Fertilization 1 Activation Changes Juxtaposition Acrosome Syngamy Membrane Potential Cortical Reaction Electrophysiology Key Concepts 2 Sexual Reproduction Fertilization Syngamy Conjugation Parthenogenesis Introduction 3
Inhibition Fertilization Artificial activation Parthenogenesis Activation 4 Physiological Changes membrane potential increase in Ca ++ increase in ph increase in permeability increase in viscosity increase in proteolytic enzymes start protein synthesis start DNA synthesis 5 Sperm morphology Surface interactions Egg cytoplasm Morphological Changes 6
Juxaposition of Gametes Chemotaxis Chemotaxins Species Specific (1) Miller (1978) sea urchins resact Capacitation 7 8 sea urchin midpiece nucleus G-actin acrosome jelly coat vitelline membrane species specific (2) 9
membrane fusion acrosome membrane sperm cell membrane 10 digestive enzymes channel jelly coat 11 G-actin polymerize F-action acrosomal tubule 12
acrosomal tubule vitelline membrane 13 gamete binding species specific (3) bindin binding sites 14 fertilization cone actin microfilaments 15
gamete fusion membrane fusion 16 Fertilization: Hermit Crab sperm midpiece nucleus oocyte 17 Fertilization: Giant Sperm Frog lung Fluke (trematode) - small eggs: 8 µm - sperm head: 20-30 µm - sperm tail: 275 µm - fertilized egg: 10 µm Drosophila bifurcata - sperm: 6 cm (male=0.3 cm) - translates to 40 yds for a 6 ft man - females: store sperm in long, thin receptacle in which sperm stretch out 18
Fertilization: Mammals bindin acrosomal tubule zona pellucida - (vitelline membrane) gamete fusion 19 Syngamy male pronucleus centriole microfilaments mitochondria flagellum 20 Syngamy nuclear membrane decondensation histones 21
Syngamy nuclear membrane decondensation histones centriole 22 nuclear membrane decondensation histones centriole --> movement Syngamy 23 Syngamy centriole spindle fibers aster 24
Syngamy spindle fibers movement pronuclear fusion 2N--> zygote 25 Syngamy centrioles mitotic spindles DNA replication 26 Resting Membrane Potential (-70 mv) ions Na + membrane permeability +20 mv fast block to polyspermy Membrane Potential 27
Cortical Reaction 28 slow block to polyspermy cortical granules golgi 15,000/egg Cortical Reaction 29 Cortical Reaction fusion with cell membrane release contents cell membrane vitelline membrane 30
Proteases Mucopolysacchrides osmotic gradient Peroxidases fertilization membrane Hyalin hyaline layer Cortical Reaction 31 fertilization membrane hyaline layer cell membrane Cortical Reaction jelly coat cell cytoplasm 32 Electrophysiology Na + influx H + efflux Ca ++ 33
Electrophysiology What is the relationship between Na+ in and H+ out? Experiment 1 -use Na+ free H 2O -fertilize eggs 34 Electrophysiology What is the relationship between Na+ in and H+ out? Experiment 2 -add Na+ back into previous experiment 35 Electrophysiology Which is more important: Na+ in and H+ out? Experiment 1 -add freshly fertilized eggs to a solution of amiloride and Na+ 36
Electrophysiology Which is more important: Na+ in and H+ out? Experiment 2 -add ammonia to the cytoplasm of the oocyte Conclusions 37 The Role of Calcium Electrophysiology Heilbrum Mazia Steinhart & Eppel ionophore A23187 Vacquier & Payne aequorin General Conclusions 38 Summary: Electrophysiology 39
Summary: Timing Event Sperm-egg binding Minor influx of sodium ions; changes in membrane potential (fast block to polyspermy) Sperm-egg membrane fusion Liberation of Ca++ from endoplasmic reticulum Cortical reaction; Na+/H+ exchange begins Conversion of NAD+ to NADP+ Formation of fertilization membrane complete Rise in oxygen consumption Timing 0 sec 1 sec 6 sec 6 sec 15-60 sec 60 sec 60 sec 60 sec 40 Summary: Timing Event Sperm entry Increase in intracellular ph Sperm chromatin decondensation Sperm nucleus migrates toward the center of the egg Egg nucleus migrates toward the center of the egg Increase in protein synthesis Activation of amino acid transport systems Fusion of egg and sperm nuclei Initiation of DNA synthesis First cleavage Timing 1-2 min 1-5 min 2-12 min 2-12 min 5-10 min 5-10 min 5-10 min 20 min 20-40 min 85-90 min 41