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BIOL 303 Study Questions Set 4

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University of Waterloo
BIOL 303
Dragana Miskovic

BIOL 303 Study Questions Set 4 Key Concepts  Acrosome: head of sperm that releases enzymes to degrade zona pellucida of egg  Capacitation: a biochemical event that results in physiological changes in the maturation of the spermatozoa so that they can fertilize an egg.  Egg or ovum: female gamete capable of being fertilized  Oocyte: developing egg that cannot bind sperm and be fertilized.  Vitelline membrane: ECM in invertebrates (sea urchin)  Zona pellucida: ECM in vertebrates  Cumulus: surrounds zona pellucida in vertebrates; a layer of ovarian follicle cells that nurture the egg at the time of its release from the ovary  Corona radiata: innermost cumulus cells  Cortex: thin gel-like cytoplasm enriched in globular actin which will polymerize into actin microfilaments upon fertilization  Cortical granules: contain digestive enzymes mucopolysaccharides to prevent polyspermy, adhesive glycoproteins, and hyaline protein (mechanical support for blastomeres).  Ampulla: where mammalian fertilization takes place.  Types of cell movement during gastrulation:  Invagination: in-folding of a sheet of epithelium cells, like an indentation  Involution: where you have a whole group of cells that are coming in and folding in back on themselves – a full sheet of cells moving in on itself and coming up  Ingression: some cells decide they are leaving the epithelium and migrate independently. Change in cells from epithelial to mesenchymal.  Delamination: a cellular sheet splits into two cellular sheets.  Epiboly: extension of a whole sheet of cells together, coming down and around.  Protostome: Animals that form their mouth regions from the blastopore, such as mollusks.  Deuterostome: the first opening (blastopore) becomes the anus while the second opening becomes the mouth 1. In addition to the maternal genetic material, what else might you expect to find in a typical egg cell?  Egg/ovum: female gamete capable of being fertilized.  Oocyte: developing egg that cannot bind sperm and be fertilized.  Also contain nutritive yolk, ribosomes (ready to make proteins), tRNA, mRNA (ready for gene transcription), morphogenic factors (paracrine and transcription factors), protective factors (UV filter, DNA repair enzymes, chemicals). 2. In comparing mammalian and sea urchin development, how is the fusion of the genetic material (i.e. formation of the diploid zygotic genome) different in these two systems?  Sea Urchin Fertilization o Female meiosis is completed before fertilization o Sperm chromosomes must undergo decondensation: removal of histones and addition of maternal histones (histones- basic proteins of chromatin). o Male and female pronuclei (haploid gamete nuclei) migrate toward one another and fuse o DNA replication takes place o Sperm centriole (involved in development of spindle fibres in cell division) is used to assemble mitotic spindle (segregates chromosomes between daughter cells) o Sperm mitochondria and flagellum disintegrate within the egg.  Mammalian Fertilization o Oocyte nucleus is arrested in metaphase II o Sperm DNA must be decondensed because it is tightly packed with protamines (compacts DNA into small space of sperm head through disulfide bonds). o Several waves of calcium (initiated by sperm entry) activates kinase that triggers completion of meiosis II division via degradation of cyclin and securin. o DNA synthesis occurs before any fusion of pronuclei. o Nuclear envelopes break down as condensed chromosomes assemble on the first shared mitotic spindle (from sperm). Mixing of genes is not established until two cell stages. 3. Considering the general interaction of the sperm and egg, what strategies are used to establish an inter-species barrier? Acrosomal reaction: reaction that occurs in the acrosome (tip of sperm cell) as it approaches the egg (from chemotaxis). The sperm must penetrate the corona radiata by releasing hyaluronidase from acrosome to digest the cumulus cells surrounding the oocyte and exposing acrosin attached to the inner membrane of the sperm. After reaching the zona pellucida through the egg jelly, the sperm binds sulfated polysaccharides on the egg jelly coat and the acrosomal reaction actually begins. Acrosin digests the zona pellucida and jelly membrane of oocyte. G-actin is going to be polymerized to F-actin (extension of acrosomal process); bind to surface of actin. Acrosomal reaction eats through matrix layer, exposing proteins