1
Animal Development
Lecture Notes
Jan 7th
8 Principles of Developmental Biology (FTQ 4)
1. Cellular continuity: Simply put, this says that cells come from cells. It’s almost a given if you have
a multicellular animal that all cells of the animal came from 1 cell. You have preexisting cells that
divide and multiply.
2. Overlapping mechanisms: Animals have more than one way to accomplish the same goal/purpose.
This is good because sometimes one way may fail and you need a backup system to fit in. This is
wise cause you don’t want a system based on one critical weakest link.
3. Cytoplasmic localization: The cytoplasm of cell is not necessarily the same everywhere. It is not
uniform. In contrast, there are certain parts in the cell that are localized. Cells by design have
regional differences.
4. The default program: Several developmental processes seem to be programmed in to automatically
happen. So unless something intervenes, the program moves to some particular end point. The
default means the automatic tendency to move towards a given destiny.
5. Induction: This is a specific term that refers to cell-to-cell communication using chemicals. Cells
will interact with other cells by sending chemical messengers that influence what the cell is doing.
6. Reciprocal interactions: When the signals are sent from one cell/tissue to another cell/tissue, the
receiving cell can send a message back.
7. Differential gene expression: When you look in cells, not all genes are on in all cells. Certain cells
have certain genes that are on that are different than another cell type.
8. Cell differentiation: Cells (most) as we typically understand will develop to a particular destiny.
They differentiate.
Developmental biology is multidisciplinary. It draws from several fields to understand how life develops.
There is a lot that we don’t understand. We will draw from cell biology, anatomy, biochemistry, histology
etc. As we go through the class some areas will be very strong in any one field.
An Overview of Animal Development:
The mature frog produces gametes by the process of gametogenesis. These eventually unite (Fertilization).
Then this cell divides (cleavage). As they divide, they start to differentiate. Germ layers develop (endoderm
etc). These are created through a process called gastrulation. Then organs are generated (organogenesis). It
continues to develop and goes the metamorphosis and you get a mature frog.
Gametogenesis:
Genesis refers to beginning. Gametogenesis is the process of gamete formation (male and female). The
process is different in males and females.
Cells come from preexisting cells. The oocyte comes from an oogonium in the process of oogenesis. This is
the specific gametogenesis in females.
Spermatogenesis is the process in males that leads to the formation of sperm from spermatogonium.
The oogonium and spermatogonium are primary germ cells (stem cells). A stem cell is a cell that has the
ability to divide and divide. In females Oogonium production happens before we are born. These are then
released at ovulation.
In males, we form spermatogonium at puberty. Then these can be drawn on.
There are also mammalian associated somatic cells. In the female, those are follicle cells and in males,
sertoli cells. These cells surround and nurse the developing cells.
Mice were genetically altered to not have sertoli cells. These mice were then infertile. This shows the
importance of these mammalian associated somatic cells.
2
Gametogenesis has 2 phases, whether male or female:
1. Meiosis:
a. Reduction division creating a haploid state
b. Genetic crossing over allowing the gametes to have different kinds of genes
c. It determines the sex. Sex chromosomes get divvied up.
2. Cell Differentiation:
The relative timing of meiosis and cell differentiation is flip flopped between male and female. In females,
cell differentiation occurs first. In men, Meiosis happens first. In men, the cell differentiation gets finished
just in time before fertilization.
An Overview of Mammalian Spermatogenesis:
Spermatogonium starts off spermatogenesis. Some of these cells differentiate to a primary spermatocyte
and has a 2N (Diploidy), 4C (Has to do with the number of DNA molecules). The primary spermatocyte
begins meiosis and we go from 2N to 1N. This secondary spermatocyte undergoes meiosis 2 to form a
spermatid (1N, 1C). This is where the sister chromatids separate. The spermatid is the product of meiosis
and is now ready to undergo cell differentiation. In the male it occurs in different parts of the body and has
different names.
1. Spermiogenesis: The process that shapes the spermatid into an elongated cell.
2. Spermiation:
The product of these processes is the spermatozoan (sperm cell)
These happen in the seminiferous tubules. When you look in the testes there are hundreds of feet of coiled
tubes. In the wall of the tubes is where we form spermatozoa. These look ready but are not. There are some
changes that have to happen. The first set of changes happens in the epidydimus. This is where they are
stored until they leave the body. This is where maturation occurs (2 weeks in humans). After maturation,
they have the potential for fertilization. They then have to undergo capacitation, which happens in the
female reproductive tract.
Lets say you are going to shop for your very first Ford Escort. You decide to go and get the best one. You
go to the Ford plant and try to get it before anyone else. You hand the money over and get in the car and
sits there. But the tank is empty. It’s not ready. He asks for some gas and there is a tank around the corner.
He pushes it there and fills the tank and then sits in the car. But then he realizes that he doesn’t have a key.
So to have a functional car, you need gas, but you also need a key.
In spermatogenesis, you have a sperm factory. Even though they are cranking out, they are empty. They
need to get filled up. This happens in during maturation the epididymus. Capacitation is where you get the
key in the female reproductive tract.
So morphologically, things stop in the seminiferous tubule.
1-9-02
Mammalian Sperm anatomy: The head
- There is a very dense area in the head, which is the nucleus, surrounded by the nuclear envelope.
The DNA (Chromatin) is very compacted. That’s why it appears very dark. In fact, the volume of
a sperm nucleus is about 5% of the average cell. The nucleus is also elongated.
- There is an organelle (orange in diagram) called the acrosome. The word some means body and
“across” refers to heights. In this case it is referring to the tip. The acrosome is a secretory
membrane bound organelle. It has inner and outer membranes. The acrosome itself is found
between the cell membrane and nuclear envelop and has 2 regions:
o Principle segment – this part is lost during fertilization
o Equatorial segment – not lost during fertilization.
