Fertilization Developmental Biology – Biology 4361 October 19, 2006Fertilization Fertilization acc omplishes two thin gs:  Sex (combining genes from two genomes)  Reproduction (initiates reactions in the egg cytoplasm that allow development to proceed) Major Even ts: 1. Con tact and recognition between s perm and eggs.  species specificity 2. Reg ulation of sperm entry into egg. 3. Fusion of genetic material of sperm and egg. 4. Ac tivation of egg metabolism to start development. Lennart NilssonFertilization Overview Sperm formation and structure Egg structure and function Model species: sea urchin & human Interactions between sperm and eggs Chemoattraction Acrosome reaction Binding and fusion Prevention of polyspermy Egg activationSperm FormationSperm AxonemeThe Egg All material necessary for the beginning of development and growth must be stored in the egg.  eggs actively accumulate material as they develop:  proteins  yolk (made in other organs (liver, fat bodies), transported to egg  ribosomes and tRNA  burst of protein synthesis after f ertilization  mRNA encode proteins for use in early development  protective chemicals  UV filters  DNA repair enzymes  antibodies  alkaloids (and other protective molecules)  morphogenic factors  direct differentiation of cells into certain types  transcription factors, paracrine factors  some localized regionally; segregated into different cells during cleavageEgg Maturation at Sperm Entry Fertilization activates the egg; including release from meiotic arrestEgg Maturation at Sperm EntrySea Urchin Egg Structure cell membrane  fusion with sperm cell membrane  regulates ion flow extracellular envelope  inverts  vitelline envelope  fibrous mat  sperm egg recognition  contains glycoproteins ( vertebrates – zona pellucida) egg jelly  glycoprotein meshwork  attract or activate sperm Volume: 2 x 10 4 mm 3 (200 picoliters) > 200 X sperm volumeMammalian Eg g cumulus – ovarian follicular cells innermost layer – corona radiataEgg Membrane Structure  cortex actin microvilli – filamentous globular (factin) Golgiderived  proteolytic enzymes ~15k/sea urchin egg  mucopolysaccharides  adhesive glycoproteins  hyaline protein Cortical granules: (gactin) cortex  egg jellyInteraction Bet ween Egg and Sperm 1. Chemoattraction of sperm to egg  soluble molecules released by egg 2. Exocytosis of the acrosome  stimulated by binding of egg molecules 3. Binding of sperm to the extracellular envelope  usually a multistep process  involves binding molecules and receptors located on each gamete 4. Passage of sperm through the extracellular envelope 5. Fusion of the egg and sperm c ell membranes General steps: sperm and egg pronuclei meet, fuse; development initiatedSea Urchin F ertilization Challenges for sea urchins (and others):  how to bring two very small cells together in a very large space  how to ensure that only sperm and eggs of the same species joinSperm Chemoattraction A. 0 sec B. 20 sec C. 40 sec D. 90 sec “resact” Arbacia punctulata  14 aa peptide  source – egg jelly  species specific  membrane resact receptors  binding: ↑ guanylyl cyclase  cGMP activates Ca 2+ channel  Ca 2+ provides directional cueSpermEgg Interaction  Sea UrchinAcrosome Reaction – Sea Urchin AR stimulated by contact with egg jelly  speciesspecific stimulatory molecules  in S. purpuratus – fucose sulfate  fucose sulfate binding to sperm receptor activates:  Ca 2+ transport channel  allows Ca 2+ into sperm head  Na + /H + exchanger  pumps Na + in/H + out  phospholipase  produces inositol trisphosphate (IP 3 )  elevated Ca 2+ and bas ic cytoplasm triggers fusion of acrosomal and cell membranes  proteolytic enzymes digest a path through jelly coat t o surfaceAcrosome Reaction – Sea Urchin  Ca 2+ influx stimulates gac tin polymerization to factin  acrosomal process adheres to vitelli ne envelope through bindin protein  bindin – speciesspecific binding to receptor on vitelline envelopeVitelline Membrane Bindin Receptors regular distribution species s pecificityFusion 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 microvilliPrevention of Pol yspermy Fast block to polyspermy  electrical  sea urchins, frogs, not in most mammals (why not??) Slow block to polyspermy  chemical, physical  most species, including mamm als More than one sperm entry into egg results in polyploid situation; eventual death.Fast Block to Polyspermy  cell membranes provide a selective ionic barrier: c ytoplasm/extracellular  seawater: high Na+, low K+ (relatively)  cytoplasm low Na+, high K+ (relatively)  ionic imbalance maintained by membrane pumps, exchangers  ionic imbalance creates an electrical potential across the membrane; ~ 70 mV  13 sec after sperm binding, membrane potential shifts positively; ~ +20 mV NOTE – transient  sperm cannot bind to eggs with positive membrane potentialSlow Block to Polyspermy Cortical granules  contained just beneath plasma membrane ~ 15,000 granules/sea urchin egg ~ 1 μm diameter Cortical granule reaction  chemical and mechanical block  active ~ 1 min aft er spermegg fusion Sperm entry – stimulates fusion of cortical granule membrane with cell membrane (like AR) CG contents released into the space between the cell membrane and vitelline envelope 1. serine protease  dissolves protein post connections between envelope and membrane  clips off bindin receptors & connected sperm 2. mucopolysaccharides  sticky compounds; produce osmotic pressure  water rushes in, envelope raises 3. peroxidases – oxidizes, crosslinks tyrosines  “hardens” fertilization envelope 4. hyaline (protein) forms a coating around the egg: hyaline layerCortical Granul e Exocytosis  1 cortical granule fusion; release of CG contents elevation of vitelline mem braneCortical Granul e Exocytosis  2 hyaline layerFertilization Envelope 10 sec Time after sperm addition: 25 sec 35 secCa 2+ Role in Cortical Granule Reaction Cortical granule