

(vertebrates – zona pellucida)

Mammalian Egg

cumulus – ovarian follicular cells

inner­most layer – corona radiata

Egg Membrane Structure

­ cortex

actin microvilli – filamentous

globular

(f­actin)

Golgi­derived; contain: ­ proteolytic enzymes

­ mucopolysaccharides ­ adhesive glycoproteins

­ hyaline protein

Cortical granules:

(g­actin)

cortex ­

egg jelly

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

General steps:

sperm and egg pronuclei meet, fuse; development initiated

Sea Urchin Fertilization Challenges for sea urchins (and others):

­ how to bring two very small cells together in a very large space

­ how to ensure that only sperm and eggs of the same species join

Sperm Chemoattraction

A. 0 sec B. 20 sec

C. 40 sec D. 90 sec

eggs produce “resact” e.g. urchin Arbacia punctulata

­ 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

Chemoattraction: eggs produce chemical attractant for sperm

Sperm­Egg Interaction ­ Sea Urchin

Acrosome reaction: fusion of acrosome and cell membranes releases acrosome contents

Acrosome contains enzymes that digest jelly layer

The exposed sperm membrane contains proteins that bind to egg receptors

Sperm acrosomal process membrane fuses with egg membrane

Ionic changes stimulate actin polymerization – 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 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

Fast Block to Polyspermy Cell membranes provide a selective ionic barrier: cytoplasm/extracellular

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

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

This ionic imbalance is maintained by membrane pumps, exchangers

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

Depolarization

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

NOTE – transient

­ sperm cannot bind to eggs with positive membrane potential

Slow Block to Polyspermy

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

Cortical granule reaction ­ chemical and mechanical block ­ active ~ 1 min after sperm­egg fusion

Sperm entry initiates fusion of cortical granule membrane with cell membrane (like Acrosome Reaction)

CG contents released into the space between the cell membrane and vitelline envelope (perivitelline space)

R. Bowen

Slow Block to Polyspermy

CG 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 ­ 1

Cortical granule fusion; release of CG contents

Elevation of vitelline envelope

Cortical Granule Exocytosis ­ 2

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 mechanism ­ at fertilization, egg cytoplasmic Ca 2+ concentration 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, fertilization envelope, etc.

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

Egg Activation – Early Responses

Egg Activation – Late Responses

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 )

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° so that sperm centriole is between the sperm and egg pronuclei

­ sperm centriole acts as a microtubule organizing center

­ forms an aster

Pronuclear Fusion

Mammalian Fertilization Sea urchin v. mammalian fertilization: many similarities, 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

Sperm Translocation and Capacitation

Sperm are deposited at the cervix; fertilize the egg at the ampulla of the fallopian tube

­ motility not sufficient to move sperm to the ampulla

­ sperm are transported by the female reproductive tract ­ uterine muscle contractions move sperm to the oviduct

The ovulated egg (surrounded by cumulus cells) is picked up by the oviduct fimbriae

­ sperm transport slows at ampulla (sperm time­release?)

­ sperm motility important within the oviduct

­ hyperactivated motility in the vicinity of the oocyte or cumulus

­ directional cues from temperature gradients (thermotaxis)

­ ciliary beating and muscle contractions move oocyte­cumulus complex into oviduct

Mammalian Sperm Capacitation

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 ­ non­capacitated sperm are “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

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

­ resulting in Ca 2+ ­mediated exocytosis of the acrosomal vesicle

­ this initiates a cascade that opens membrane Ca 2+ channels

­ crosslinking activates specific G­proteins in sperm membrane

Mammalian Gamete Fusion Mammalian sperm enter egg tangentially

­ contact takes place 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

­ N­acetylglucosiminidase cleaves part of ZP3 carbohydrate chain

­ ZP2 is also clipped; loses ability to bind sperm

Equatorial region

Mitochondria

cortical granules

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)

Pronuclear Fusion, cont. In mammals DNA synthesis occurs separately in male and female pronuclei

­ male and female pronuclear chromatin condenses into chromosomes that orient themselves on a common mitotic spindle

­ a true diploid nucleus in mammals is not seen in the zygote, but at the two­cell stage

(NOTE – sea urchins produce a common zygote nucleus)

Sperm contribution to zygote:

­ several sperm proteins and mRNAs for transcription and paracrine factors 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

nucleus, centriole, mitochondria, cytoplasm (minor)

Egg Activation Pathway

Early responses Late responses