The acrosome covers about 3/4 of the nucleus.
The job of the sperm is to deliver its genetic material to the egg.
3
Mammalian Sperm Anatomy: The tail
- This is the motor to deliver the DNA. It has 4 parts
o Connecting piece: It connects the tail to the head.
o Middle piece: This is the actual motor part of the tail. It is encased by mitochondria.
o Principle piece: Characterized by being covered with a sheath (protective). When you cut
it in half, you see 2 ribs that encircle the tail.
o End piece: This is not covered with a sheath and is the most slender part of the tail.
How does the tail actually move (FTQ 2b)?
If you look at a cross section of the tail you see a combination of microtubules arranged in a special way.
Taken together, these are called the axoneme. You can actually see individual microtubules and the
protofilaments with electron microscopy. There are different parts to the axoneme. There is a 9+2
arrangement of microtubules. The 9 refers to 9 doublets/pairs of microtubules that surround the axoneme. If
you look closely at the pairs, you have a complete one and an incomplete one. In the center of the axoneme,
you have two singlets. There are molecules that connect the central microtubules to the surrounding
doublets. There are also dynein arms mediate the movement of the sperm tail. As they interact with the
microtubules of the axoneme you get movement.
Each of the nine doublets has dynein arms that can reach out and bind to the neighboring doublet. In some
unknown way, the dynein arm binds the other microtubule doublet and then retracts in a specific way. This
causes the tail to bend. They don’t all grab each other at one side. There is sort of a “wave” of binding and
releasing, which causes movement of the tail. Dynein arms require ATP to work. This is why you need the
mitochondrial sheath to provide the ATP necessary.
Some men have a mutation and are don’t have dynein arms and are infertile.
There are also nine dense fibers that surround the axoneme and extend through the middle and principle
pieces, but not the end piece. These dense fibers provide rigidity to the tail. The evidence for this is that
when you look at mammals, animals with longer sperm have thicker fibers. One species of fruit fly has the
longest sperm tail.
CELL DIFFERENTIATION
Spermiogenesis.
- The nucleus moves off center and we call it to an eccentric nucleus. It then looses its nuclear
pores. There is no biosynthesis.
- There is nuclear elongation via the manchette and dynactin. The manchette is a cage of
microtubules that surround the nucleus. We believe that there are connections between the
microtubules of the manchette via dynactin. Dynactin mediates movement. It helps stretch the
nucleus. This is another role microtubules play in development.
- Chromatin condenses. It does this in a three step way:
o The histones of the chromatin are chemically modified. Histones are part of the
nucleosome that you wrap the helices around. This can be accomplished in 3 ways:
 Hyperacetylation
 Phosphorylation
 Ubiquitination
o Replacement by transition nuclear proteins and protamines. Protamines are effective at
binding DNA and condensing it down. They are rich in arginine and cysteine. Arginine is
positively charged and basic. DNA has a negative charge. So Arginine is effective in
helping binding the DNA. Cysteine residues can form disulfide bridges and helps in
compaction of the chromatin.
A rooster was taken and proteins were extracted over time. Lane 1 is the most immature
sperm. As the sperm matures, there is a reduction of histones and an increase in
protamines. This has also been examines with transgenic mice (FTQ 3). A transgenic
mouse is a genetically engineered mouse. In this case, the genetic engineering can be two
ways. Some researchers were asking that if we change the expression of protamines, how
would that change chromatin condensation.
You harvest embryonic stem cells and introduce genes. This engineered stem cell is
reintroduced into a normal embryo. This embryo now has a mixture of modified and
4
unmodified cells. This is introduced into uterus of a surrogate mother. These mice are
then called chimeric mice. Then you are looking for chimeric mice that will introduce the
new gene into its offspring. If you can get 2 heterozygous transgenic offspring and cross
them, you can get a homozygous transgenic mouse. In the case of protamines, genes were
inserted that caused early expression of protamines. As the proteins were made
prematurely, condensation of the chromatin happened early. This supported the fact that
the condensation was in some way controlled by protamines.
1-14-03
Transgenic mice have been used as a tool to study all sorts of things like cancer etc.
We were looking at the ability to change protamine expression by using transgenic mice. The altered gene
expressed protamine prematurely and caused early compaction of the DNA.
Knockout mice: A mice with a gene removed. It is similar to transgenic mice but involves a little more
manipulation. You take a gene and clone it. You insert a drug marker into it. This mutated gene no longer
works. This is introduced into a stem cell. In the genome of the mouse, there is a normal copy of the gene.
Then you force crossing over to happen in an artificial way. This crossing over introduces the mutant copy
of the gene into the genome of the mouse. You now have a genetically modified mouse with the gene no
longer working. You then follow the same procedure – putting the stem cell into an egg and injecting it into
surrogate mother. Then you breed chimeric to wild type in the same way. If you get 2 mice with a mutated
gene, when you cross it over it is transferred like an allele. Then you get a homozygous mouse with both
copies of the gene knocked out. The knockout mouse was used to knockout the protamine from mice. The
problem is that there are multiple genes for protamine. They couldn’t knockout all of them so they knocked
out 1 important one. What they noticed was that the chromatin was not as compacted. There was less
binding to the chromatin of DNA. This shows the importance of protamines.
There was a study done to actually see the compaction of the DNA. A bead was attached to a DNA
molecule. The bead is in a solution that was flowing. Then they added protamine to watch what would
happen to the DNA. They actually saw the DNA compacting. Also with more protamine, there was more
compaction.
Where does the acrosome come from?
Derived from the Golgi complex. The axoneme is believed to be built from the centriole. A centriole is a
region where microtubule gets organized.
SPERMIATION
A syncytial nest: This is a group of sperm cells that are developing in unison. They are interconnected and
share intercellular bridges. They actually grow up in families. The spermation is the release of sperm cells
from the nest.
