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Lecture 10

Lecture 10

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University of Toronto Scarborough
Biological Sciences
Ian Brown

LECTURE 10 Vitellogenesis: Differentiation of the amphibian egg; We can use a signal developmental system to show gene expression. Have used different model systems to see levels of gene expression. This system can be used to look at many levels of gene expression in the egg yolk of the amphibian. Oogenesis: Development of the female egg; Functions of the egg: One of the prime functions of the egg is to provide half of the genetic information required for fertilization. Process of meiosis, female cells start as diploid cells, but reduce to produce haploid egg cells. The haploid female and haploid male combine to give a diploid cell with the blending of genetic material. Eggs functions as vehicle to provide cytoplasmic factors. After fertilization, these factors segregate to different daughter cells in the embryo to turn on different patterns of gene expression. These are the transcription factors. Egg has a polarity. The frog egg has an animal pole and vege (?) pole. The animal pole is where the nucleus is and the vege pole is where the egg yolk protein accumulates. Birds, amp and reptiles have a large amount of yolk. Mammalian eggs have a lot less yolk and are smaller than other species. The egg also stores energy for the developing embryo. The yolk is an energy source for the developing embryo. Egg yolk protein: Researchers thought there were two (2) main proteins; Phosvitin and Lipovitellin. It was thought they were processed by different genes. However, it has been found that they come from the same protein (Vitellogenin), which is made in the liver, transported in the blood stream and is taken up by the developing egg in the female and makes phosvitin and lipovitellin. Vitellogenin: This is a precursor protein. It is not a zymogen. It is a precursor protein that is cleaved into two (2) products. Egg yolk is a food source for the developing embryo. It can vary between species. Yolk protein can be concentrated into a yolk platelet in the species that have a large amount of yolk (amphibians). In the platelets the yolk crystallizes. They are found in high concentrations in the mature egg. As the embryo ages, the yolk is used up by the developing embryo. KNOW THE TWO (2) PROTEINS 1 Types of species that have high amounts of yolk are birds, reptiles and fishes. In addition to large amounts of yolk, they have large eggs. This is because embryonic development occurs outside the female. There is no nourishment being provided by the mother. The lone egg needs a source of food to continue development until the species can get its own food. This is not the case in mammals as the egg is smaller and has less yolk because the placenta is used to provide energy to the developing fetus. Spermatogenesis meiosis was completed before adult sperm had started. Cells went through meiosis to make the haploid cell before the cell starts the biochemical changes to look like an adult sperm. In the development of the egg, reduction development starts and stops at prophase. In species producing yolky eggs: Prophase I of oogenesis is divided into three (3) stages; 1. Previtellogenesis 2. Vitellogenesis 3. Postvitellogenesis Previtellogenesis: This is before the development of egg yolk starts. This is when it stops at prophase. This is when meiosis starts and stops. Vitellogenesis: Development and deposition of yolk. Postvitellogenesis: After yolk development and meiosis starts up again and completes to produce a haploid cell. This stage is also called maturation of the egg (completion of meiosis) is triggered by progesterone. This triggers egg maturation which completes meiosis. Two (2) ways to make the yolk: There are two (2) ways the protein can be made; 1. Autosynthesis (making vitellogenin protein within the egg itself) 2. Heterosynthesis (vitellogenin made outside the egg in the liver and transported by the blood) Some organisms use one or both of these ways. Sometimes it starts in the egg, but the main bulk is made in the liver. This is what happens in the frog egg. After synthesis in the liver: 2 Vitellogenin precursor protein goes under post translational modification. The protein is modified after synthesis by enzymes adding phosphate and lipid groups to vitellogenin. 