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Lecture 10

BIOC19H3 Lecture Notes - Lecture 10: Puromycin, Ovulation, Exocytosis


Department
Biological Sciences
Course Code
BIOC19H3
Professor
Ian Brown
Lecture
10

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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
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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:
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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.
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