These cells develop in this nest. This keeps it in sync and allows it to happen together. Also these bridges
allow for the communication of gene products. This is important because if one cell has an X chromosome
and needs some Y chromosome products, it can get this along these bridges.
Notice that the transition of the round spermatids to the sperm cells involves the loss of cytoplasm. This is
seen as residual bodies left in the seminiferous tubules.
In many animals when the sperm cells are released, they are ready for fertilization. But in mammals, there
are 2 more things that have to happen
- Maturation: Occurs in the epididymus. This takes about 2 weeks in humans. Part of the
spermiation process involves the loss of cytoplasmic droplet that is left over from spermiation.
Cholesterol levels increase in cell membrane. Cholesterol is a molecule that stabilizes the
membrane. It buffers changes in the fluidity of the membrane. Sperm membranes during
maturation are extra stable. This is because we don’t want them to undergo events prematurely.
One of the ways to keep them from doing this is to give them a stable cell membrane. In the walls
of the epididymus there are proton pumps into the fluid. This gives an acidic pH, which promotes
quiescence. There is also another factor that promotes quiescence is the decapacitation factor. This
turns on a pump that pumps Ca2+ outside of the cell. This lower calcium level also promotes a
quiet state of the cell.
5
-
Capacitation: Occurs in the female’s body in mammals. When the sperm is introduced into the
female body it undergoes this process for about 5 hours. The membrane cholesterol levels
decrease. Motility goes up, accompanied by respiration to promote “hyperactivation”. The reason
for this is because it will be moving through some secretions that can be fairly viscous. Having an
extra active tail will help it go through that mucous and also some of the layers of the egg cell.
Calcium levels go up because DF is gone. In humans it has been demonstrated that the sperm are
attracted to chemicals released by the follicle (chemotaxis). Most sperm, depending on what phase
of the cycle the female is in, get stuck to the wall of the fallopian tube and stay there until it is time
to be released (when an egg is ready). Studies have also been done that show that during
capacitation, the membrane hyperpolarizes due to K+ leaving. This hyperpolarization is believed
to prepare the sperm for the future exocytosis that it will experience. The sperm is designed to
deliver its DNA to the egg, and this is done via exocytosis during fertilization. Cholersterol levels
dropping, as mentioned above, also aids in this process. Bicarbonate also comes in to neutralize
the acid pH.
Sperm competition: In many animal species you have males that fight with other males in order to
inseminate a female. This rivalry can occur at the cellular level.
1. Primate testes size correlates with social group breeding. As you look at primates you see different
approaches. There is a chimpanzee approach, which is more promiscuous. Then you have the
gorilla that dominates. When you look at chimpanzees you see that the size of the testes are 4
times more than gorillas. The idea here is if chimpanzees can make more sperm, you can outcompete other males. This correlation with breeding behavior and sperm production is one line of
evidence.
2. Sperm motility correlates with degree of male competition. The species with more competition
have faster swimming sperm.
3. Strategic ejaculation. Sperm volume changes with degree of competition. Expts have been done
with a pair of rats, male and female. They were allowed to live together and the amount of sperm
ejaculated from the male. If you take this same female and put it around other males and put it
back with the first male, the amount of sperm ejaculated increases.
4. Miscellaneous:
a. In butterflies, males fill the female up with cheap sperm so that the female doesn’t go
with other males. Once the females are full, they won’t go with any more males.
b. Inhibitory semen
Consequences: May shorten the lifespan of the male.
Sperm Cooperation: In the common woodmouse, 5% of its body weight consists of testes. The idea here is
that these mice a promiscuous breeders. Some researchers noticed that the sperm do not operate as
individual cells. They hook up and form a sperm train. There is a little hook at the end of the head of the
cells. When the first sperm is introduced into the female’s body. Shortly after insemination, the hook of one
sperm hooks around the tale of the other. Then you get a whole mass of the cells hooked together and form
a train. As the cells are moving towards the egg in the reproductive tract of the female they are working
together. Sperm in the train moves faster than they would as individuals. They will work together as a
group to find the egg. Once they are close enough to the egg, they will unhook and try to find the egg on
their own. The last thing that they noticed was that not all the sperm in the train are capable of fertilizing
the egg. They are sacrificing themselves to help the ones that can fertilize the egg. This is termed altruism.
OOGENESIS
Some terms:
- Oviparous: A female that is laying a fertilized egg/early embryo. Chickens etc. The chicken grows
up outside the female body
- Ovuliparous: Lays unfertilized egg
- -Viviparous: Live born e.g. in humans
- ovoviviparous: eggs hatch immediately upon laying e.g. sharks, snakes.
6
In the formation of the oocyte, cell differentiation happens first and then meiosis. It parallels the formation
of sperm. You have primary oocyte undergoing meiosis I to form the secondary oocyte and the polar body.
Then the secondary oocyte undergoes meiosis II to form an ovum and a polar body. Cell differentiation
happens when the egg is in the oocyte stage. In humans, women are born with primary oocytes that are
“frozen”. Cell differentiation happens to the primary oocyte. At puberty ovulation starts. Ovulation is the
release of secondary oocytes. There is also an arrest until fertilization. Meiosis II is not completed until
after fertilization.