7.7% of the protein is lipid in content and 1.3% of the protein is phosphate. All of most of the addition of the lipid groups happen at one end of the molecule and the phosphate and added to the other end. When it is cleaved, phosphate content becomes phosvitin and the end that has lipids becomes lipovitellin. (diagram) Shows the molecule weight of the precursor protein. When it’s cleaved we can see the molecule weights of the two (2) proteins. During the act of cleavage, some of the amino acids are removed to leave 55 and 290 MW, while the precursor protein was 500MW (molecular weight). The post translational modification happens in the liver before being released into the blood stream. Summary of overall sequence of events of vitellogenesis: (diagram) Ovaries make progesterone (this is a seasonal/environmental cue) Egg production in the frog happens at a specific season. At the right time of the year the ovaries make estrogen and it is released into the blood stream. This affects the liver because it binds to estrogen receptors on the liver. This changes the pattern of gene expression in the liver. The liver is making albumin and is released into the blood stream. In response to estrogen binding the albumin release is turned off, at this point the vitellogenin gene is turned on. The message is translated into the vitellogenin protein. This protein will then undergo post translational modifications. The modified precursor protein is released into the blood stream and is taken up by the developing egg cells by endocytoses. In the egg cells a protease enzyme cleaves the protein to phosvitin and lipovitellin and they accumulate in the yolk platelets and crystallize in the yolk. This builds up the energy resource material in the egg. After fertilization, this egg yolk can be broken down and used for the developing embryo. Why use vitellogenesis as a model system? We can study five (5) levels of gene expression in one system. Vitellogenin is a stable protein that is easy to detect. Vitellogenin is enduced by a single hormone, estrogen. Vitellogenin is a well characterized protein and has antibodies so we can use Western Blotting. There is a high level of post translational modifications. We can induce vitellogenin synthesis in males, where normally there is no vitellogenin produced. Vitellogenesis enables us to study: We can study the effects of hormones on gene expression and coordinated hormones on gene expression. In this system we will see how hormones will work together in gene expression. 3 The two (2) different places where this is found is in the liver and the jelly coat layers around the egg. As the egg moves down the oviduct the egg must be coated by the jelly coat. This must be done at the right time. Other proteins need to be triggering the liver to make other proteins. Hormones act to co-ordinate these events so they happen at the right time. We can study transcriptional control by seeing how estrogen causes changes in the liver. Estrogen turns on the vitellogenin gene, but at the same time it’s turning off the albumin gene. Posttranslational control, the precursor has to have the lipid and phosphate groups added. How long does this take? There is the control of the secretion and the control of the export. It must move from the liver into the blood stream. There is no zymogen control. There is cleavage of the precursor protein to make two (2) product proteins. What systems can we used to study vitellogenesis?: We can study in three different systems; In the female frog, in males, where it normally doesn’t occur and in tissue culture. All of these systems have unique processes to each one of them. Female frogs: This is where it normally occurs biologically. The advantage is in the female frog we can see the whole series of events (process) from beginning to end. We understand the process is triggered by estrogen, which is made in the ovaries, which travels through the bloodstream to the liver where it turns on vitellogenin in the liver. The vitellogenin precursor protein is turned on by the liver. Vitellogenin is cleaved (modified) and exported back into the bloodstream by the liver. The vitellogenin is taken up into the developing egg cells. Lipovitellin and phosivitin are derived from vitellogenin (made from one gene). At early stages of egg development, some of the vitellogenin can be made in the oocyte, but is usually made in the liver and transported by the blood and taken up by the developing egg cells. Male frogs: We inject the male with estrogen. The estrogen interacts with the liver to activate the vitellogenin gene in the male. Some of the steps are observed in the male frog. The vitellogenin gene is turned on and begins to make the vitellogenin protein (synthesis) and sends it out into the bloodstream (it builds up), but there is no target organ to absorb this protein out of the bloodsteam. Therefore, vitellogenin replaces the normal serum proteins in the bloodstream. There is no further study in the male because there are no ovaries (no target organ). Why do we do this in the male frog? We can study the time course in the liver and the process of the transport in the blood with a zero baseline as this gene has never been expressed. 4 We can look at single injections of estrogen and watch the time course as there is a control of the estrogen being a
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