CHAPTER 7 FERTILIZATION BEGINNING A NEW ORGANISM ZOO3603C The human infant preformed in the sperm as depicted by Nicolas Hartsoeker 1694 Preformation An embryo develops from a complete miniature version of the organism Epigenesis An embryo develops progressively from an undifferentiated egg cell de novo Fertilization Fertilization is the process whereby the sperm and the egg fuse together to create a new individual 1 2 3 4 Contact and recognition Regulation of sperm entry Fusion of genetic material Activation of egg metabolism Structure of the Gametes Sperm The egg Recognition of egg and sperm Structure of mammalian sperm Haploid nucleus Acrosome contains enzyme to lyse the outer of the egg Axoneme motor portion of flagellum emanating from the centriole Flagellum 9 2 microtubule arrangement The motile apparatus of the sperm 9 2 microtube Tubulin interconnected protofilments Dynein ATPase Capacitation occur inside the female reproductive tract Axoneme in flagellum Structure of the sea urchin egg at fertilization The cytoplasm includes Proteins Yolk proteins Ribosomes tRNA mRNA Morphogenic factor Protective chemical Egg jelly outside Stages of egg maturation at the time of sperm entry in different animal species Second Metaphase Completed Most Vertebrates Sea Urchins Female pronucleus is not haploid in all animals Sperm entry occurs before the egg nucleus has completed meiosis The sea urchin egg cell surface Sea Urchin Cell membrane Vitelline envelope Microvilli help sperm entry into the cell Cortical granule homologous to the acrosome in sperm Hamster eggs immediately before fertilization Hamster Zona pellucida Vitelline envelope in mammals Cumulus nutrition by ovarian follicular cells Microfilament polymerized actin necessary for cell division Recognition of egg and sperm 1 2 3 4 5 Chemoattraction of sperm Exocytosis of acrosome Binding to the extracellular envelope Passage through the extracellular envelope Fusion of cell membranes Sea Urchin Exocytosis Endocytosis Recognition of egg and sperm Sea Urchin External fertilization Mouse Internal fertilization External Fertilization in Sea Urchins Sperm attraction Acrosome reaction Species specific recognition Fusion of cell membrane Problems of marine organisms on the fertilization How can sperm and eggs meet in such a dilute concentration How can sperm be prevented from trying to fertilize eggs of other species Sperm chemotaxis in the sea urchin Arbacia punctulata Injection of Resact Resact Sperm chemotactic peptide 14 amino acid peptide isolated from the egg jelly 1 sec 20 sec 40 sec 90 sec Sperm migrate into the region of the injection and congregate there Model for chemotactic peptides in sea urchin sperm Resact a sperm activtion peptide Signal transduction induced by resact in sperm RGC receptor guanylyl cyclase Ca2 levels in sperm 0 sec 1 sec The acrosome reaction in sea urchin sperm 1 2 3 4 5 6 Calcium transport channel Sodium Hydrogen exchanger Phospholipase enzyme Intercellular Ca2 increase RhoB activated Polymerizing actin Species specific binding of acrosomal process to egg surface in sea urchins Bindin the acrosomal protein mediating this recognition Species specific bindin receptor should be on the egg vitelline membrane Bindin receptors EBR1 on the egg Beads were coated with EBR1 Bindin in sperm and it receptor on the beads showed species specific binding The prevention of Polyspermy The fast block to polyspermy The slow block to polyspermy Calcium as the initiator of cortical granule reaction Abnormal development in a dispermic sea urchin egg Each haploid nucleus contain 18 chromosome centrioles come with sperm Early death of embryo Aberrant development in a dispermic sea urchin egg At the first cleavage There are 4 centrosomes Egg should prevent the polyspermy How The presentation of polyspermy Fast Block Within 1 3 seconds Chemically Electric potential Transient for a minute Slow Block Within 30 seconds Physically Fertilization envelope Permanent Membrane potential of sea urchin eggs before and after fertilization Resting membrane potential about 70 mV Open Na Channel 70 mV By K leak channel Formation of the fertilization envelope and removal of excess sperm Slow Block 10 seconds Sperm surround the egg 25 seconds Starting at the point sperm entry 35 seconds A fertilization envelops is forming A fertilization envelops is complete Formation of a fertilization envelope The components of the cortical granules fuse with the vitelline membrane to form a fertilization envelope From cortical granules Mucopolysaccharides Peroxidase enzyme Cortical granule exocytosis Exocytosis is caused by the rise of free Ca2 Formation of hyaline layer Hyaline layer is coating around the egg Calcium as the initiator of the cortical granule reaction Within 30 second Released from the endoplasmic reticulum ER Cortical granules fuse with the cell membrane above the rise of Ca2 Calcium Wave in Sea Urchin Fertilization The calcium ion release is monitored by green dextran which fluoresces in the presence of calcium ions Activation of Egg Metabolism in Sea Urchins Early responses Late responses Resumption of protein and DNA synthesis Postulated pathway of egg activation in the sea urchin Postulated pathway of egg activation in the sea urchin IP3 inositol triphosphate Postulated pathway of egg activation in the sea urchin A burst of protein synthesis at fertilization uses mRNAs stored in the oocyte cytoplasm No transcription pH increase and Ca2 elevation act together to stimulate new DNA and protein synthesis 1 2 3 4 5 Fertilization eIF4E degradation Removal of maskin Translatable mRNA Mitosis promoting factor MPF Nuclear events in the fertilization of the sea urchin The fusion form the diploid zygote nucleus Mammalian Fertilization Getting the gametes into the oviduct Translocation and capacitation In the vicinity of the oocyte Hyperactivation thermotaxis and chemotaxis Recognition at the zona pellucida Gamete fusion and prevention of polyspermy Fusion of genetic material Major differences from External Fertilization Fertilization happens in the ampulla the region of the oviduct Sperms are not mature when they are ejaculated Need the capacitation Hypothetical model for mammalian sperm capacitation Translocation 1 2 3 4 5 Uterus contractions Oviduct region before ampulla slows down sperms Hyperactive in vicinity of the oocyte Temperature and chemical cues Long trek from vagina to ampulla Capacitation What does provide the sperm with direction