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Unformatted text preview: MCDB 112 F10 STUDY QUESTION SET #2 - FERTILIZATION page 1 Fertilization serves as an excellent model for studying several basic biological phenomena, including the molecular basis of cell-cell recognition, signal transduction pathways, and activation and control of the cell cycle. Thus, we spent a little extra time in lecture going over some of these aspects in detail, to serve as reference points for similar phenomena that occur during later stages of development – the same general principles will apply. These study questions are designed to help you self-learn these key principles. 1. When sea urchin sperm contact the jelly of an egg of the same species, they undergo the acrosome reaction (AR). It is thought that a small, glycosylated peptide molecule in the egg jelly binds a receptor on the sperm surface, triggering a signal transduction cascade that leads to the exocytosis of the sperm acrosomal vesicle and thus the AR. Suppose that you have cloned and sequenced the cDNA encoding this receptor protein from 3 different species of sea urchin. All 3 have a similar domain structure; the protein appears to be a single pass, type I transmembrane protein, meaning that the N-terminus is extracellular and the C-terminus is cytoplasmic. This structure is represented schematically here (the small, black box is the transmembrane or “TM” domain): N C 1A. Based on the function of this protein, how do you think the primary structure (amino acid sequence) of this protein might compare in each of its domains among each of the three species? Explain your answer. 1B. Suppose that your colleague does a very careful sequence analysis of this protein in all 3 species. She finds that the extracellular domain has a stretch of 7 amino acids that is highly variable. The rest of the protein is highly conserved in all three species. Is this consistent with your answer to 1A? Explain. 1C. Based on the given information, state a hypothesis that relates the specific primary structure of the receptor protein to its biological function in the sperm. 1D. Describe (in general terms) one experiment that directly tests your hypothesis in the above question. Explain your rationale, and discuss the possible outcomes of your experiment in terms of whether the results would support or refute your hypothesis. [Note: If you are having trouble with this, it might mean that your hypothesis needs revision – keep it simple and testable]. 2.Suppose that you are studying fertilization in sea urchins. You add sperm to normal eggs or to eggs from which you have physically removed the jelly coat. Then, you count the eggs and calculate the percent fertilized. Explain these results: experiment % eggs fertilized normal eggs + sperm 99 dejellied eggs + sperm 12 mock dejellied + sperm (control) 92 What additional experiment might you do to extend or confirm your explanation? 3. The slow block to polyspermy involves a permanent modification of the egg surface, usually through exocytosis of the cortical granules. In the 1970s, Dave Epel carried out experiments that led to the conclusion that a Ca2+ rise in the egg is both necessary and sufficient to trigger cortical granule exoctyosis (CGE). For each experiment below, describe it as correlation, loss - MCDB 112 F10 STUDY QUESTION SET #2 - FERTILIZATION page 2 of-function or gain-of function. Then, indicate the predicted result and finally, describe at least one control for each experiment. Experiment (a) – Dr. Epel injects the egg with an indicator dye that fluoresces when Ca2+ binds to it. In unfertilized eggs, there is no fluorescence and no CGE. However, when sperm are added to the eggs, he observes fluorescence and then CGE. Experiment (b) – Dr. Epel injects a small amount of Ca2+ into unfertilized eggs and observes CGE almost immediately. Experiment (c) – Dr. Epel injects a calcium chelator (called EGTA; chelators act as specific “sinks” or “magnets” for ions) into unfertilized eggs and then adds sperm. The sperm bind and fuse with the eggs, but no CGE occurs. 4. Sperm-egg interactions leads to rapid, profound physiological changes in the egg. The interaction leads to a release of calcium from the egg’s endoplasmic reticulum (ER) and much work has focused on how this occurs. There are two main hypotheses or models for how sperm egg interaction triggers the signaling pathway(s) leading to calcium release at fertilization. Clearly describe these two models, focusing on the differences between them. 5. Now suppose that you conduct an experiment that addresses the hypothesis of the calcium release pathway trigger (question 4). You isolate a sperm protein (don’t worry at this point how you did that) and purify it. You make an antibody against the protein to use as a specific probe. Here are some experiments and the data: (a) incubate eggs in buffer + purified sperm protein (no sperm added) (b) incubate eggs in buffer + any random protein (no sperm added) (c) incubate eggs in buffer + antibody against sperm protein, then fertilize (d) incubate eggs in buffer + random antibody, then fertilize 100 % eggs with CGE 0 conc of protein or antibody (a) (d) (b) (c) 5A. Based on these results, what do you hypothesize about the function of the sperm protein? Which trigger mechanism is favored? Explain. 5B. Based on your hypothesis in 5A, what do you expect to see if look at the localization of the protein in sperm? Do you expect a difference in non-AR compared to AR sperm? Explain. 5C. Describe one experiment (with controls) that rigorously tests your hypothesis in 5A. ...
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