1-16-03
Preparation for fertilization
Fertilization
Before birth
“puberty”
Oogoniu ------- > 1o oocyte --------- 2o oocyte ------ > ovum
Ovulated
Arrested in
Meisosis I
Meiosis II complete
Meiosis II arrest
Resumption and
Completion of Meiosis I
Cell diff happens here
If you look closely at a frog egg, you would notice that not everything is uniformly distributed. This is the
principle of cytoplasmic localization. In the frog the yolk tends to accumulate away for the nucleus. In the
egg, the nucleus is called the germinal vesicle. This is usually at the animal pole. The yolk tends to be near
the vegetal pole. The yolk is the food for the egg and developing embryo. The early embryo in frogs draws
on this for support it. Ribosomes tend to be towards the animal hemisphere and less in the vegetal
hemisphere in a gradient. If you stain living frog eggs for mitochondria, you see that there cloud of
mitochondria around the germinal vesicle. When we look at oogenesis, especially in cell differentiation is a
time of stockpiling. The egg is accumulating all sorts of resources to build the embryo. The quantity of
these resources is much more than in normal cells.
YOLK AMOUNT AND DISTRIBUTION
Amount:
- Alecithal: These eggs don’t have any yolk. Mammals are an example of this kind. They don’t need
yolk because the placenta is feeding the developing child.
- Microlecithal: very little
- Oligolecithal: small amount of yolk. E.g. marine invertebrates.
- Mesolecithal: moderate amounts. Amphibians.
- Megalecithal: lots of yolk. Birds.
Distribution:
- Isolecithal: evenly distributed. E.g. echinoderms
- Telolecithal: largely in vegetal pole. E.g. fish, amphibians
- Centrolecithal: concentrated in center. E.g. insects
RNA is massively stockpiled in the oocyte. Once fertilization happens in the frog, the genome is silent in
the nuclei of these cells until the frog embryo is about 4000 cells in side. Any RNA and protein in this stage
has to come from the mother until the genes are turned on.
RNA is a product of transcription. The majority of RNA in cells is ribosomal RNA. If you look in frogs,
ribosomal RNA is the dominating type. In the nucleus is where Ribosomal RNA is produced.
Mechanism of RNA synthesis:
- Increased transcription. This is happening during meiosis I. This is when the cells are diploid. It
has twice the complement of DNA to be transcribed.
7
-
Gene families. This is a collection of sequences that encode similar products. This is seen in the
gene for 5S rRNA. This is a kind of mass production of the same proteins.
Gene amplification: Certain parts of the genome are selectively replicated. These copies can then
be transcribed. E.g. the gene for 18S rRNA.
THE HORMONAL BASIS OF YOLK PRODUCTION
There can be seasonality to breeding in animals. Part of the seasonality is tied in with the production of
yolk as those oocytes are getting ready to be fertilized. The types of cues that the animal can sense is
temperature, photoperiod etc. The animal can pick these cues up, and the brain produces gonadotropic
hormone. This is a hormone that moves to the gonad and influences the follicle cells to produce estrogen.
This estrogen has many affects. One of these is influencing the liver to produce a yolk precursor
(vitellogenin). This is secreted into the blood stream. It moves to the ovary and is taken up by
micropinocytosis. It is then used to make yolk platelets. The liver is really the origin of yolk in frogs.
When you compare this to fruitflies, you see a similar kind of scheme. There is still a neural structure
sensing an environmental change. Its corpus allatum produces JH. The JH then influences the ovary to
produce ecdyson, which influences the fat body to produce vitellogenin, which is taken into the oocyte and
results in the production of yolk platelets.
General scheme. Brain  hormone  ovary  hormone  vitellogeninic organ  yolk.
The yolk in different organism is very similar in different organisms.
OOCYTE-ACCESSORY CELL INTERACTION
Accessory cells contribute to the success of the oocyte.
- In mammals: Oocytes are surrounded by follicle cells (somatic). In the ovary, you have graafian
follicles that are forming. There is usually only 1 Graafian follicle that eventually ovulates. The
oocyte is released and the left over are the follicle cells. The fluid in the follicle cavity is secreted
by the follicle cells. Hormones are also produced by follicle cells.
- Fruitflies: They have nurse cells. They are derived from the germ line. The presence of nurse cells
in oogenesis is called meroistic oogenesis. Nurse cells are derived from the oogonium. This
oogonium starts to undergo mitosis. The oogonium undergoes 4 rounds of cell division to yield 16
cells that are clustered together in an ovariole part of the ovary of a fruitfly. In the fruitfly, there
are lobes in the ovary called the ovariole. As the cytoplasm cleaves, the cytoplasm leaves at least 1
connection (ring canals) with another cell in the 16 cell cluster. One of these 16 cells is the actual
oocyte. The rest are nurse cells. The ring canals allow for the nurse cells to send components from
there cytoplasm to the developing oocyte. There is a cytoskeletal structure called the fusome.
Whatever cell inherits that is designated to become the oocyte. The rest become the nurse cells (by
default). The components know to move to the oocyte because of the fusome. The fusome is
actually a complex of microtubules. The fusome is only important at the early stage when the
nurse cells are sending their components to the oocyte. It is the lost.
Nurse cells are polyploid. They can have up to a thousand copies of chromosome. Several
experiments have been done to show the RNA movement. A mRNA that fluoresces was injected
into one area of the nurse cells. Eventually, the fluorescence moves into the oocyte. Also, a beta
galactosidase marker showed a lot of beta galactosidase in the nurse cells. In late oogenesis, the
beta galactosidase is seen in the oocyte.
8
1-21-03
Oocyte Maturation
- How progesterone triggers the events from the end of prophase I to ovulation.
These details are from frogs.
The job of the oocyte is to finish meiosis and have cell differentiation occur. During the arrest period (in
prophase I), cell differentiation happens. In humans this happens before birth and continues until puberty.
Meiosis I resumes due to hormonal influence and the egg is eventually ovulated. We now have a secondary
oocyte. It starts meiosis II but then there is a metaphase 2 arrest. It stays in this arrest until fertilization
occurs when meiosis II goes to completion.
Oocyte maturation is the period when the oocyte gets ready for fertilization. It involves a couple of things:
- In the frog, this is triggered by progesterone. This initiates oocyte maturation.
- The sperm ends maturation.
