6/25/14 The Sea Urchin Fertilization Lab

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Purpose | Hypothesis | Materials | Procedure | Background Information | Observations
Purpose: top
To observe and analyze the processes of fertilization and development in sea urchins.
Hypothesis: top
The procedure will allow the viewing of the physical processes of fertilization, cleavage, blastulization, gastrulization,
and further development and specialization.
Materials: top
Female sea urchins
Male sea urchins
Hypodermic needles
Potassium Chloride (KCl) solution
4% salt (NaCl) solution
Depression slides
Petri dishes
Pipettes
Microscope and image capture hardware and software
Ice
Procedure: top
1. Place live sea urchin specimens over a sterile cup filled half way with salt water solution.
2. Fill hypodermic syringe with .5 to 1.5 cc of the salt solution. Insert the needle into the oral region of the
urchins, and inject the solution.
3. Hold urchin over the cup upside down, allowing the gametes to drip into the cup.
4. Observe gametes. If they are white, the urchin is a male; if they are orange, the urchin is a female.
5. Remove the ova (female gametes) first and place them on the slide. Observe and diagram the ova.
6. Add sperm carefully to the slide with the ova. Observe closely for the events of fertilization and the formation of
the fertilization membrane.
7. Monitor the developement of the new zygote. Watch for cell divisions.
Background Information: top
The fertilization process witnessed in this lab is typical of fertilization in all sexually-reproducing animals. The sea
urchin life cycle is almost entirely diploid. This means that all somatic cells (every cell except gametes), the nucleus
contains two slightly different copies of each chromosome. The process that is essentially the converse of fertilization
is meiosis. By this process, cells of a diploid somatic lineage replicate their DNA, and each proceeds to divide by
two separate processes into four haploid sperm (in males) or one haploid ova (in females).
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The sperm and egg (or ovum) cells are the contribution of each organism to the next generation. Because sea urchins
exist as diploid adults, fertilization is necessary to combine the haploid genomes of the sperm and ova into a single
diploid zygote. In order to fully understand the physical and chemical processes of fertilization, a brief look at the
structure of the sperm and ovum is required.
A mature sperm is a truly miraculous
union of structure and functionality.
With each emission from the male,
millions upon millions of sperm are
released. Each time, only one can
fertilize the egg. This fact dictates the
evolution of sperm; any advantage of
one structural variation over another will
provide a much greater likelihood for
that sperm to fertilize the egg and
hence pass its genome on to offspring.
The sperm is highly streamlined, and
thus suited to swim through the fluids
of the female reproductive tract (in
species with internal fertilization) or
other fluids (in species with external
fertilization, such as most aquatic
species including the sea urchin). It has a long flagellar tail, connected to a middle piece. The middle section of the
sperm contains a very long and spiral-shaped mitochondrion. It serves to metabolize high-energy nutrients (especially
fructose) contained in semen, the milky fluid that the sperm are conveyed to the female in. The ATP produced by the
mitochondrion serves the vital function of driving the movement of the flagellum. The midsection is connected by a
very small neck to the thick head of the sperm. The head of the sperm is the part that actually plays a direct role in
the fertilization of the egg. It contains a haploid nucleus, as well as an acrosome. The acrosome is a membranous
organelle that contains enzymes to break down a jelly-like coat surrounding the egg.
In contrast to the sperm, the egg is a generally stationary cell. However, it, too, contains several features key to the
process of fertilization. The egg is surrounded by three primary layers: the plasma membrane common to all cells as
well as two others. The first unique layer, immediately surrounding the plasma membrane, is known as the vitelline
layer. It is composed primarily of glycoproteins. Surrounding the vitelline layer is a much thicker coat of a jelly-like
substance serving to protect the egg.
The process of fertilization in sea urchins is physiologically complex, but conceptually simple. It begins as the sperm
approaches the egg, having swum through ocean water after being released from the male. At this point, the sperm
has used almost all its energy, but has enough remaining to carry through the procedure of fertilization. As the head
of the sperm comes in contact with the jelly layer of the egg, the acrosome in its head releases its store of enzymes.
The enzymes digest a cavity in the jelly layer. The sperm head continues into the cavity. When it comes in contact
with the vitelline layer, specific proteins on its head bind to receptor molecules on the vitelline layer. In species (like
the sea urchin) with external fertilization, this step is key; the proteins are species-specific and hence prevent the
fertilization of an egg of species A by a sperm of species B.