that can bind specific receptors; releases enzymes. Sperm membrane has receptors for specific sulfated polysaccharides of egg jelly coats. These receptors are highly species specific. The binding of sulfated polysaccharides initiates the acrosomal reaction (above). Another level of species-specific interaction occurs when sperm acrosomal process contacts the vitelline envelope (sea urchin). An acrosomal protein called bindin binds to de-jellied eggs (exposed vitelline envelope) in species-specific manner. Bindin receptors are thought to be aggregated into complexes on the vitelline membrane (invertebrates – sea urchins) so it limits the number of sperm that can bind to the vitelline membrane. 4. What is the consequence of more than one sperm entering the egg? How does the sea urchin egg prevent this from happening?  Polysperm will occur. If two sperm enter, there will be three sets of chromosomes (triploidy). Each set of chromosomes is bringing in its own centriole. The centriole is used to set up spindle fibers. If you have 3 haploid nuclei in a cell, and 2 spindle apparatuses, you end up with a mess – multi polar spindle apparatus pulling on each other with a random number of chromosomes. Will be aneuploid- will not have a full set of chromosomes. Fast block: electric charge in egg membrane. Ion pumps maintain a resting membrane potential at about -70mV (more Na+ externally in sea urchins). There is an influx of Na+ immediately following the binding of the first sperm and the membrane potential shifts to +20mV. This prevents further sperm-egg fusion because bindin protein cannot bind to a positively charged surface. 5. Describethe cortical granule reaction in sea urchin eggs (ie how is it triggered? What structure results and what are the contents of a cortical granule?).  Slow block (cortical granule reaction): slower but a mechanical block to polyspermy in sea urchins and mammals. Sperm entry causes cortical granules to fuse with egg membrane, releasing the granule contents (proteins). Cortical granule serine protease cleaves protein “posts” that connect vitelline envelope to egg membrane. Glycosoaminoglycans (GAGs) bind to vitelline envelope and absorb water, pushing the envelope away from egg membrane, causing a fertilization envelope. Hyalin forms a coat around the egg for structural support.  Cortical granule reaction is triggered by an increase in cytoplasmic Ca2+ that triggers the egg/cortical granule membrane fusion. Ca2+ is released from endoplasmic reticulum (where it is stored) at the point of sperm entry. It is released by IP3 (from PIP2  IP3 + DAG. DAG activates protein kinase C which activates Na+/H+ pump) Mammalian  No fast block. Only slow block (fusion of cortical granules with egg membrane).  Zona pellucida is modified so it cannot bind any more sperm.  Removal of N-acetylglucosaminidases (NAGs) by ZP3 protein.  Cleaved ZP2 is important to prevent polyspermy as well.  Experiment: Mouse eggs expressing a mutant ZP2 (these are genetically altered mice) that cannot be cleaved by ovastcin result in polyspermy.Cleaved ZP2 is necessary for the block to polyspermy in mammals. Eggs with mutant ZP2 (cannot be clipped by ovastacin) are unable to get rid of sperm by the same stage, also polyspermy occurs. 6. What type of experiment easily demonstrates that Ca required for the cortical reaction resides inside the egg?  Use aequorin protein. It becomes luminescent upon binding Ca2+. 7. In what structure does mammalian fertilization take place? How do sperm locate the egg?  Ampulla: segment of mammalian oviduct near the ovary where fertilization takes place.  Oviduct: picks up ovulated oocytes, transporting gametes, providing suitable environment for fertilization and early development, and transporting embryos to the uterus.  Mammalian oocyte is covered with cumulus cells. They are required for the oocyte to be picked up by fimbriae and then transported to ampulla by cilia and muscular contractions  Sperm are motile but that is not enough for them to find the egg. o Uterine muscle contractions get the sperm into the oviduct to reach the ampulla region so fertilization can occur. o Direction cues from temperature gradients along oviduct o Chemical cues from cumulus cells 8. What differences in the sperm behaviour are observed following capacitation?  Capacitation: a biochemical event that results in physiological changes in the maturation of the spermatozoa so that they can fertilize an egg.  Transient: the oviduct may bind and capacitate sperm and release packets of them a
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