Maturation results in a metaphase II-arrested oocyte that can be fertilized.
Relies on an internal response triggered by an external stimulus. It utilizes the regulatory system of cell
division: MPF.
What is MPF?
It stands for maturation promotion factor. It was first discovered in an experiment. They took progesterone
and they treated an oocyte with it. It started to undergo oocyte maturation. They took out some of the
cytoplasm from the progesterone-treated oocyte and transferred it to a second oocyte that never saw
progesterone. The second oocyte lacks the stimulus for oocyte maturation. That oocyte started to undergo
oocyte maturation. So there was something about the cytoplasm of the 1 st egg that change due to
progesterone. They then purified out the MPF factor from this cytoplasm.
Progesterone activates MPF. It is found in the cytoplasm in its inactive state. Via c-mos (and other factors),
progesterone activates MPF. MPF has 2 subunits. The regulatory part of the complex is the cyclin protein.
Its job is to help regulate MPF’s activity. The actual catalytic part is called p34 (based on its 34kDA size).
It is also called cdc2 kinase. Cdc2 was first described in the yeast. It is short for control cell division. It is a
kinase, which is an enzyme that phosphorylates targets. During the activation of MPF, the inactive MPF is
phosphorylated to become active. MPF allows Meiosis I to continue. In meiosis I you have the formation of
the polar body. The nucleus has its first meiotic division, which involves the breakdown of the germinal
vesicle.
C-mos is believed to be very important in the metaphase II arrest. In transgenic mice with the gene for cmos knocked out, there is no Metaphase II arrest. Without that arrest, the oocytes pretend as if they were
fertilized. This phenomenon is called parthenogenesis.
In oocyte maturation in the frog, the pituitary gland senses some trigger to start the yolk production.
Gonadotropic hormone is then produced. It is sensed by the follicle cells in the ovary, and the follicle cells
produces progesterone. Progesterone is then sent to the oocyte. There are receptors on the surface of the
oocyte. The progesterone doesn’t need to enter the cell to exert its effect. Once the receptor is activated, the
c-mos protein is translated. In the frog egg, during oogenesis, c-mos RNA is stockpiled in the cytoplasm.
When progesterone comes along, the RNA is translated. This activates MPF. The MPF, phosphorylate
several targets.
- H1 kinase is one of the proteins phosphorylated. H stands for histone. Phosphorylation of this
enzyme causes it to phosphorylate H1. This can result in changing of compaction.
- Lamins are also phosphorylated by MPF. Lamins are part of the nuclear lamina, which is part of
the nuclear envelope. The major component of the cytoskeleton is called Lamins. This results in
the breakdown of the germinal vesicles.
- Myosin is also phosphorylate. This may be involved in maintaining metaphase arrest.
- RNA polymerase is also phosphorylated. RNA polymerase is the enzyme that makes the RNA
from transcribing DNA
- Poly(A) polymerase is also phosphorylated. Poly(A) polymerase is involved in the translation of
RNA.
These last two results in the inhibition of transcription and translation.
9
Xenopus Oocyte Maturation
The red pathway is the pathway that promotes maturation. The green pathway opposed the red
pathway and it is the pathway that is triggered by fertilization. It acts to stop maturation and allows the
oocyte to continue developing.
Red:
It all starts with progesterone released by the follicle cells. Progesterone promotes the translation of cmos RNA. It does that by adding a poly(A) tail of the RNA (polyadenylated). The RNA is then
translated into c-mos, which is a protein kinase. C-Mos then phosphorylates different proteins. It has 3
targets:
1. Inactive MPF. The c-mos attaches a phosphate to p34 of the inactive MPF, which result in the
MPF being active. When MPF is active it phosphorylates its targets and promotes maturation.
2. c-mos can also activate a MAP kinase pathway. C-mos phosphorylates MAP kinase kinase. This
kinase then converts inactive MAPKK to the active MAPPKK. It then activates MAPK. MAPK
can phorsphorylate MPF. So the phosphate on the active MPF can come directly from c-mos and
also indirectly through MAPKK. Also, with decreased [cAMP] (this is a result of decreased
adenylate cyclase activity), which happens as a result of progesterone can also result in the
activation of MAPKK.
As long as the red pathway is dominant, metaphase arrest is happening and maturation is promoted.
Green:
This is triggered by the sperm. Fertilization happens. During fertilization there is a massive release of
Ca2+ from the oocyte. The calcium acts to inactivates MPF. It seems to be the signal.
Calcium acts in 2 pathways:
1. It activates calpain II. When this is activated, it goes to work. It specifically attacks c-mos and then
degrades it. Without c-mos around, oocyte maturation is not going to work very well
*
3. C-mos also causes cyclin protease to be inactive. When c-mos is degraded, cyclin protease is now
active. Calpain II hurts MPF indirectly by hurting c-mos.
*
2. Calmodulin is also activated by calcium. The calmodulin calcium complex takes an inactive CAM
kinase II to and active CAM kinase II. Its target is PUC. PUC stands for proteosome and
Ubiquinated complex. Part of the PUC is a proteosome, which is a molecule that breaks up
protein. It looks for things that have been tagged for destruction. Ubiquitin is the marker that is
recognized by PUC. MPF is ubiquinated by PUC. So PUC tags MPF and breaks it down.
Summary:
The Biochemical Basis for Oocyte Maturation (see handout p.6)
For each pathway, red and green, there are different pathways. Nature has built in backup systems. The
figure is showing the period of oocyte maturation. It shows that cyclin B, MAP kinase and MPF levels
increase during maturation.
1-23-03 (late)
Ovulation and State of the Ovulated Oocyte
Mammals: Follicle rupture involves smooth muscle contraction (involving prostaglandins) and follicle wall
weakening due to the plasmin pathway.
The combination of muscle squeezing the follice and weakening the walls with collagenase is involved in
ovulation.