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After this initial phase, the sperm proceed through the
vitelline layer. The plasma membrane of the sperm then
fuses with that of the egg, and the two cytoplasms
combine. Less than one second after this process occurs,
the plasma membrane of the egg cell becomes
impermeable to other sperm. Just after this, the vitelline
layer hardens and separates from the plasma membrane
of the egg. The space between the vitelline layer and
plasma membrane fills with water, and the vitelline layer
becomes a fertilization membrane. These steps are
essential in the process of fertilization, for they prevent the
fertilization of the same egg by multiple sperm (which
would result in a polyploid zygote). The photograph to the
right (obtained from Biology: Concepts and Connections)
shows an egg covered in sperm, just before or after fertilization.
The final phase of fertilization occurs in preparation for the growth of the embryo. The egg's metabolic machinery,
nearly dormant since oogenesis, gears up suddenly. Finally, the haploid protonuclei of the sperm and egg fuse into a
single, diploid zygote nucleus. The entire process of fertilization is shown below:
After the process of fertilization, the development of the new sea urchin begins with cleavage. The zygote begins by
mitotically dividing into two cells. Each of these cells divides to produce four, which split into eight, and so on.
However, although DNA replication and mitosis are occuring rapidly and cytokinesis is spliting the cells, little
transcription occurs during cleavage, so the zygote does not actually grow. Instead, each of the daughter cells after a
division is almost twice as small as the parent cell. Deeper into cleavage, each cell is much smaller than the original
zygote after fertilization. Every division in a sea urchin takes approximately twenty minutes. As cleavage continues, a
hollow cavity containing fluid forms in the middle of the group of cells. This cavity, the blastocoel, becomes the center
of the embryo and is important in gastrulation, discussed later. By the end of cleavage, the embryo is a ball of cells
with the hollow blastocoel in the center. This ball is called a blastula. In a sea urchin, completion of cleavage takes
approximately 3 hours.
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Gastrulation, the second major phase in the development of the embryo, is set up by chemical interactions during
cleavage. Near the end of cleavage, regulatory chemicals localize in certain cells, activating the transcription of
certain genes and beginning the process of cell specilization. In gastrulation, this process is continued as three
distinct layers of tissue take form: endoderm, ectoderm, and mesoderm. These three cell layers will give rise to all
parts of sea urchin. As stated above, gastrulation is actually begun by the blastula. In the blastula, there are cells of
different sizes. The larger cells are concentrated at the bottom of the blastula, called the vegetal pole. The smaller
cells are found at the top of the blastula, the animal pole. The cells of the animal pole are smaller because they have
been dividing more rapidly than those of the vegetal pole. The size and location of cells are important in determining
what type of tissue they will become. The small cells of the animal pole will become the ectoderm, the mid-sized,
central cells will form the mesoderm, and the larger cells of the vegetal pole will give rise to the endoderm.
The actual process of gastrulation begins when a groove in the blastula, known as the blastopore, forms on one side
of the embryo. This is where the cells of the inner tissue layers, the endoderm and mesoderm, will migrate to the
middle of the embryo. The ectoderm cells from the animal pole begin to spread out across the surface of the blastula,
filling in the areas left behind by the migrating cells of the other tissue layers. Next, the three cell layers continue to
migrate to their respective areas and are able to be seen as distinct layers. The endoderm, which gives rise to the
digestive tract, forms a simple digestive cavity called the archenteron, and the blastocoel shrinks as it is filled with
cells from the mesoderm and endoderm. The mesoderm and endoderm continue to move inwards as the ectoderm
migrates to cover the entire outside of the embryo. The completion of gastrulation occurs when the three tissue layers
can clearly be seen. The ectoderm covers the entire gastrula, as the embryo is now known, except for the entry to
the digestive cavity formed by endodermal cells, called the yolk plug. This will become the anus of the sea urchin.
The blastocoel is gone, but the archenteron has replaced it. Cells of the endoderm cover the archenteron, which are
then covered by mesoderm tissue. All organs and organ systems arise from these three tissue layers, as seen next.
The next step in the development of sea urchins is the differentiation into tissues and organs by the three cell layers.
