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Aaron Ye
March 2, 2016
C01
TA: Christiana
Yellow Group 3
Lab Partners: Michael, Robert

The Role of Calcium and MAPK in terms of
Sea Urchin Egg Fertilization Processes

Abstract
Sea urchins, like many animals, have sperm egg fertilization processes
largely dependent upon downstream protein mechanisms and pathways
...
Calcium is one of the
significant signaling molecules involved
...
However, exact mechanisms or effects are not entirely
known
...
By understanding these effects, we
can more confidently deduce calcium’s specific role within sea urchin egg
fertilization
...
In addition, the influx is only partly sufficient to cause
MAP kinase inactivation, and fully sufficient to induce PLC activation
...

Introduction
Downstream pathway of proteins is no
surprise in many different life forms that exist
...
Since
this type of pathway is so common, it is
expected that this mechanism plays a role in
terms of sexual fertilization and reproduction
...
The
process has been mapped out in some detail
for sea urchins
...
The sperm’s plasma
membrane
fuses
with
the
acrosomal
membrane in order to release enzymes to
digest the jelly coat
...
Next, the
cytoplasm of the sperm fuses with that of the
egg, releasing its contents
...
The
increase in calcium levels cause granules to
migrate to the membrane and break the
connection between the vitelline membrane
and the plasma membrane
...
We
know that calcium is a very important
intermediate mechanism for almost all species’
fertilization
process
(Tokmakov,
2014),
including the sea urchin
...
We attempt to answer five major
questions:
1) Is Ca2+ influx sufficient to induce
activation in an unfertilized egg?
Activation occurs when the vitelline
membrane and the plasma membrane
separate, allow water to flow in via osmosis
...
Although
activation normally precedes cell division, it
may not cause it
...


2+

2) Is Ca
influx sufficient to induce
activation in an unfertilized egg?
Sperm induces a downstream event
responsible for the initial steps of cell division
...
In normal
somatic cells, MAP kinase activation leads to
cell division and entering into the S, G2, and
Mitosis phases
...
This is the case
even though meiotic cells enter two successive
M phases after the synthesis of DNA
(Tachibana et al, 1997)
...

Although MAP kinase inactivation is a crucial
step that leads to cell division, calcium may
have the ability activate one and not the other
...

What exactly is the mechanism to MAP
kinase inactivation? It is not very clear on
exactly what the fate of MAP kinase is when it
is disabled
...

4) Is
MAP
kinase
inactivated
degradation or dephosphorylation?

by

We believe that it is either one or the
other processes mentioned in question 4
...

Therefore, it is logical to assume that it is
inactivated by dephosphorylation
...
By this mechanism,
an enzyme would assist by digesting or
breaking down of MAP kinase
...

Of course, MAP kinase activity alteration
is only one step in the grand scheme of

fertilization
...
Upon
fertilization, PLC acts on PIP2 by cutting it into
IP3 and DAG
...
The other half, DAG, causes calcium
influx from internal storages
...
However, in a
real organism, a blend of the process may
occur, in which positive or negative feedback
loop could affect any of the processes along
the way
...

Sea urchin eggs were split to four
groups: seawater, fertilized, A23187, and
DMSO
...

A23187 is a calcium ionophore that can induce
an inward flow of calcium from the external
seawater environment
...
However, because
DMSO could possibly affect the cell on its own,
the last group was required and had DMSO by
itself as a solvent control
...
By introducing each egg with the
desired conditioned, we can observe the
potential percent activation and division for
each group under a light microscope
...
Primary antibodies
are obtained from rabbits after an immune
response was induced
...

Finally, the last experiment measures the
activation of PLC via ELISA
...
This procedure serves to answer question
5
...
A stop solution of LiCl was stopped
IP1 from reacting to inositol
...
Since many biochemical mechanisms
are relatively similar between organisms,
studying sea urchin gametes can ultimately
lead to advancements of medicine for humans
in the future
...

2ml of egg each for the western blot)
...
The
A23187 group was created by addition of 5µl of
1mM stock of A23187 dissolved in DMSO
...
All of these reagents are per
ml of egg, so the western blot would require
double the reagents
...
After a brief observation, sperm
was introduced into the drop by dipping a
pipette tip, and percent egg activation was
noted by observing the fraction of cells that had
a separation of the vitelline and plasma
membranes
...
After, all
samples were incubated again for 2½ hours,
then a drop of each was placed under a light
microscope and observed under a 10x
magnification
...

