Gametogenesis & Fertilization
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What is wrong here? BIMA82: Developmental Biology and Genetics Gametogenesis & Fertilization Stefan Baumgartner Lund University Sept. 11, 2018 What is Developmental Biology? Phylogenetic tree showing the positions of some models organsims used in developmental biology ”Developmental Biology is at the core of all biology. It deals with the process by which genes in the fertilized egg control cell behaviour in the embryo and so determine its pattern, its form, and much of its behaviour.” Wolpert 2015 ”For animals, fungi, and plants, the sole way of getting from egg to adult is by developing an embryo. Whereas most of biology studies adult structure and function, developmental biology finds the study of the transient stages leading up to the adult to be more interesting. Developmental biology studies the initiation and construction of organisms rather than their maintenance.” Gilbert and Barresi 2016 1 Developmental Biology Model Systems Comparative developmental milestones for the six model systems System Advantages Disadvantages gastrulation segmentation Yeast Very good genetic system. Easy to Unicellular organism. Limited manipulate. Short generation time. cell-cell communication. Limited set of genes. hatching/birth Very good genetic system. Easy to Simple organism. 959 cells. C. elegans manipulate. Short generation time. Specialized genome. last molt/ metamorphosis Excellent genetic system. Excellent Drosophila tools for manipulation. Short No vertebra. generation time. No genetics. No mutants. Xenopus Vertebrate. Eggs easily accessible. Transparent embryos. Ectopic studies. Limited phenotypic studies. Zebrafish Vertebrate genetic system. Embryos New system. Genetically not well easily accessible. Transparent embryos. developed. Long generation time. Chicken Vertebrate. Eggs easily accessible. No genetics. No mutants. Ectopic studies. Limited phenotypic studies. Mammalian genetic system. Good tools Complex organism. Long generation Mouse for experimental manipulation. time. Embryos inaccessible. Expensive. Historical aspects I Historical aspects II Early Scientific Period: Zeus Ernst Haeckel 1834-1919 Thomas Hunt Morgan William Harvey 1651 liberating Book: “Generation of “Entwicklungsmechanik” 1866-1945. “ Model systems”: living Drosophila. Nobel price 1933 Animals” beings weak magnifying glasses Nikolas Hartsoeker future 1695 “Homunculus” structure is already Marcello Malphigi present at 1673 chicken egg 1st stage primitive microscopes of development 1800-1880: Era of “Recapitulation” or Karl von Baer Charles Darwin 1920 - 1950 1945 - 1985: 1985 - : Era of Molecular “Embryological parallelism” Johann W. Goethe Ernst Haeckel Era of Biochemistry Era of Cell Biology Biology and Engineering 2 Concepts in Developmental Biology Germ Layers Axis Fate Map Developmental Commitment not committed committed 3 Cytoplasmic Determinant Induction Lateral inhibition Stages of development presented in the course Week 2: Fertilization, cleavage, C. elegans Stefan Baumgartner Week 3: Early Development I: Early development in invertebrates: Drosophila Udo Häcker Week 4: Early Development II: Early development in vertebrates: amphibians & fish Udo Häcker + Labs Week 5: Early Development III: Early development in vertebrates: mammals and birds Udo Häcker + Labs Week 6: Neurogenesis and eye development Per Ekström Week 7: Paraxial mesoderm / Limb development and regeneration Edgar Pera Week 8: Organ development / Influence of the environment on development Edgar Pera, Stefan Baumgartner 2, 8 3, 4, 5 6 7, 8 4 Week 2: Gametogenesis, Fertilization and Cleavage Week 3: Early development in invertebrates: Drosophila A-P axis formation in the oocyte A-P axis formation in embryos Segmentation mutants Maternal genes Gap genes Pair-rule genes C. elegans Segment polarity genes Hox genes gametogenesis fertilization Establishment of D-V-axis cleavage Gene targeting cloning iPS cells gastrulation Week 4: Early development of vertebrates: amphibians and fish Week 5: Early development of vertebrates: mammals and birds Spemann experiment Formation of the Spemann Organizer Xenopus and axis formation zebrafish Gastrulation in amphibia and zebrafish Convergent extension PCP pathway Left-right symmetry 5 Integrated in weeks 3-5 : signalling events during early development Week 6: Neurogenesis and eye development Wg (Wnt) Birth and migration signalling of neurons: pathway Stratification Hedgehog Neural crest cells signaling pathway growth cones trophic factors Shh and Bmp in neural patterning RTK-signaling pathway Sevenless pathway Notch pathway Hippo pathway Neural induction eye development, eye field specification Cyclopia due to defective Shh signaling TGF β pathway Neurulation Formation of lens, cornea, retina Week 7: Paraxial mesoderm / Limb development and regeneration Week 8: Organ development & Influence of the environment on development temperature and Contergan (Neurosedyn) sex determination salamander planaria Organs: Bisphenol (BPA) • (endoderm) • pancreas alcoholism hydra • kidney Epimorphosis: adult structures dedifferentiate, grow and respecify into new structures apical ectodermal ridge AER, zone of polarizing activity ZPA Morphallaxis: existing structures repatterns, little regrowth Zika virus 6 relative sizes of eggs Structure of the human egg Stages of oogenesis Structure of the sea urchin egg from first menstruation onwards ! 