The first step in this process is the development of the nervous system. The ectoderm begins to form the neural plate,
a raised, pronounced area. In the middle is a neural fold, which is rolled as the neural plate moves inward. The neural
tube, the rolled up neural plate, detaches from the ectoderm and will become the brain and spinal cord, the central
nervous system, of the sea urchin. Although a sea urchin has no true central nervous system because it is radially
symmetrical, the neural tube will become the nerve ring and radial nerves of the organism. Another major step in
development is the the development of somites from the mesoderm. These will give rise to segmental structures of
the sea urchin. Next to the somites, the mesoderm forms the coelom, the body cavity. In echinoderms, this is a
coelom that is from the digestive tube.
There are other major stages in the development of tissue from the three cell layers of the gastrula. They can not be
discussed in detail, but it is known which layer of cells forms which tissue. The ectoderm forms the epidermis and its
sense receptors (cells that signal the nervous system), the lining of the mouth and anus, the cornea and lens of the
eye, the adrenal medulla (a member of the nervous system important in stress), and tooth enamel. The endoderm
forms the lining of the digestive tract, the lining of the respiratory system, many of the major digestive and endocrine
glands (the pancreas, liver, thyroid, parathyroids, and thymus), the lining of the urethra and urinary bladder, and the
reproductive systems. The mesoderm produces many of the main systems in the body, including the skeletal,
muscular, circulatory, excretory, and reproductive systems. Also, it produces the dermis (under the ectoderm
epidermis), the adrenal cortex, and the coelom and its lining, which protects and supports the major organs of the
body. In this way, the major body organs are formed in the sea urchin, and other techniques lead to further
differentiation and specialization.
Many changes at the cellular level are very important in the development and growth of the sea urchin. One of these
is change in cell size and shape. An example of the importance of this is the formation of the neural tube. In order to
form this tube, the cells grow and become wedge shaped, forming a curve. These cells elongate and others grow in
and replace them to form the neural tube. Cell migration is also very important, especially in gastrulation. Cells use
pseudopodia to crawl towards their destination, the inside of the gastrula for the mesoderm and endoderm and the
outside for the ectoderm. Once a cell has migrated, proteins produced by the cell allow it to signal and find other
cells of its type. These cells secrete glyoproteins to bind in place when they find that they are near other cells of their
type. Another important process later in development is programmed cell death. Suicide genes code for proteins that
produce the cell's death. These proteins also send out signals to adjacent cells to make them phagocytic, engulfing
the dead cell. This process is important for the nervous and immune systems of the sea urchin. Another very
important cellular aspect of development is the influence by a group of cells on an adjacent group, discussed below.
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Induction is the process by which one group of cells stimulates the development of a group of adjacent cells. This is
accomplised through signal transduction. A group of cells activate genes that code for a secretory molecules. This
molecule binds to a receptor in the plasma membrane of a nearby cell and begins a signal transduction pathway.
This pathway will eventually lead to the transcription of one or more genes in the host cell, producing proteins that
help in differentiating the cell. The secretion of these signal molecules by many cells can produce an effect on a large
number of cells. Induction is very important in relationships between nearby groups of cells and cell differentiation.
Another factor in determining specialization is pattern formation. In pattern formation, the sea star's major body parts
are formed by master control genes. These genes respond to chemical signals from cells and respond by telling a
cell where it is in relation to other cells of the embryo. This is very important in forming groups of cells that are alike in
the fact that they are producing the same proteins and belong to the same tissue. Not much is know about these
master control genes, but studies with bird wings have proven that chemical signals released from these master
control genes, which are found in certain places, tell a cell where it is in relation to anterior, posterior, dorsal, ventral,
proximal, and distal ends by the number of chemical signals that the cell receives. The more signals, the closer the
cell is to the master control gene, used as a point of reference on the wing.
Although major development of the sea urchin occurs above, development and growth continue until birth and beyond.
The formation of the organs for each organ system and growth of the embryo occur later in development. But in this
amazing process, a single cell, the zygote, undergoes cleavage, gastrulation, organ and tissue formation, cellular
changes, induction, and pattern formation in order to become a living organism.
Observations: top
Thi s page copyri ght 2000, the Bi o-Web Group, Si dwel l Fri ends School , Washi ngton, D.C.