BIBC

103
B06 Lab Report Huayi Zhao

Relationship between Ca2+ influx,

MAPK inhibition and PLC activation of
Sea Urchin Eggs at Fertilization
Huayi Zhao, Yuhang Zhou, Jiajun Zhou















Cellular division and mitosis are the basis of life. It is known that the upon
fertilization, MAPK are inactivated in order for mitosis to happen. In this
experiment, we are trying to determine the factors that can activate sea urchin egg
division and is that only caused by a calcium influx into the cell or does it involve
other factors that contribute to the fertilization of the egg cell to achieve its post-
cleavage state. At the end, we found that despite calcium influx do cause an
activation of the sea urchin egg but a calcium influx alone do not cause the
dephosphyrlation of the MAPK to begin egg mitosis and it is undetermined whether
the MAPK are inactivated by degradation or dephosphyrlation.
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B06 Lab Report Huayi Zhao

Introduction:
It is known that cellular mitosis is induced through the fertilization of an egg.
Once the sperm hits an egg, it leads to a signal transduction pathway that starts with
Phospholipase C and causes an influx of Ca2+ from the endoplasmic reticulum through a
secondary messenger called IP3. This then leads to a fusion of plasma membrane with the
cortical granule to form a fertilization envelope, which can be seen as a sign of activation.
During the egg’s arrested state, mitogen-activated protein kinase (MAPK) inhibits the
cell from dividing through activation by MEK and when MAPK becomes inactive,
cellular mitosis begin and cleavage appears, this is when the cell is fertilized. In this
experiment we are trying to determine three questions: First is can induced calcium ion
influx induce activation in an unfertilized cell? To achieve this, we induced calcium
influx to an unfertilized sea urchin egg to see its effect compared to a fertilized egg and
found out that calcium influx can induce activation. Second and third are is influx of
calcium ion enough to cause MAPK inactivation and is MAPK inactivated by
dephosphorylation or be degradation? These are determined through the means of a
Western Blot to compare the Total MAPK and phosphorylated MAPK upon each
treatment and an ELISA analysis to determine the activation of other divergent pathways
upon fertilization. And we found out that calcium influx is not sufficient to inactivate
MAPK but we cannot conclude whether MAPK is degraded or dephosphorylated due to
experiment error.
Materials and Methods:
Observation of Sea Urchin eggs and Egg activation:
Adult Sea Urchin eggs are provided in seawater, 1ml of eggs will be
obtained for each 2ml microfuge tube using micropipette. Incubate for 10 min and then
treat each 1ml egg with 4 treatments (1. None 2. Sperm 3. 5mM A23187 dissolved in
DMSO 4. DMSO). Incubated for 2.5 hours and Placed 1 drop of each sample to view
under microscope to view its percent activation and percent post-cleavage.
SDS-PAGE gel Electrophoresis:
Obtained egg for each sample in separate microfuge tubes, incubated 10
min and added twice the volumes of treatment according to table. Prepared a buffer with
90 mul MAPK lysis buffer with inhibitor cocktail. Centrifuged sample and removed
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B06 Lab Report Huayi Zhao

supernatant, added l MAPK lysis buffer with inhibitor and centrifuged in cold-room
microfuge for 10 min. MAPK was present in the supernatant, Store and label sample in
freezer. Then set up gel in electrophoresis chamber and add buffer. Determined the
protein concentration of the lysate with a Bradford assay and added total protein worth of
sample volume. Then added water to sample to obtain a volume 16 mul and then add 5X
SDS-PAGE sample buffer. Heat sample for 3 min at 95 degree Celsius. Loaded each
sample onto gel with a Molecular standard. Ran the gel at 120V until the tracking dye is 1
cm from the bottom.
Western Blotting:
Obtained a sheet of nitrocellulose and two transfer stacks. Removed gel
from plate, placed one transfer stack on the electro blotting unit and then added on the
nitrocellulose paper, then the gel and then the last transfer stack on top. Ran blotting and
then place nitrocellulose into pre-labeled container and added TBST with 5% bovine
serum albumin. Incubated overnight at 4 degree Celsius and then added anti-MAPK (total
or phosphorylated). Incubate, then washed the sheet with PBS for 4 times and added goat
anti-rabbit IgG fluorescent-tagged antibody. Was 4 times with PBS and then capture
image through UV trans illuminator.
ELISA Determination of IP1 Levels:
Took two-egg sample, added 0.5M LiCl and incubated for 10 min and
gave them experimental treatments according to the table. Incubated for 35 min at 16
degree Celsius. Centrifuged for 30s post incubation, removed supernatant and added
ELISA lysis buffer to the pellet. Then placed them on ice and freeze. When conducting
ELISA, thaw sample and added IP1-peroxide conjugate to ELISA well and anti-IP1
antibody to each well, Incubated wells on orbital shaker for 2 hours. Vacuumed wells
with vacuum aspirator consisting of a glass Pasteur pipette. Added lysate and then wash
buffers, repeat for 5 times. After the last wash, added TMB to each well and incubate for
20min. Then added stop solution to each well and transfer from each well onto a 96-well
plate fro absorbance reading at 450 nm. The absorbance data were then analyzed based
on an IP1 standard absorbance vs concentration curve.

