1. Fertilization: Part 2: Prevention of Polyspermy
Gilbert - Chapter 7 pp

2. Today’s Objectives
Identify the following important components of the process of fertilization: gametes, spermatogonia, acrosome, flagellum, tubulin, oocyte, pronuclei, vitelline membrane, zona pellucida, resting membrane potential, capacitation, cortical granule reaction
Identify the structure of an oocyte
Recognize the harm of polyspermy
Describe various ways that polyspermy is inhibited
Recognize that fertilization is species-specific

3. Fertilization: 4 major events
Sperm and egg make contact and must recognize each other as the same species
ONE (and only one) sperm enters egg
Fusion of the genetic material
Activation of egg to begin development
Fuse membranes
Prevent further sperm from entering

4. Polyspermy Monospermy is the norm Polyspermy is disastrous
Restores the diploid chromosome number
Sperm centriole becomes the mitotic spindle
Polyspermy is disastrous
Results in triploid nucleus
Multiple mitotic spindles form

7. Dispermic Sea Urchin egg
Dispermic Human Egg -Note 4 centrioles

8. Prevention of Polyspermy
Usually done by preventing multiple sperm from entering the egg
Sea Urchins have 2 mechanisms:
Fast Block
Involves a change in egg cell membrane potential
Slow Block
Involves exocytosis of the cortical granules in the egg

9. Fast Block to Polyspermy (Sea Urchin model)
The egg has a different ionic concentration from the seawater in which it exists
Egg has lower sodium ion concentration; higher potassium concentration
This is maintained by sodium/potassium pumps in the egg cell membrane
The difference in charge across the egg membrane can be measured as -70mV and is called the resting membrane potential

10. Sodium-Potassium Pump
Pumps Sodium Out of cell
Pumps Potassium Into Cell

11. Fast Block to Polyspermy (Sea Urchin model)
1-3 seconds after first sperm binds, the membrane potential shifts to +20 mV
Sperm can no longer fuse to the egg
Experimental evidence - Polyspermy can occur if*:
Eggs are supplied with an electrical current that keeps charge at -70mV
Fertilization occurs in water with a low sodium ion concentration
***This fast-block is only transient and lasts only for about a minute
*Jaffe 1976

13. Slow Block to Polyspermy - Sea urchin model
Slower, mechanical, permanent block
Occurs about a minute after sperm-egg fusion
Upon sperm entry cortical granules fuse with the cell membrane and release several molecules

14. Cortical Granule Molecues
Cortical granule serine protease
Releases vitelline membrane from its anchors to the cell membrane
Clips off bindin molecules
Mucopolysaccharides
Cause osmotic gradient
Water rushes into space between vitelline envelope
Vitelline envelope expands (lifts) and becomes the fertilization envelope
Peroxidase
Hardens the fertilization envelope
Hyaline
Forms a coating around the egg, protects during early embryonic development

18. Formation of fertilization envelope in sea urchin

19. Mammalian Cortical Granule Reaction
Does not form a fertilization envelope
Does modify Zona Pellucida so sperm cannot bind
In mice, cortical granules cleave an essential portion of the ZP3 molecule

20. Calcium and the Cortical Granule Reaction
Upon fertilization, intracellular concentration of Calcium ion in the egg increases
This is necessary for the fusion of cortical granules with the cell membrane
Calcium comes not from outside the egg, but from inside the egg itself
The fusion begins near the site of sperm entry and continues in a wave across the egg
A similar wave of calcium ion release can be observed

21. Egg injected with a dye that fluoresces when it binds calcium ion

22. Calcium experiments - Ca2+ is directly responsible for cortical reaction
A23187 is a calcium ionophore
Transports Ca2+ across lipid membranes
Placing sea urchin embryos in sea water containing A23187 results in cortical granule reaction & fertilization envelope to rise (without presence of sperm)
If Ca2+ chelator is injected into egg, no cortical reaction occurs

23. Fertilization: 4 major events
Sperm and egg make contact and must recognize each other as the same species
ONE (and only one) sperm enters egg
Fusion of the genetic material
Activation of egg to begin development