This happens in mammals.
Dressing the oocyte: The ovulated oocyte is not naked:
10
-
-
Zona pellucida in mammals. On top of that layer there is the corona radiata. This is the edge of cells
that surround the oocyte and may be involved in transporting the oocyte. It can also have a protection
type of role.
Jelly coat in frog
Shell in the chick.
FERTILIZATION
In echinoderm (sea urchins). These are marine animals. The gametes are shed into the marine environment
and have to find each other. These gametes have never met before. It’s kind of like a blind date. They have
to find each other. There has to be some built in mechanism to bring them together. The process of being
united does 2 things:
- Restoration of the diploid state. This is the normal state. The gametes are haploid.
- Activation of the developmental sequence. Cell division etc.
Overview: The 6 steps of fertilization:
1. Activation of sperm motility. In mammals, this is part of the capacitation process.
2. Sperm attraction to oocyte. The sperm senses and move towards the egg,.
3. Sperm penetration into oocyte.
4. Sperm-oocyte cellular fusion.
5. Fertilization responses.
6. Pronuclear fusion Beginning of the developmental sequence: the 1st cell division.
More detail:
- Step 1: Sperm motility activation. In the testes of the sea urchin, the sperm are inactive. They become
active as they are shed into water. There are different ionic events in fertilization. Ionic event #1
happens here. The sperm is in the sea water. The reason we believe the sperm is inactive to begin with
is because the pH in the tail of the sea urchin is too low (due to high CO2 concentration). The dynein
arms inside of the axoneme are inactive at a low pH. These pH sensitive dynein respond to the
increased pH in the seawater (more alkaline than testis). Another way to increase the pH of the sperm
is through encountering the oocyte. The oocyte is surrounded by a jelly coat. In the coat, there is a
molecule called speract, which is small diffusible peptide. It stands for sperm activation. On the sperms
tail, there is a receptor for speract. If speract is around in the environment, it will bind to the receptor,
causing an increase in cGMP in the tail of the sperm. This will lead to a loss of K+ ions from the tail of
the sperm. This is the 1st ionic event. This leads to a hyperpolarization of the tail, which triggers an
ionic exchange where proton leaves and Na+ ions enter. This will increase the pH in the tail, leading to
sperm motility. This is kind of a second messenger system. Now we have an active sperm.
- Step 2: Sperm attraction to oocyte (chemotaxis). Resact (respiration activation) is the chemical
involved in chemotaxis. Embedded within the jelly coat, there is also the resact peptide. Only sperm of
the same species will respond to the specific resact peptide. The sperm has a resact receptor. The
binding to the receptor ativates an increase in cGMP, which results in Ca2+ coming in, which is ionic
event #2. It doesn’t take very much resact to trigger this. So, the sperm is activated, it has found the
oocyte.
- Step 3: Sperm penetration. Penetration is getting through the jelly coat and getting access to the cell
membrane so that fusion can take place. There are 2 events that accomplish this:
- Acrosome reaction: This is an exocytosis reaction that had to be supressed during the maturation of the
sperm. This is the fusion of the membrane of the sperm with the cell membrane. This is triggered by
the jelly coat. The membrane fuses with the cell membrane. In the acrosome, there are lots of enzymes
that help break down the jelly coat. The jelly coat itself is what triggers it. This is a species-specific
triggering. There are sugars on the jelly coat that is recognize by the head of the sperm. If you take the
sugars from different species, only sugars from the same species of sperm will trigger the acrosome
reaction. So, the sperm swims to the oocyte. The extracellular matrix of the egg has the particular
sugars. There are receptors on the sperm for those sugars. The membrane of the sperm fuses with the
oocyte, the enzymes are released and start to break down the jelly coat allowing the sperm to move
through it. The exocytosis is triggered by ion flow. We believe that the entrance of Ca2+ in exchange
for K+ promotes membrane fusion and exocytosis.
11
-
-
Acrosome process formation: This is a structural change at the front end of the sperm. The acrosome
process is a result of the polymerization of actin. Its built from g-actin that is stored behind the
acrosome that can be polymerized at the right time. This polymerization is triggered by a pH change.
The increase in pH triggers this. So there are 2 ways in which pH change is involved in fertilization –
motility of the sperm and the polymerization of the actin to form the acrosome process. This is also
involved in the penetration.
Step 4: Once the sperm arrives at the cell membrane, there is going to be sperm oocyte fusion. The cell
membrane of the oocyte and the inner acrosomal membrane are the ones that are going to fuse. After
the acrosome reaction, bindin comes on the outside membrane of the sperm. Bindin is a protein that
recognizes a protein on the surface of the egg. The binding will bind to a species-specific receptor on
the surface of the egg. We believe that the egg actively sucks up the sperm. What is sucking up the
sperm is the fertilization cone. When the sperm comes in and you get fusion, the sperm is partly drawn
in. Then there will be actin polymerization inside of the egg. This actin starts to push out a bulging
mound called the fertilization cone. This cone will swallow the head of the sperm. The actin in the
oocyte is found localized right under the cell membrane. There is a localized enhancement of
polymerization at the point of sperm contact.
1-27-03 (lab)
There are a variety of responses in response to fertilization
- Step 5: Fertilization responses. They have 2 purposes:
1. Prevent polyspermy.
2. Activate 1st division.
Two Types of responses:
1. Early response: Occurs within the first minute
2. Late response: Takes at least a minute or two.
Both of these responses have similar goals – preventing polyspermy. It is kind of a double assurance.
The two methods are called the fast block (temporary) and the slow block (permanent).
The Fast Block.
This is the influx of calcium and sodium ions (Ionic event #5). Calcium comes in first producing a
smaller depolarization, followed by sodium coming in – sodium being the major player. This results in
an increase in membrane potential. Extra sperm cannot fuse with the egg when the membrane potential
is depolarized. After a few minutes the membrane potential goes back down to its resting membrane
potential.
