Transcript Gamete Handling and Fertilization

Lithium Chloride Interferes with Fertilization in Arabacia punculata
Elizabeth M. Ziegler
York College of Pennsylvania
(Cavanihac 2000)
(ThinkQuest 2001)
Lithium has been shown to disturb development in
sea urchin embryos, but its effects on fertilization
have remained unexplored. To address this, sea
urchin eggs (Arabacia punculata) were incubated
for 10 minutes in varying concentrations of Lithium
Chloride (LiCl); 0.01M, 0.1M, 0.3M, and 0.5M.
After the incubation period, fertilization was
attempted. Increasing concentrations of the LiCl
showed a decrease in fertilization rates. Control,
untreated eggs showed a 100% fertilization,
whereas fertilization among LiCl treated eggs
ranged from 50% (0.01M LiCl) to 7.41% (0.50M
LiCl). LiCl treated eggs also showed a decrease in
the number of 4-celled embryos, with an increased
incidence of amorphous cells (0% amorphous in
Control embryos versus 55.0% in 0.10M LiCl).
Trypan Blue Staining showed that LiCl had no
effect on the viability of the eggs, since all samples
showed 100% stain exclusion. These data
suggest that LiCl disturbs the fertilization process
in sea urchin gametes, however, the continued
viability of the cells may indicate that these effects
are localized to the jelly coat and/or the osmolarity
by affecting the sodium gradient within the cells
such that the plasma membrane remains intact.
Lithium is one of the most commonly prescribed
drugs for the treatment of bipolar disorder, is a
leading anode material for batteries, and a coolant
in nuclear reactors. It is also used on spacecraft to
remove carbon dioxide from the air (Encyclopedia
Britannica). Lithium has been shown to have a
major effect on the development of many different
organisms, including sea urchins, Xenopus, and
zebrafish, and even fruits such as tomatoes, by
acting on Lithium sensitive enzymes such as GSK3β (Klein and Melton, 1996). Lithium Chloride
(LiCl) has been shown to progressively block the
cell cycle of sea urchin embryos by inhibiting the
phosphoinositide signal pathway, an important
regulator of embryonic cell division (Becchetti and
Whitaker, 1997). LiCl has also been shown to alter
nuclear β-catenin, an event which coincides with
an increase in mesoderm and endoderm (Logan,
et al., 1999). LiCl has also been suggested to act
through the inhibition of glycogen synthase kinase3β (GSK-3β), a regulator in cell fate determination
in many organisms (Klein and Melton, 1996).
Although the effects of LiCl on embryonic
development seem extensive, its effects on
fertilization have remained virtually unexplored.
Results
1.) To examine the effects of LiCl on fertilization of sea
urchin (Arabacia punculata) oocytes.
Increasing concentrations of the LiCl solution resulted
in decreased fertilization rates (100% for the control
group, 50% in 0.01M, 10% in 0.1M, 24.14% in 0.3M,
and 7.41% in 0.5M).
2.) To monitor the effects of LiCl on subsequent embryo
development and compare them to previous studies.
Materials and Methods
LiCl also showed a decrease in the number of 4-celled
embryos (readings were taken on all embryos present:
90.91% in the control group, 66.67% in 0.01M,
15.38% in 0.1M, and 0.0% in 0.3M and 0.5M) and an
increased number of amorphous cells (0% in the
control group, 9.38% in 0.01M, 55.0% in 0.1M,
24.14% in 0.3M, and 37.04% in 0.5M).
Artificial seawater was used during the collection,
incubation, and fertilization of gametes. All experiments
were performed at 24˚C.
Trypan Blue staining showed that the LiCl had no
effect on the viability of the oocytes (all samples
showed 100% cell viability before fertilization).
3.) To examine the effects of LiCl on cell viability of sea
urchin oocytes.
Lithium Chloride Solution
21.20g LiCl was dissolved in 100mL of ASW to obtain a
5.0M stock solution. This was then diluted down to achieve
molarities of 0.01, 0.10, 0.30, and 0.50.
Figure 2.
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Gamete Handling and Fertilization
Gamete shed was induced by injecting KCl into the soft
tissue around the mouth. Eggs were collected by placing
the female aboral side down on top of a 250mL beaker filled
with ASW, while sperm was pipetted off the male sea urchin
and placed in a dry Petri dish. Equal portions of eggs were
placed in beakers containing either ASW or LiCl solutions.
Fertilization was achieved by diluting 3 drops of sperm in
ASW and then by placing 3 drops of that solution in the egg
containing beakers, after the eggs had been incubated for
10 min.
Lithium Chloride caused a decrease in fertilization rates as
concentration increased. This may be due to interference with
the jelly coat of the egg, which attracts the sperm to the egg for
fertilization. LiCl showed a dose-dependent effect on the
development embryos, as well as a teratogenic effect
(Becchetti and Whitaker, 1997), an event supported by these
data. The LiCl could also have affected the membrane
potential of the oocytes leading to a change in the osmosis,
making the oocytes hypotonic, and the integrity of the
membrane, making it more permeable to ASW attributing to a
change in osmosis or the pressure within the oocytes.
As in previous studies LiCl incubation interfered with cell
division in fertilized embryo, preventing embryos from cleaving
to the 4-cell state. This has been attributed to interference with
the phosphoinositide pathway, a block that occurs during
metaphase and cytokinesis (Becchetti and Whitaker, 1997), and
with the inhibition of GSK-3 (Logan, et al., 1999).
Observations made in previous studies are not comparable
because many were specifically looking at the activity of an
enzyme or pathway of interest and did not report the effects on
developmental rates.
The continued viability of the oocytes, in concert with the
decrease in fertilization may be due to effects on the jelly coat
or on the permeability of the cell which could have lead to
changes in the osmotic pressure due to changes in the
concentration of ASW or perhaps sodium within the cell while
leaving the cell membrane intact.
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Future Research
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Percent 4-cell Embryos
Introduction
Objectives
Percent Fertilized
Abstract
Discussion
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Control
0.01
0.10
0.30
0.50
Control
0.01
0.10
0.30
0.50
LiCl Concentration (moles/liter)
Figure 2. Mean percentage of fertilized and
4-cell embryos. Error bars indicate the
standard error of the mean (SEM). A one-way
ANOVA Test determined that the differences in
SEM were significant.
Control
How is cell viability maintained, while fertilization is inhibited
and cells become amorphous?
Literature Cited
Becchetti, A., and Whitaker, M. 1997. Lithium blocks cell cycle
transitions in the first cell cycles of sea urchin embryos,
an effect rescued by myo -inositol. Development
124:1099-1107.
Logan, C., Miller, J., Ferkowicz, M., and McClay, D. 1999
Nuclear β-catenin is required to specify vegetal cell fates
in the sea urchin embryo. Development 126: 345-357.
Klein, P. and Melton, D. 1996. A molecular mechanism for the
effect of lithium on development. Developmental Biology
93: 8455- 8459.
Encyclopedia Britannica. Lithium. Available from:
http://www.britannica.com/eb/article?eu=49668
Accessed 2002, August 30.
0.01M
Picture Cited
Inject Urchins with 4cc
0.5M KCl
Collect gametes
Incubate oocytes in LiCl
0.1M
0.3M
Figure 1. Flow chart of key steps in methods.
0.5M
Fertilize the gametes
Jean-Marie Cavanihac. 2000. http://www.microscopyuk.org.uk/mag/artjul00/urchin1.html
ThinkQuest team J0111704 . 2001March 14. Available from:
http://library.thinkquest.org/J0111704/picturegallery/pic14/pic14.
html
Acknowledgments
Dr. Ricker, PhD Department Chair