Fertilization - Part 1 Gilbert - Chapter 7 pp. 181-186

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

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)

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.

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

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

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

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

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?

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

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

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

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

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

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

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

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

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