FertilizationBiology 4361 – Developmental BiologyJune 24, 2009FertilizationFertilization accomplishes two things:Major Events:1. Contact and recognition between sperm and eggs.- must be species-specific2. Regulation of sperm entry into egg.3. Fusion of genetic material of sperm and egg.4. Activation of egg metabolism to start development.Lennart NilssonReproduction (initiates reactions in the egg cytoplasm that allow development to proceed)Sex (combining genes from two genomes)Fertilization OverviewNOTE – Tremendous variation among species- models: sea urchin, mouse, chickSperm formation and structureEgg structure and functionInteractions between sperm and eggsChemoattractionAcrosome reactionBinding and fusionPrevention of polyspermyEgg activationPronuclear fusionMammalian fertilizationSperm FormationSperm AxonemeThe EggAll materials necessary to begin development are stored in the egg.Proteins- yolk (made in other organs (liver, fat bodies), transported to eggRibosomes and tRNA- burst of protein synthesis after fertilizationProtective chemicals- UV filters- DNA repair enzymes- antibodies- alkaloids (and other protective molecules)Morphogenic factors- initiate differentiation- e.g. transcription factors, paracrine factorsmRNA- encode proteins for use in early development- some localized regionallynanosmRNAnucleusbicoidmRNAEgg Maturation at Sperm EntryMost eggs are not fully mature at the time of fertilization; - sperm entry activates metabolism and relieves meiotic arrestEgg Structure – Sea Urchin Volume: 2 x 10-4mm3(200 picoliters)(>200 X sperm volume)egg jelly- glycoprotein meshwork- attract or activate spermvitelline envelope- extracellular (inverts)- fibrous mat- sperm-egg recognition- contains glycoproteinsegg cell membrane- binds sperm- fuses with sperm cell membraneEgg Membrane Structure- cortexlayeractinmicrovilli –filamentous (f-actin)- proteolytic enzymes - mucopolysaccharides- adhesive glycoproteins- hyaline proteinCortical granules: Golgi-derivedcortex –globular(g-actin)egg jellyFertilization OverviewSperm formation and structureEgg structure and functionInteractions between sperm and eggsChemoattractionAcrosome reactionBinding and fusionPrevention of polyspermyEgg activationPronuclear fusionMammalian fertilizationInteractions Between Egg and Sperm1. Chemoattraction of sperm to egg- soluble molecules released by egg4. Passage of sperm through the extracellular envelope5. Fusion of the egg and sperm cell membranes2. Exocytosis of the acrosome- stimulated by binding of egg molecules3. Binding of sperm to the extracellular envelope- usually a multi-step process- binding molecules and receptors located on each gamete Pronuclear fusion: sperm and egg nuclei (pronuclei) meet, fuse; development initiatedSea Urchin FertilizationChallenges for sea urchins (and others):1) Bring two very small cells together in a very large space.2) Ensure that only sperm and eggs of the same species join.Sperm ChemoattractionChemoattraction: eggs produce chemical attractant for sperm, e.g. - 14 aa peptide- source – egg jelly- species-specific- A.p. sperm - membrane resact receptors- binding: ↑ guanylyl cyclase- cGMP activates Ca2+channel- ↑Ca2+iprovides directional cuesResactArbacia punctulataeggs produce “resact”A. 0 sec B. 20 secC. 40 sec D. 90 secresactSea Urchin Acrosome ReactionAcrosome reaction: fusion of acrosome and cell membranes- releases acrosome contentsAcrosome contains enzymesthat digest jelly layerExposed sperm membranecontains proteins that bindto egg receptorsSperm acrosomal process membrane fuses with egg membraneIonic changes stimulate actin polymerization; formsacrosomal processEgg jelly stimulates the spermacrosome reactionAcrosome Reaction – Sea UrchinAR stimulated by contact with egg jelly- species-specific stimulatory molecules- in S. purpuratus – fucose sulfateFucose sulfate binding to sperm receptor activates:- Ca2+transport channel- allows Ca2+into sperm head- Na+/H+exchanger- pumps Na+ in/H+ out- phospholipase - produces inositol trisphosphate (IP3)- elevated Ca2+and basic cytoplasm triggers fusion of acrosomal and cell membranes- proteolytic enzymes digest a path through jelly coat to egg surfaceAcrosome Reaction – Sea UrchinCa2+influx stimulates g-actin polymerization to f-actinAcrosomal process adheres to vitelline envelope viabindin proteinBindin – species-specificbinding to egg receptor on vitelline envelopeActinmicro-filamentsBindinVitelline Membrane Bindin ReceptorsNote: regular sperm distribution species specificity- suggests regular bindin receptor distributionFusion of Sperm and Egg Membranes- membranes fuse (fusogenic protein?)- causes egg actin polymerization- fertilization cone formed- actin from both gametes form connections- sperm nucleus and tail pass through cytoplasmic bridge- acrosomal process adheres to egg membrane microvilliAcrosome reactionFertilization OverviewSperm formation and structureEgg structure and functionInteractions between sperm and eggsChemoattractionAcrosome reactionBinding and fusionPrevention of polyspermyEgg activationPronuclear fusionMammalian fertilizationPrevention of PolyspermyFast block to polyspermy- electrical- sea urchins, frogs- not in most mammals (why not??)Slow block to polyspermy- chemical, physical- most species, including mammalsMore than one sperm entering an egg results in polyploidy; usually eventual deathWhy?Tim WatkinsFast Block to PolyspermyCell membranes provide a selective ionic barrier:- seawater: high Na+, low K+(relatively)- cytoplasm: low Na+, high K+(relatively)This ionic imbalance is maintained by membrane pumps, exchangersresting membrane potentialSecondsNaKKKKKNaNaNaNaNaNaNaKNaKIonic imbalance creates electrical potential across the membrane; ~ -70 mV = -70 mV (inside)plasmamembraneFast Block to PolyspermyIonic imbalance creates electrical potential across the membrane; ~ -70 mVCell membranes provide a selective ionic barrier:This ionic imbalance is maintained by membrane pumps, exchangersSperm binding (or fusion) causes Na+ influxNaKKKKKNaNaNaNaNaNaNaKNaKNaNa1-3 sec after sperm binding, membrane potential shifts to ~+20 mV- sperm cannot bindto eggs with positivemembrane potentialDepolarizationSeconds- seawater: high Na+, low K+(relatively)- cytoplasm: low Na+, high K+(relatively)Fast Block to PolyspermyIonic imbalance creates electrical potential across the membrane; ~ -70 mVCell membranes provide a selective ionic barrier:This ionic imbalance is maintained