Slide 1Spotted MicroarraysSlide 3Spotted DNA MicroarraySlide 510_15_DNA.microarrays .jpgSlide 7Slide 8Slide 9Slide 1010_02_cell_sorter.jpgSlide 12Slide 13Slide 14Slide 15Slide 16FertilizationSlide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Figure 7.1 The Human Infant Preformed in the Sperm, as Depicted by Nicolas Hartsoeker (1694)Slide 26Development of a Human Embryo From Fertilization to ImplantationSlide 28Slide 29Slide 30Slide 31Slide 32Slide 33Slide 34Phospholipase C activates two signaling pathways How can fertilization activate Phospholipase C?Phospholipase C activates two signaling pathwaysSlide 37Slide 38Slide 39Slide 40Slide 41Slide 42Slide 43Experiments to test importance of Ca++Slide 45Microarrays (gene chips) for assaying global gene expression patterns(some slides courtesy of J. Lieb)Spotted Microarrays•PCR products (direct from genome, or from cDNA clones), or oligonucleotides are spotted by capillary action onto a glass microscope slide.•Up to ~44,000 features per slide •Typically hybridized with 2 differentially labeled samples simultaneously.The Fordham Hall DNA MicroarrayerSpotted DNA Microarray18,000 PCR-amplified cDNA clone insertsPrinted on poly-lysine coated 1 X 3 inch glass slide Each spot corresponds to a different genecirca 1998TumorPool of Cell LinesA Typical DNA Microarray ExperimentReference or ControlExperimentalSampleLower in TumorHigher in Tumor10_15_DNA.microarrays .jpgMicroarray technologyincreased expressiondecreased expressionthese genes have higher expression in normal tissue than breast cancersthese genes have lower expression in basal-like breast cancers than in normal tissue or luminal breast cancersExpression profiling:overall patterns of gene expression can be used in diagnosisEach column is from a different tumor.Each row represents one gene. Rows are clustered by similar expression pattern.Chu et al., Science 282: 699-705 (1998)Transcription response during yeast sporulation (1116 out of ~5000 genes changed)Iyer et al., Science 283: 83-87 (1999) Transcriptional response of human fibroblasts to serum(~6% of genes on microarray changed)10_02_cell_sorter.jpgUsing a fluorescence-activated cell sorter (FACS) to separate cellsBirnbaum et al., Nature Methods 2, 615 - 619 (2005)Birnbaum et al., Science 302: 1956 -1960 (2003) ~4,000 genes have cell-type-specific expression patternsEach column shows expression of one geneGlobal expression map depicting major patterns of gene activity in Arabidopsis root cell typesBrady et al., Science 318: 801-806 (2007) Global expression map depicting major patterns of gene activity in Arabidopsis root cell types(Each row represents a group of genes with similar expression pattern)Sorted cell typeSuppose that in a particular developmental state, 500 genes are expressed.Which genes are important for specifying that developmental state?Coordinated regulation of multiple genesDevelopmental functionsFertilizationHow does fertilization occur?What events does fertilization initiate?Meiosis(Oogenesis occurs before birth in humans.)(from Gilbert, Developmental Biology)Oocyte formation in ovarian follicle(from Gilbert, Developmental Biology)Ovulation in rabbit ovaryCell cycle progression in maturing frog oocyteThe immature oocyte is arrested at G2 of meiosis I until stimulated by hormone.Cell cycle progression in maturing frog oocyteProgesterone stimulates meiosis I (oocyte maturation). The mature oocyte arrests again at metaphase of meiosis II.It stays arrested until fertilized by a sperm.SpermatogenesisFigure 7.1 The Human Infant Preformed in the Sperm, as Depicted byNicolas Hartsoeker (1694)The sperm: a stripped downmachine for delivering DNADevelopment of a Human Embryo From Fertilization to ImplantationFertilization: a multistepprocess(ECM)chemotaxis of spermContact between the sperm and the egg Zona pellucidaSperm galactosyltransferase binds ZP3protein(from Gilbert, Developmental Biology)Releases ZP-degrading enzymesSperm entry into hamster egg(from Gilbert, Developmental Biology)(from Gilbert, Developmental Biology)Fusion with egg plasma membraneFertilization induces cell cycle progression, and blocks extra fertilization eventsSperm nucleus and centriole enter egg.Egg activation:Egg finishes meiosis II.Sperm and egg nuclei migrate together to begin the first mitotic cell cycle.Prevent polyspermy:Additional fertilization events are blocked.A wave of increased calcium can be visualizedmoving across the egg from the point of sperm entry(from Gilbert, Developmental Biology)(Ca++ wave occurs in less than one minute.)Phospholipase C activates two signaling pathwaysHow can fertilization activate Phospholipase C?Phospholipase C activates two signaling pathwaysMammals: Sperm-derived PLC-zeta activates pathway.Echinoderms: A kinase activates PLC-gammaCa-dependent Ca channels in ER propagate the Ca++ wave.Egg activation:Ca++ activates Anaphase Promoting Complex (APC).Egg finishes meiosis II.Sperm and egg nuclei migrate together to begin the first mitotic cell cycle.Anaphase-promoting complex (APC)Ca++Egg activation after fertilizationHow to prevent too many sperm from fertilizing?(from Gilbert, Developmental Biology)The cortical reaction(from Gilbert, Developmental Biology)The cortical reactionSperm Galactose tranferaserecognizes ZP3Acrosomal reaction degrades Zona PellucidaMembrane fusion Cortical reaction mucopolysaccharide swells protease cleaves ZP2 peroxidases cross-link ZP proteins glycosidase modifies ZP3 to prevent sperm bindingDelivery of sperm nucleusHow to test importance of Ca++ ?Experiments to test importance of Ca++Inject Ca++Inject IP3Inject EGTA (chelates Ca++)In each case, would the cortical reaction occur?Experiments to test importance of Ca++Inject Ca++ YesInject IP3 YesInject EGTA (chelates Ca++) NoIn each case, would the cortical reaction
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