Inducing Artificial Parthenogenesis & Developmental Staging In
the Sea Urchin.
Abdulwahab Ahmed, Itamar Carter, Vikas Bakshi, Asaud Afzaal and Dr. Arne K. Christensen
BIO452 Developmental Biology, Department of Biology, York College, City University of New York, Queens, NY 11451, USA
Figure 2: Sea urchin embryo at 24 hours of developmental
Abstract:
The purpose of this experiment, performed in Developmental
Biology (Bio 452) Lab, was to induce parthenogenesis in sea
urchin eggs by agitation and observe normal fertilization stages.
We attempted to induce parthenogenesis, the development of an
egg without fertilization, with vigorous shaking. To study the
developmental sequence of the sea urchin embryo at different
intervals of time under fluorescent microscope, we stained sea
urchin embryos with propidium iodide and DiOC6(3), which are a
nucleic acid and membrane stain, respectively. We found that
parthenogenesis was not induced by shaking, but the fluorescent
probes we used to stain previously prepared embryos were useful
for resolving morphological details.
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Introduction:
The study of sea urchin development provides valuable
information on features of fertilization and development that apply
to many organisms, from jellies to humans (Gilbert, 2010). For
example, as in vertebrates, the production of gametes (i.e. eggs and
sperm) and subsequent fertilization in the sea urchin is a means to
sexual reproduction. Sea urchin eggs and sperm are similar to
human gametes in regards to shape and size. Sea urchins and
humans are also both deuterostomes, meaning that the mouth
arises at a site distant from the site of gastrulation (Gilbert,
2010). Because of these similarities, embryonic development in
the sea urchin provides a model for understanding early
development in many species. It is important to observe early
embryonic development because it is during this time that
patterning of the organism originates.
Spawning is a term used to describe the release of eggs and
sperm into the water column. Under a very special set of
circumstances, spawning is followed by the uniting of a male and
female gamete in a process called fertilization. A successfully
fertilized egg is called a zygote, and is the first step in the creation
of a new individual (Gilbert, 2010). As you might expect, the
beginnings of such an important event is quite complex and
involves a suite of processes that all must take place sequentially
in order to be successful. By studying the eggs, sperm, and zygotes
of sea urchins, scientists have begun to understand fertilization at
both the cellular and molecular levels (Gilbert, 2010). The main
purpose of this lab experiment is to observe and gather valuable
information on sea urchin developmental stages, as well as attempt
to induce parthenogenesis in sea urchin eggs by agitation. For the
parthenogenesis component, the main question being asked is; can
agitation of eggs by shaking can induce parthenogenesis?
Figure 2: 24 hour stage sea urchin undergoing gastrulation; A)
membranes stained with DiOC6(3), B) DNA stained with
propidium Iodide, C) merge.
Figure 3: Sea urchin embryo at 48 hours of developmental
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Discussion and conclusion:
Figure 3: 48 hour stage sea urchin at late gastrulation; A)
membranes stained with DiOC6(3), B) DNA stained with
propidium Iodide, C) merge. Note the invagination of the gut at
this stage with the surrounding mesenchyme cells capping it.
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Figure 1: Induction of parthenogenesis
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Figure 1: Autofluorescence; A) control egg that was not
agitated for parthenogenesis, B) egg that was shaken to
induce artificial parthenogenesis, but the image shows that the
egg is not induced, but highly disrupted.
For the induction of parthenogenesis, we first collected
pool of eggs by injecting 1-2 mL of isotonic potassium
chloride solution into the perivisceral cavity of a female green
sea urchin (Lytechinus variegatus), thereby inducing gamete
expression. We washed the eggs and placed them in an
eppendorf tube. We then tried to induce parthenogenesis by
vigorous shaking of the eppendorf tube with eggs in it. We
centrifuged the eggs for 2 min at 1000 RPM, removed the
water and replaced it with 1 mL of 4 % formaldehyde and
rocked the sample for 30 min at RT. After that we centrifuged
the eggs for 2 min at 1000 RPM, removed the formaldehyde,
added 1 mL PBS with 0.1% triton X, and rocked the sample
for 5 min at RT. We then centrifuged the eggs for 2 min at
1000 RPM, removed the PBS with 0.1% triton X, replaced it
with 1 mL PBS, and stored the embryo at 4oC.
For the sea urchin developmental series, fixed purple
sea urchin (Strongylocentrotus purpuratus) embryos were
provided by Dr. Cesar Arenas Menas from The College of
Staten Island / CUNY. We collected embryos from 24, 48, and
77 hours of development from the instructor and pelleted them
in a centrifuge (2 min at 200 RPM). We also separately
centrifuged the stored parthenogenesis embryos. We then
resuspended the pelleted embryos in 100 l PBS with 0.1%
triton X, after that we added 20 l propidium iodide stock
(300 uM) and 2 l DiOC6(3) stock (0.5 mg/ml) to each
sample. We then rocked at RT for 30 min and then pelleted the
embryos in a centrifuge (2 min at 200 RPM). We then washed
in 1 mL PBS with 0.1% triton X then pelleted the embryos in a
centrifuge (2 min at 200 RPM). We resuspended the embryos
in mounting media, transferred them to a slide, and observed
them by fluorescence microscopy
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Figure 4: Sea urchin larvae at 77 hours of developmental
Results:
Methods:
We hypothesized that we could induce parthenogenesis
in the sea urchin eggs through agitation. After performing the
lab and staining the eggs the results did not support our
hypothesis. The experiment produced two results, one result
showed that the eggs were intact, but no fertilization was
observed. In the second case the eggs were completely
disassembled (Figure 1). The failure to achieve our results is
directly related to our approach. I believe our approach to
inducing pathogenesis in the eggs was too aggressive. In the
near future the results could be improved by avoiding overly
shaking of eggs. In addition to parthenogenesis we also
stained embryos from 24, 48, and 77 hours of development
with fluorescent probes that bind nucleic acids (red: propidium
iodide) and cell membranes (green: DiOC6(3)). We can see
that at the 24 hour stage there is early gastrulation with
primary mesenchyme cells capping a thickening of the vegetal
cells (Figure 2). At the 48 hour stage we saw the invagination
of late gastrulation at the vegetal plate to form the early gut.
Surrounding the gut we see a collection of cells capping the
gut called secondary mesenchyme (Figure 3). The last stage
we looked at was 77 hour stage that showed the gut
developing at the center (Figure 4), we also see the prism
larval stage of embryo with spicules around the edge of
membrane. If our attempts at parthenogenesis were successful,
we might have expected to see these progressive stages of
development (e.g. morula and blastula) in our treated eggs.
Reference:
Tyler, Mary S. Developmental Biology, A Guide For Experimental
Study . Sunderland,Massachusetts: Sinauer Asscoiates, 2010.
Figure 4: 77 hour stage sea urchin as prism larvae; A)
membranes stained with DiOC6(3), B) DNA stained with
propidium Iodide, C) merge.Note the prominent gut tube at this
free-swimming stage.
Acknowledgements:
We are grateful to the students (e.g. Yewande Jegede and
Michelle Yun) of Bio452 who provided some of the images to use
in our poster.