The Slow Block:
This takes a minute or two to happen. It is based on the cortical reaction, which is exocytosis of
cortical granules. The barrier produced is called the fertilization membrane.
If you look at the cortical region of the sea urchin egg, you see the cortical granules. These granules
will fuse with the cell membrane in response to fertilization. The egg is surrounded by a vitelline layer.
The contents of the cortical granules transforms the vitelline layer into a hard envelope. This moves
out physically and is farther from the cell membrane in addition to being extra tough. Ovoperoxidase is
an enzyme in the cortical granules that uses hydrogen peroxide to crosslink amino acids within the
vitelline layer, reinforcing the fertilization envelope making it hard for any sperm to penetrate the egg.
When oxygen is taken up as part of respiration, one of the byproducts is hydrogen peroxide. When
eggs are fertilized, they experience a burst of respiration with hydrogen peroxide being a byproduct.
So, the hydrogen peroxide that is needed for the fertilization membrane crosslinking is a result of the
respiratory burst in response to sperm penetration.
We believe that there are second messengers released in response to the sperm. When the sperm binds
to the egg, it releases IP3 and cADPR. Their job is to mediate the cortical reaction. They bind to the
endoplasmic reticulum of the egg, resulting in the release of calcium (Ionic event #6), triggering the
cortical reaction. The fertilization membrane is then established. If you inject IP3 into an egg, it causes
a rise in internal calcium levels. If you fertilize eggs in the presence of an inhibitor of IP3, there is no
increase in calcium level. So it turns out that IP3 is necessary. The calcium that is released is released
as a calcium wave. What they did was take a sea urchin egg and loaded it with a die that will fluoresce
12
white where calcium is present. When a sperm comes along and initiation, you see a release of calcium
initiated at the site of sperm penetration and then you see an increase in calcium as a wave along the
egg. The lifting up of a fertilization membrane is also initiated at the point of sperm fusion.
1-28-03
-
Step 6: Pronuclear fusion. During spermiogenesis, the nuclear envelope loses its pores. The
nuclear envelope breaks down in this step. This may be tied into phosphorylation of lamins and
then a new envelope is built around it from material in the egg. This envelope has pores. The
chyromatin decondenses due to 3 factors:
1. Histone phosphorylation via phosphokinase in the cytoplasm of the egg. This reduces the
affinity of the histone for the chromatin of the sperm.
2. Action of nucleoplasmin. This protein has the ability to pull of protamines from chromatin
and add histones in their place.
3. Glutathione is a reducing agent that helps reduce disulfide linkages in protamines. This helps
loosen up the structure of the protanine so that the nucleoplasmin can do its work.
These all help decondense the chromatin of the sperm. There is another interesting thing that
happens during this process. The 2 nuclei have to find each other. The way they do this is through
the action of the sperm aster. This is a cytoskeletal structure, made up of microtubules, that
somehow through a change in pH brings the sperm and the egg pronuclei together. This change in
pH is dure to external Na+ influx and internal H+ efflux, which is ionic event #7. This is called
fertilization acid.
So there are 7 ionic events. The first 4 has to do with the sperm, the last 3 has to do with the egg.
The first one is the change in pH that triggers sperm motility. The last one is a change in pH that
brings the pronuclei together.
So, the sperm is responsible for building the sperm aster. In the tail there is the axoneme that was
built from the centriole. This same centriole builds the sperm aster during fertilization. It has been
shown that some roots of male infertility in humans can be traced to a problematic centriole.
Without the sperm aster the 2 nuclei never come together.
How the developmental sequence is activated (sea urchin).
The sperm comes along and fertilizes based on some receptor. It is probably not the bindin receptor. It’s not
clear what it is. There is then a signal that activates PLC. There is good evidence that this signal is nitric
oxide. PLC then hydrolyzes phosphatidyl inosital, which produces diacylglycerol and IP3. This IP3 is the
same IP3 involved in the slow block to polyspermy. This IP3 binds to the ER, resulting in the release of
calcium. This calcium feedbacks and promotes further activity of PLC. This is positive feedback. The
calcium also has the ability to activate various kinases. On target is PKC and NAD kinase. When NAD
kinase is activated, it activates NAD and you get NADP. This NADP acts as a cofactor or activator of and
oxidase. This oxidase contributes to increased respiration, which results in H2O2 production, which
promotes the crosslinking of the fertilization envelope.
The DAG also stimulates PKC. This is another way of accomplishing the same purpose. PKC also
phosphorylates ion exchangers in the cell membrane, which causes H+ to leave and NA+ to come in,
resulting in a rise in intracellular pH, resulting in pronuclear migration.
One of the goals of fertilization is to activate the first cell division. The way the sperm triggers that is
through 2 actions:
1. Increased levels of calcium
2. Increasing pH.
These activate enzymes that lead to the first cell division.
Comparison to humans
- Step 1: sperm motility. Mammalian sperm also need to be motile. It was found that there is an ion
channel in the cell membrane of the tail in mice (also in humans). The protein is called CatSper
(cation channel of sperm) that mediates motility of the sperm. It ties in with calcium levels in the
13
-
-
tail. In knockout mice for this protein, these sperm do not increase the levels of calcium. An
experiment was done to demonstrate that. They loaded the sperm with a dye that fluoresces when
calcium is present. Once you treat the sperm with cAMP, the sperm light up. This showed that
mice sperm can respond by increases calcium levels. When you knockout the CatSper gene, there
is little or no increase in Calcium levels. These sperm have been shown to not swim very well.