Western Blot
Each sample was split in half to be
performed twice (32 in total)
...
Initial
steps involved 10-minute incubations in 16°C
water before introducing the various conditions,
followed by a 35 minute incubation
...
Centrifugation for 30 seconds
then served to pellet the eggs, which were
resuspended in 28µl of MAPK lysis buffer with
inhibitors
...
Finally,
a 12µl of SDS-PAGE sample buffer were
added to each sample, and after a brief heat
incubation at 95°C, the solutions were frozen
down
...

The volume equal to 5µg of protein was added
along with 4ul of 5X SDS-PAG sample buffer
and water to a total volume of 20µl
...

When the gels have finished, the
samples were analyzed using western blot and
immunodetection for the presence of MAP
kinase
...
1% Tween-20) with 5% bovine
serum albumin was added as a blocking agent
in order to cover surfaces of the nitrocellulose
not bound by protein so that antibodies will not
stick to irrelevant areas
...

ELISA
100µl of 0
...
After a 10 minute
incubation at 16°C, egg treatments were
introduced, then another 35 incubation
occurred
...
Another 50µl
was
added
before
the
eggs
were
homogenized
...

To begin the assay, we added 60µl of
dilution buffer to each egg solution after the
samples were no longer frozen
...

Wells were aspirated with a vacuum
suction and washed 5 times with wash buffer
...
It is
important to prevent light from irritating the
solution during this time
...
175µl of this solution was
transferred to a 96-well plate for absorbance
measuring at 450nm
...
Initial inspection yielded 95
...

After introducing the various conditions
for the different groups, the percent activation
and cleavage was noted
...
As can be seen, it
appears that the A23187 condition was met
with activation of the egg when introduced
...


Sample  #  
1  
2  
3  
4  
5  
6  
7  
8  
9  
10  
11  
12  
13  
14  
15  
16  

Condition  
Seawater  
Fertilized  
A23187  
DMSO  
Seawater  
Fertilized  
A23187  
DMSO  
Seawater  
Fertilized  
A23187  
DMSO  
Seawater  
Fertilized  
A23187  
DMSO  

%  
Activation  
0%  
100%  
98%  
0%  
0%  
100%  
87%  
0%  
0%  
93%  
100%  
0%  
0%  
100%  
92%  
0%  

%  
Cleavage  
0%  
100%  
0%  
0%  
0%  
79%  
0%  
0%  
0%  
95%  
0%  
0%  
0%  
95%  
0%  
0%  

Fig
...
Percent activation and cleavage of
sea urchin eggs under the various testing
conditions
...
Presence of the secondary antibody
initiated a reaction catalyzed by a peroxidase,
which gave us a band that indicated the
presence of our target protein
...


Blot C shows the amount of
phosphorylated MAP kinase present within the
samples
...
Fertilized
eggs have much less MAP kinase due to the
lighter band, suggesting that MAP kinase has
been inactivated
...
Perhaps some MAP kinase has
been dephosphorylated, but most remain
active
...
Since the fertilized condition has a
band that is just as dark as seawaters, we can
conclude that MAP kinase is not degraded
...


Fig
...
Western blot immunodetection for
phospho-MAPK and total-MAPK of each
condition
...


ELISA
The ELISA competitive assay was
performed in order to detect the presence of
IP3, and ultimately, the activity of PLC
...
This oxidized form, along with sulfuric
acid stopping the reaction at a certain point,
produces an absorption peak at 450nm
...

The table on the right is our data
collected for the concentration of IP1
...

The standard deviation of the fertilized
group seems to be very large, with one sample
over 2,000 nM of IP1 (this one was excluded
and marked as an outlier)
...
Means
for each group were collected
...
Seawater and DMSO had
relatively the same amount, with A23187 sitting
at a value in between seawater and fertilized,
but much closer to fertilized conditions
...
59  
168
...
69  
303
...
95  
53
...
00  
   
Average  (Sewater)  
277
...
77  
1294
...
44  
103
...
83  
118
...
55  
723
...
59  
 (n  =  8)  
A23187  
3  
194
...
00  
A23187  
7  
384
...
67  
A23187  
11  
293
...
11  
A23187  
15  
933
...
15  
Average  (A23187)  
466
...
80  
106
...
64  
506
...
97  
   
DMSO  
16  
489
...
48  
 (n  =  6)  
Fig
...
Calculated IP1 concentrations for
each of the four conditions via ELISA
competition assay
...
4
...
Standard deviations are portrayed by error bars
...