Recipes
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B06 Lab Report Huayi Zhao

MAPK Lysis Buffer
• 1% NP-40
• 20mM HEPES, pH 7
• 15mM EGTA
• 150mM NaCl
Inhibitor Cocktail (10x)
• 2mM Pefabloc
• 10mug/ml pepstatin
• 100mM beta-glycerophosphate
• 4mM NaF
• 2mM Na3VO4
Result:
1) Treatment of Sea urchin Table
Group Sample Experimental Treatment Percent Percent
Condition (Per ml of Activation post-
eggs) (%) cleavage
(%)
1 1 Seawater None 0 0
2 Fertilized Add 1mul 88 71
sperm
2 3 5mM A23187 Add 5mul 94 0
of 1mM
stock in
DMSO
4 DMSO Add 5mul 0 0
DMSO
3 5 Seawater None 0 0
6 Fertilized Add 1mul 95.4 92.5
sperm
4 7 5mM A23187 Add 5mul 90.3 0
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B06 Lab Report Huayi Zhao

of 1mM
stock in
DMSO
8 DMSO Add 5mul 0 0
DMSO
5 9 Seawater None 0 0
10 Fertilized Add 1mul 92 72.5
sperm
6 11 5mM A23187 Add 5mul 60 0
of 1mM
stock in
DMSO
12 DMSO Add 5mul 0 0
DMSO
7 13 Seawater None 0 0
14 Fertilized Add 1mul 100 95
sperm
8 15 5mM A23187 Add 5mul 86 0
of 1mM
stock in
DMSO
16 DMSO Add 5mul 0 0
DMSO

A21387 (ionophore)
• A compound that facilitates diffusion of an ion across cell membranes
• Lipid soluble molecules that enter the membrane and allow calcium to
move across the membrane down its concentration gradient.
• Dissolved in DMSO as a 1mM stock
BIBC 103
B06 Lab Report Huayi Zhao

To answer question 1, we can see from the percent activation and percent post-cleavage
where A23187 treatment and sperm treatment of the sea urchin egg can lead to a very
high percentage of activation but only sperm can make the eggs achieve post-cleavage
while all other treatment cannot. Therefore, we can conclude that cytoplasmic calcium
influx is not sufficient to produce cell division in an unfertilized egg.

2) Western Blot:
To determine the band intensity of activated MAPK and total MAPK present
in the sample to determine whether inhibition of MAPK is done through
dephosphyrlation or degradation.

SDS-PAGE Western Blot of MAPK (phosphorylated vs. total)

BIBC 103
B06 Lab Report Huayi Zhao

The Western blow is done to determine the presence of total MAPK vs. P-MAPK after
each treatment. A stronger band indicates a much bigger concentration of MAPK.
Difference in band can tell us the effect of calcium influx on MAPK inactivation
compared to control. We will answer the third question first from the Western Blot. As
we can see from Gel A, which is our only good, visible lanes gel. A visible lane can be
observed at the A23187 treatment and DMSO but not a visible lane for fertilized egg.
This can indicate that at fertilization, P-MAP kinase is no present while with only an
influx of calcium (treatment with A23187), it is clear that P-MAPK is still present in the
zygote, which might explain the 0% cleavage percent in all A23187 treatment. Therefore
we can conclude that a calcium influx is not sufficient to inactivate MAPK in sea urchin
eggs as we can see from the anti-P-MAPK gel. However, we cannot conclude that MAPK
is inactivated by degradation as the gels for total-MAPK is not clearly visible.

