1. Fertilization - Part 1
Gilbert - Chapter 7 pp

2. Fertilization
“There is perhaps no phenomenon in the field of biology that touches so many fundamental questions as the union of the germ cells in the act of fertilization; in this supreme event all the strands of the webs of two lives are gathered in one know from which they diverge again and are rewoven into a new individual life-history. . . ” F.R. Lillie

3. Fertilization
Fusion of two gametes to create a new individual, with a genome different from both parents
HAS 2 MAJOR GOALS:
Joining of genetic material to create new variations (sex)
Creation of a new organism (reproduction)

4. 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
Vary from species to species, but in general
Tonight, we’ll look at some examples of each of these steps. We’ll begin by looking at the unique structure of the gametes that allow and enable these steps, then move on to each step in detail. Your experiment this evening will allow you to watch fertilization in the sea urchin model system, and examine some of the factors important for successful fertilization.

5. 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

6. Recognition of Sperm and Egg
Chemoattraction of sperm to egg by soluble molecules
Exocytosis of acrosomal vesicle of sperm to release its enzymes
Binding of sperm to extracellular envelope (vitelline membrane or zona pellucida)
Passage of sperm through extracellular envelope
Fusion of egg and sperm membranes
NOTE: In mammals, step 2 and 3 are reversed
5 Basic steps
Step 2 & 3 are reversed in mammals
After these 5 steps are finished - nuclei can fuse

7. SEA URCHIN Sperm activated by egg
Egg jelly triggers acrosomal reaction
Sperm binds to Vitelline membrane
Sperm makes hole in membrane
Sperm membrane fuses with egg membrane

8. MOUSE
Sperm must first bind (species-specific molecules) then can release acrosomal contents.
Sperm is capacitated, attracted and activated in female reproductive tract
Sperm binds to ZP before releasing acrosome contents
Acrosome contents digest ZP
Sperm adheres to cell membrane, fuses with egg cell membrane

9. Step 1 - Chemoattraction - Sea urchin model
Some species (marine animals) release eggs & sperm into vast environment
Often other species are present
How do correct egg & sperm find each other at dilute concentrations?
How are sperm and egg modified so that only same species can successfully fertilize?

10. Two mechanisms have evolved to address these issues:
Species-specific attraction of sperm
Species-specific sperm activation
Example:
Sea urchins have resact peptide in the egg jelly of the egg
14 amino-acid peptide
Can diffuse in sea water
Attracts sperm to egg
Acts as a sperm-activating peptide
Increases sperm motility
Increases mitochondrial production of ATP

12. Step 2: Acrosome Reaction (Sea Urchin Model)
Initiated by species-specific compounds in the egg jelly
Compounds bind to receptors on sperm cell membrane
Calcium channels open; calcium flows into sperm head
This induces fusion of the acrosomal membrane with sperm cell membrane

14. Extension of Acrosomal Process (Sea urchin model)
After acrosomal vesicle fuses, an acrosomal process is extended.
Occurs by polymerization of globular actin molecules into actin filaments
Acrosomal process is important for species-specific recognition
In S. purpuratus the acrosomal process contains a protein called bindin
This bindin can bind to the surface of S. purpuratus egg but not A. punctulata

17. Experimental evidence of the species-specific role of binding in fertilization
Biochemical studies show other species of sea urchin sperm contain bindin molecules that are closely related, but different
Immunohistochemistry shows
bindin on the tip of the acrosomal process

22. Mammalian Gamete Binding and Recognition
Zona pellucida plays the role of vitelline membrane
Sperm binds at ZP; ZP initiates acrosomal reaction
There is some species-specificity, but this is not as necessary if fertilization occurs internally

23. The Mouse Zona Pellucida
Made up of 3 proteins: ZP1, ZP2, ZP3
ZP3 is the protein that initially binds the sperm, according to several lines of evidence
Carbohydrate moieties on ZP3 molecule bind to proteins on sperm cell membrane
ZP3 also initiates the acrosome reaction, allowing sperm to traverse the thick zona pellucida
This is mediated by an influx of calcium ions, as in Sea Urchins
Sperm must then bind ZP2 in order to get across the zona

24. Immunofluorescent staining for mouse ZP3 binding proteins
Radioactively labeled ZP3 binds to capacitated mouse sperm
Immunofluorescent staining for mouse ZP3 binding proteins
Radioactively labeled ZP3 binds to capacitated mouse sperm

25. Fusion of Egg and Sperm Membranes
Mechanisms vary between species and are not well understood
Mammalian sperm fusion occurs on the side of the sperm rather than the tip
Cell adhesion molecules, such as integrin seem to be important
Female mice with gene knockout of integrin associated CD9 molecule are infertile
Sperm can’t fuse with their eggs
Can be reverse by microinjecting the sperm into the egg

27. 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