When you try to use these sperm in vitro fertilization you see that these sperm are infertile,
probably due to the inability to penetrate the zona pellucida. The speculation was that now that we
have a target on the sperm that is needed for fertilization, if we can design drugs to knockout the
action of catspur it can be a contraceptive for men. The increase in calcium parallels ionic event #2
in the sea urchin, which resulted also in sperm motility.
Step 3: Sperm penetration into oocyte. The zona pellucida must be penetrated. Surrounding that is
a layer of cells that came from the cumulus oophorus and is called the corona radiata. So the
sperm must get through this layer first. The sperm has some big challenges. There are 2 ways it
gets through this.
1. Acrosome reaction.
2. Some sperm can get through the corona radiata without undergoing the acrosome reaction.
This tool is believed to be a cell surface enzyme called PH-20. Its localization changes during
the fertilization process. It is an enzyme on the membrane of the sperm that has hyaluronidase
activity, meaning that it breaks down hyaluronic acid. This is a protein that’s present in the
ECM that helps hold the cells together. If you treat ovulated oocytes with PH-20, it breaks
down the corona radiata. This enzyme (PH-20) is believed to act before the acrosome
reaction.
The acrosome reaction is very important and is very similar to how it happens in the mouse. The
outer membrane of the acrosome fuses with the cell membrane, releasing its contents. At the end,
the inner acrosomal membrane is exposed. In the sea urchin, jelly coat sugars were the stimulus to
trigger the acrosome reaction. This is the same in mammals. Components of the zona pellucida,
specifically ZP3 triggers the acrosome reaction. The ZP3 is recognized by some protein on the
sperm cell membrane. There are 3 candidates:
1. p95 hexokinase
2. sp56
3. galactosyltranferase.
The binding of the protein results in the H+ efflux and Na+ influs, which increases the pH,
resulting in an increase of internal Ca2+ (due to calcium channels in the cell membrane opening).
This calcium results in PLC activation, which results in an increase in diacylglycerol, which
enhances membrane fusion.
In the sea urchin, the acrosome reaction had to do with calcium levels increases. The same thing
happens here in mammals. The calcium influx has to do with calcium channels in the cell
membrane. Also, the hyperpolarization that happens in capacitation aids in the opening of calcium
levels. In the seminal fluid of mammals, there is caltrin, which also aids in the increase in calcium
levels.
When men are being treated for high blood pressure, the drugs act by modifying calcium channels.
This helps lower blood pressure. However, these same drugs affect the calcium channels in sperm,
compromising the acrosome reaction, leading to infertility.
When hamster sperm is treated in a little dish and loaded with the fluorescent dye that responds to
calcium levels. Calcium levels are shown to rise within only .6 seconds of when ZP is applied to
the sperm.
The pathway was tested with knockout mice that are PLC-. They did not respond to zona
pellucida.
1-30-03
Step 3: The zona pellucida is an extracellular matrix secreted by the oocyte, and is made up of
proteins and carbohydrate residues. The proteins are specifically ZP1, ZP2 and ZP3. We think that
the ZP3 is the protein that triggers the acrosome reaction. It is believed that binding the
carbohydrate residues on ZP3 that triggers the acrosome reaction.
14
In transgenic mice experiments, there was an overproduction of the galactosyltransferase are
hypersensitive to ZP. The over react in response to the zona pellucida and undergo a premature
acrosome reaction by responding to smaller levels of zona pellucida.
In knockout mice, the gene for galactosyltransferase was knocked out. These sperm don’t bind to
ZP3 as well. The next question was whether or not those sperm could fertilize eggs and the answer
was yes. They fertilized eggs without the acrosome reaction. There is no explanation for this thus
far.
This evidence suggests that galactosyltransferase is important in binding to the zona pellucida and
triggering the acrosome reaction.
There is now a ZP vaccine with antibodies that bind to the zona pellucida, blocking sites for sperm
to bind and doesn’t seem to be species specific. This study has been done on elephants. They
injected the females with pig zona pellucida. The elephants then produced antibodies against zona
pellucida that binded to their zona pellucida and stop them from getting pregnant.
In some other experiments, the genes for zona pellucida proteins were knocked out. When they
knocked out the gene specifically the gene for ZP3, no zona pellucida formed and the oocytes
would not become fertilized.
In mammals, as the egg is fertilized and is ready to divide, the zona pellucida is still their until
around day 5 or 6, an enzyme breaks down the zona pellucida and causes the eggs to “hatch” out
of their case and get implanted into the walls of the ovary. In mutants with a messed up zona
pellucida, they hatched prematurely and implanted early.
-
Step 4: When a sperm comes to the egg, it doesn’t come in tip first like in the sea urchin. It comes
in at an angle and the back half of the head starts the fusion. Eventually, the whole head gets
involved. There are 2 parts to the acrosome: Principle and equatorial parts. Of these 2 parts the
principle part is shed in the acrosome reaction, not the equatorial part. The equatorial part is seem
to be involved in the initial fusion of the two membranes. What causes the membranes to fused.
We be leave that there are 3 mechanisms that are important:
1. The interaction between a membrane protein on the sperm called fertilin that binds to a
surface protein called integrin on the surface of the egg. Integrin is associated with another
protein called CD-9. This complex is believed to be involved in the fusion of the gametes.
When the CD9 gene on the oocyte or the fertilin gene on the sperm were knocked out, fusion
was compromised.
2. Involves the complement system. A complement protein (C3) seems to be involved in the
binding of the 2 oocytes.
3. ZP2 has been shown to bind with a protein released from the acrosome called acrosin. This
interaction may help hold sperm to ZP facilitating penetration and fusion.
In an experiment, tests were done to see where fertilin was found. They took sperm from different parts of
the epididymus of gunnea pig and showed that the distribution of fertilin was found in the back half of the
head of the sperm. Exactly where we would expect it to be since there is where fusion takes place first.
Copyright © 2003, Leslie Samuel Productions.
All Rights Reserved.