Fertilization
When Can Eggs Be Fertilized?
• Meiotic State of the
Egg
–
–
–
–
–
Young primary oocyte
Fully grown oocyte
Metaphase I
Metaphase II
Pronucleus Stage
When Are Sperm Capable of
Fertilization?
• State of Sperm
– Meiosis completed
– Competent in testis (most animals)
– Finish maturation in male ducts and capacitate
in female tract (mammals)
– Fraser Molec Reprod Devel 77: 197-208 (2010)
What Are Barriers to the Sperm?
• Egg Vestments
– Coats made during oogenesis by follicles
– Potential Barriers
• tough keratinaceous shells (insects, fish,
cephalopods)
– micropyle
• soft gelatinous shells (sea urchin, frog, mammals)
– sperm acrosomal enzymes
Sperm Competition I
Sperm Competition II
Most wily?
Fastest?
Success!
Some Membrane Fusions
• Fusion of Gametes
– in most species triggers completion of meiosis
– result: haploid male and female (pro)nuclei in
common cytoplasm
• fusion of nuclear membranes (syngamy)
– mixing of chromosomes in new nuclear
envelope = zygote nucleus
Events at the Egg Surface
How is the Sperm Activated?
• I. Metabolic Activation
– sea urchin from pH 7.2 testis to pH 8 sea water
– H+ out/Na+ in
• increases internal pH to 7.6
– stimulates motility and respiration
• pH sensitive dynein
• uses up ATP to swim
• activates mitochondria to produce more ATP
through respiration (oxidative phosphorylation)
How is the Sperm Activated?
• II. Acrosomal Reaction
– induced by egg jelly
– influx of Ca++ triggers two things
• fusion of granule with p.m. secreting proteases
• actin polymerization from behind the granule to
make acrosomal filament
– plasma membrane covered filament contacts
the egg and fuses
Acrosomal Reaction
How Do Sperm Recognize Eggs?
• Sperm
– species specific binding to egg vitelline by tip of the
acrosomal filament
– expt: isolate acrosomal vesicles
• major protein: 30,000 MW
• make antibody which shows where it is and blocks binding of
sperm to egg
• protein is called bindin
– pure bindin agglutinates eggs, species specific
– important barrier to mixing of species
• Very rapidly evolves
Bindin
Localization
Agglutination
How Does Egg Bind Sperm?
• Egg
– sperm binding sites can be detached with
proteases
– ~1500 per egg
– detach 70 kDa fragment and purify and make
antibody
• Antibody blocks gamete binding
– recognizes 350 kDa protein on blot
– sperm receptor is integral membrane protein
What Does Egg Do When Sperm
Fuses?
Cawave.mov
Release of Free Ca++
What Does Ca++ Trigger?
Raising of vitelline layer – conversion into fertilization envelope
Secretion of hyaline of cortical granules
How Does Egg Prevent Multiple
Fertilizations?
• Polyspermy lethal in most species
• Two levels of control in sea urchins
– slow: physical
– fast: electrical
How Does the Slow Block Work?
• Slow Block
– secretion of cortical granules converts
vitelline to fertilization membrane (f.m.)
or envelope
– sperm pushed from surface by f.m. ->
barrier
• expt: remove f.m., re-fertilize -> polyspermy
– isolate c.g. products
• remove vitelline, fertilize and collect in sea
water
• find trypsin-like proteases
SU Fert.mov
What Is the Role of Cortical
Proteases?
• cortical proteases have two roles
– 1. Elevation of f.m. (delaminase)
• eggs + sperm or parthenogenetic (chemical)
agent -> f.m.
• eggs + sperm and SBTI (inhibitor) -> rosettes
• eggs + cortical proteases, then SBTI and
parthenogenetic -> f.m.
• Interpretation: cortical proteases sever p.m.v.l. bonds which permits lifting of f.m.
What Is the Role of Cortical
Proteases?
• cortical proteases have two roles
– 2. Detachment of sperm (receptor hydrolase)
• eggs + cortical proteases, then sperm -/-> f.m.
– sperm don’t attach to vitelline
• eggs - v.l., then sperm -> polyspermy
• eggs + sperm and SBTI -> rosettes and sperm
remains on surface of f.m.
• Interpretation: cortical proteases detach sperm
binding sites from v.l. releasing excess sperm