3-8 months of embryonic ~ 400-800 000 growth oocytes 7 Maturation of the follicle. The hormonal cycle of women. oviduct = fallopian tubules LH FSH Pregnancy test: mab against hCG (human Chorionic Gonadotropin, 244 aa) estrogen progesterone at birth Spermatogenesis Stages of spermatogenesis at puberty: primary Meiosis spermatocytes during secondary spermatocytes ca. 24d embryogenesis birth spermatogonia ca. 35d capacitation mouse: ca. 35 d total ca. 60d 8 Maturation of sperm: ”capacitation” occurs during the travel to the oocyte. Structure of the sperm adenylate kinase important issues during capacitation: • removal of cholesterol • certain proteins and sugars lost • membrane potential more negative • protein phosphorylation takes place Location of sperm production: seminiferous tubules Formation of syncytial clones of germ cells. mitotic divisions meiotic phase differentiation Leydig cells secrete testosterone 9 Sex determination in mammals Sex Determination Inititation of primary sex determination in mammals: Differentiation of human gonads, shown in transverse section the testis and ovary pathway. testis: Sry, Sox9 ovary: Wnt4, Rspo1 XX mouse + Sry transgene: but males do not Which sex? form functional sperm ! 10 Development of the gonads and their ducts in mammals. II Development of the gonads and their ducts in mammals. I development of urogenital system in male embryos comparative 3D analysis of male and females embryos development of Müllerian MD and Wolffian WD duct From: Tridimensional Visualization and Analysis of Early Human Development. Cell 169, 161-173 (2017) 3 Methods: - whole mount immunostaining - 3DISCO clearing - light-sheet imaging A duplication in Sox9 leads to intersex in deer. What are the primary sex determinants in the animal kingdom? XX-XY type XX-XO type unbranched antlers insects: bugs, cockroaches & grasshoppers ZW-ZZ type How can this observation be explained? mammal, insects: Drosophila birds, fishes, reptiles, some insects mammals: Y determines male ZO-ZZ type small testicles Drosophila: ratio retro-posed between sex penis testicles chromosomes / autosomes bees, moths, butterflies 11 The moment of fertilization Fertilization The acrosomal reaction ca. 7µm Prevention of polyspermy: fast block (only in sea urchin) sperm: 1µm/min 12 Film: the cortical reaction and elevation of the vitelline Film: sperm entry and cortical reaction => slow block (sea urchin) envelope (fertilization membrane; sea urchin) cortical granules serine proteases cortical granule exocytosis Sperm-egg binding models in mammals involving ZP3 (old) and ZP2 (new) The slow block system: cortical reaction (mammals) old model new model mammals Localization of Ovastacin in cortical granules Ovastacin=Astl cleaves ZP2 13 Cleaved ZP2 is critical for inhibiting polyspermy (mammals) Humans have 4 ZP proteins (mice only 3), and only ZP2 binds sperms (mammals) ZP2Mut cannot be cleaved anymore Spermatozoa-ZP Assay: interaction is mouse eggs largely species- transgenic species specific. for each of the 4 human Human spermatozoa ZP proteins. bind only to ZP from Human humans and hominoid sperm species (gibbon, added gorrilla), but not to other sub-hominoid species only ZP2 ⇒ (baboon, rhesus binds monkey, squirrel sperms. monkey). From: Gahlay et al., 2010 From: Baibakov et al., 2012 Acrosome-reaction can already occur in the cumulus layer Juno – Izumo binding is essential for fertilization and may and acrosome-reacted sperm can bind the ZP (mammals) contribute to the membrane - block to polyspermy (mammals). Juno- females are infertile GPI- anchored Note: reaction takes place at plasma membrane Rapid shedding Sperm with GFP = acrosomal of Juno = reaction not yet white arrow possible block completed wins the race! to polyspermy, (yellow arrows) but probably not a fast block. From: Jin et al., 2011 from: Cell Cycle Vol. 13, issue 13 (2014) 14 Juno is the egg Izumo receptor and is GPI anchored. Table of events during sea urchin fertilization. recombinant recombinant Izumo1 protein + Izumo1 protein juno cDNA preincubation with antibody staining Juno ab’s + using Juno ab’s recombinant Izumo1 protein PIP lipase C treatment from: Bianchi et