Fertilization
has
a
concentration
average 2 fold greater than that of seawater
...
One explanation for this is that
calcium influx has a positive feedback for PLC
activity
...
As a
result, there are two sources of stimulation
...

Discussion
Introduction of calcium via artificial influx
had conclusive effects to the sea urchin eggs
in terms of activation and cleavage
...
1, A23187 was sufficient to produce
separation of the vitelline membrane and the
plasma membrane, with percent activation as
high as 98% to 100%
...
This quantitative observation allowed
us to conclude questions 1 and 2
...
However, the same process is not
sufficient in producing cleavage
...
As can be seen in blot
C, the fertilized lane still contained a dark band
indicating the presence of total MAP kinase,
similar the seawater band
...

Meanwhile, Blot D allows us to spectate
the inactivation of MAP kinase in the various
conditions
...

This is expected, since phosphorylated MAP
kinase should remain active
...
Finally, we compared the
A23187 sample to the two control groups
...

However, the thickness of the band is much
less extreme, and does not seem to overflow

the lane like the center two seawater and
DMSO lanes
...
There is
not any sufficient evidence to help decide if the
A23187 lane is more similar to the positive or
negative control
...
Perhaps
there were some mistakes regarding the
amount of A23187 added
...
Perhaps this would
be sufficiency and match up to the regular
calcium influx amount of the fertilized condition
...
A second experiment that would
give us more information would be to test the
necessity of calcium in terms of MAP kinase
inactivation
...
Inhibitors BAPTA with
CaCl2, or ETGA with pipes would be utilized to
prevent the rise in intracellular calcium
concentrations
...
Results of this
experiment suggests that influx of calcium is
very necessary for inactivation of MAP kinase,
since inhibiting the influx resulted in MAP
kinase staying in the active state, and the cell
remaining under arrest in the G1 phase (Carroll
et al, 2000)
...
Once a cell enters the S phase, its
fate is already determined to enter the second
growth phase and then, ultimately, cell division
...
Inactivation can
occur in one of two ways
...
Since the latter

is equally as effective as the former, we can
assume that MAP kinase has an equilibrium
point in which the rate of phosphorylation is
equal to that of dephosphorylation, and that it
is the imbalance or ratio between the two that
either arrests the cell in G1 or pushes it to the
S phase
...
Research has also
shown that inactivation of MAP kinase is both
sufficient and necessary to promote the
entering into the S phase and the inception of
cleavage
...
Inhibition of calcium influx proved
fatal to MAP kinase’s dephosphorylation
processes and cell division, while an
unfertilized egg still had the power to inactivate
MAP kinase
...
Like
sea urchins, humans have properties that also
hold egg cells in mitotic arrest until fertilization
occurs
...
Studying
sea urchin eggs may be one of our best
opportunities to understanding fertilization of
humans
...
Sea urchins offer a
great alternative to this limitation
...
This study can
even clear up some procedures that could
improve the lives of many, such as in vitro
fertilization and culturing of zygotes
...
, Diana T
...

Hoang, Forest J
...
Foltz
...
"
Developmental Biology 217
...

Kumano, Maya, David J
...

Denu, and Kathy R
...
"Calcium-Mediated
Inactivation of the MAP Kinase Pathway in
Sea
Urchin
Eggs
at
Fertilization
...
1 (2001): 244-57
...
Foltz
...
" Development,
Growth and Differentiation Dev Growth Differ
45
...

Rottinger, E
...
" Development 131
...

Tachibana, K
...
"
The EMBO Journal 16
...

Tokmakov, Alexander, Vasily Stefanov,
Tetsushi Iwasaki, Ken-Ichi Sato, and Yasuo
Fukami
...
" IJMS
International Journal of Molecular Sciences
15
...




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