3) ELISA:
ELISA Standard and Sample Table:
(See Next Page)
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B06 Lab Report Huayi Zhao

Standard IP1 Abasorbance curve @

450nm vs Log10 of Protein
Concentration
1.8
1.6
1.4
Absorbance @ 450nm

1.2
1
0.8 Standard IP1
0.6 Poly. (Standard IP1)
0.4
0.2
y = 0.1285x3 - 0.6919x2 + 0.2167x + 2.1792
0 R² = 0.99576
-0.2 0 1 2 3 4
Log10 of Protein Concentration
BIBC 103
B06 Lab Report Huayi Zhao

Log10 of Protein vs Standard IP1

Abasorbance curve @ 450nm Log10 of
Protein Concentration (reversed for
calculation)
4
Log10 of Protein Concentration

3.5
3
2.5
2 Standard reversed
1.5
y = -1.4345x3 + 4.2521x2 - 4.6339x + 3.671 Poly. (Standard reversed)
1 R² = 0.99007 Poly. (Standard reversed)
0.5
0
0 0.5 1 1.5 2
Absorbance @ 450nm
BIBC 103
B06 Lab Report Huayi Zhao

Average IP1 Concentration according to

abs @450nm for Seawater, fertilized,
A23187 and DMSO with error bar (From
google doc absorbance)
3000
2500
IP1 Concentration (nM)

2000
1500
DMSO
1000
500 Seawater Seawater
Fertilized A23187
0 Fertilized
-500
A23187
-1000
-1500 DMSO
Sample
Seawater 227.7305114
Fertilized 39.46179628
A23187 54.49739393
DMSO 825.6091533

Average IP1 Concentration according to

abs @450nm for Seawater, fertilized,
A23187 and DMSO with error bar (From
Google doc calculated Concentration
Table)
600
Average IP1 Concentraiton (nM)

500
400 DMSO
300 Fertilized A23187
200 Seawater Seawater
100
0 Fertilized
-100
sample
A23187
Seawater 99.531202
DMSO
Fertilized 138.8111372
A23187 185.746102
DMSO 243.6874851
BIBC 103
B06 Lab Report Huayi Zhao

From the ELISA data, we can see that at fertilization, the PLC pathway is activated due
to ligand receptors on the cell membrane and a treatment of A23187, which causes a
cellular Ca2+ influx also activates PLC pathway. However, there is an inconsistency with
the DMSO treatment, causing the data to be relatively much higher than that of other
treatment. This might happen because of the misreading or possible contamination during
the loading process onto the 96-well plate.

Discussion:
The result we obtained from experimental data is that calcium influx is sufficient
to induce sea urchin egg activation but not fertilization. It is also clear from the Western
Blot that Ca2+ influx is also not sufficient enough to induce MAPK inactivation. We
were unable to determine whether MAPK inactivation is caused by dephosphyrlation or
degradation. These results do comply with our expectation in that we understand
activation of eggs can be obtained through multiple pathways such as PLC and other
while fertilization only happens due to sperm releasing specific nucleus into eggs to
induce cellular mitosis.
My conclusion that calcium influx is not sufficient is not consistent with the result
of the experiment conducted by David J.Caroll’s experiment in which he states that
calcium influx insufficient to inactivate MAPK (The Relationship between Calcium,
MAP Kinase, and DNA Synthesis in the Sea Urchin Egg at Fertilization). Another
experiment that can be designed to determine the necessity of the Calcium influx would
be to treat 2-egg lysate sample with 10muM U0126 (a MEK inhibitor) (1,4-diamino-2, 3-
dicyano-1, 4bis[2-aminophenylthio] butadiene; Promega) to stop the phosphorylation of
the MAPK. Then one sample is introduced to 1.7 muM free Ca2+; we then observe the
resulting P-MAPK concentration comparing to the sample with only MEK inhibitor
treatment. Then we can determine the necessity of the Ca2+ influx.
One possible flaw in our experiment is that during the running of Western Blot,
there is no method for us to ensure the integrity of our MAPK during lysis. It is possible
that we can denature the protein into single units, causing the gel to have multiple bands
within one lane, decreasing band intensity, creating an under-estimation of MAPK
BIBC 103
B06 Lab Report Huayi Zhao

concentration. A way to solve this is to add reagent that is able to protect the MAPK from
the lysis buffer. Another factor that is not controlled is the binding specificity of the
secondary antibody directly to IP1; this can cause a difference in absorbance, leading to
false data. A way to solve it is to have a control with only IP1 conjugate and IP1-
peroxidase and the secondary antibody as a control.

Reference
Maya Kumano,*, † David J. Carroll,‡ John M. Denu,§ and Kathy R. Foltz*,1 (2006)
Calcium-Mediated Inactivation of the MAP Kinase Pathway in Sea Urchin Eggs at
Fertilization 236, 244–257

David J. Carroll,1,2 Diana T. Albay,1 Kenneth M. Hoang, Forest J. O’Neill, Maya

Kumano, and Kathy R. Foltz3 (2000) The Relationship between Calcium, MAP Kinase,
and DNA Synthesis in the Sea Urchin Egg at Fertilization 217, 179–191