Fertilization: Part 2: Prevention of Polyspermy Gilbert - Chapter 7 pp. 187-192

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

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

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

Dispermic Sea Urchin egg Dispermic Human Egg -Note 4 centrioles

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

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

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

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

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

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

Formation of fertilization envelope in sea urchin

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

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

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

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

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