Name _____________________________
Lab: Sea Urchin Embryology
BACKGROUND
Studies of sea urchins provide information on fertilization and development that apply
to all organisms from jellies to humans. These eggs, then, provide a model embryo for
understanding development in all forms. It is important to observe early embryonic
development because it is during this time that the patterns of development of the
organism originate.
Sea urchin gametes are the same size as human gametes and development is very
similar through the gastrula stage. Both sea urchins and humans are deuterostomes,
meaning that patterns of cleavage are radial and the mouth arises at a site distant from
the site of gastrulation. The name deuterostomes means second mouth. In contrast
protostomes follow a spiral cleavage pattern and the mouth forms near the blastopore,
the origin of gastrulation.
MATERIALS
0.5 M potassium chloride solution
dissecting scope
1 gamete collecting cup
1 plastic petri dishes
1 pipette
syringe with needle
seawater
hand lenses
microscopes
concavity slides
paper towels
PROCEDURE
Day 1: Gamete Release and Observation of Fertilization
1. Load the syringe with 1.5 mL of 0.5 M solution of potassium chloride. Carefully
insert the needle through the soft tissue surrounding the urchin's mouth and inject
the solution into the central cavity. Swirl the urchin gently to mix the solution
inside. Within 10 minutes, gametes will release from the gonopores in the aboral
(“not oral”) side.
2. You will be able to differentiate male and female urchins when you see the gametes.
Eggs are yellow, and sperm is white. Eggs need to be collected in seawater, and sperm
need to be collected "dry." One approach is to invert the urchin over a dry cup and,
when you hear something begin to plop into the cup, quickly take a pipet and
examine the contents under a microscope. If it is a female, there will be many eggs,
and you should immediately add about 50 mL of seawater to the cup.
If the urchin is male, there will be lots of active sperm and you will
add no water to the cup. If the solution is clear, wait; it was just a drop
of water and the gametes have not yet been released.
3. Label each cup with the contents (sperm or egg) and students' names.
Label each pipet to match each cup.
4. After the gametes have been shed and the urchins' sex is known, there
will be enough sperm and eggs so that every student can observe
gametes and the fertilization process.
5. To witness fertilization, place a drop of eggs on a concavity slide and
add a drop of diluted sperm to it. (To dilute sperm, add a drop of sperm to 75-100
mL of seawater, but do not do this until just before use of the sperm. Using fullstrength sperm suspension would likely cause polyspermy followed by abnormal
development of the zygote). Within a couple of minutes of adding the sperm to the
eggs, you should see the fertilization coat lift from the egg. Continue to observe the
slide.
Storing Gametes:
6. The eggs in the cups will settle to the bottom. The seawater can be decanted or the
eggs can be pipetted into fresh seawater. This process is called washing the eggs.
Leave the egg cups out, at room temperature.
7. Collect the sperm "dry" in a pipet or eyedropper and place it into a small test tube.
[Seawater "activates" the sperm reducing their life span from days to minutes!
Keep concentrated until just before use.]
8. After you have collected all that you can, place the tube into a refrigerator (not
freezer) or an ice bucket with ice. Sperm will keep at 4 C for 2-5 days.
Day 2: Observing Development
1. Fill a petri dish half full of seawater.
2. With a pipet, add 1 squirt of washed eggs. The eggs should not cover more than
1/8 of the bottom of the dish.
3. Prepare a dilute sperm solution (see procedure from Day 1). Add 1 drop of dilute
sperm suspension to the eggs.
Keep the dish covered at room temperature (22-25°C) and observe the contents with a
microscope every 15-30 minutes for evidence of fertilization and development. The first
zygotic division should begin about 80 minutes after adding the sperm suspension.
Development will continue through a series of divisions. Though the rate of
development may vary due to environmental conditions in the laboratory, generally, the
blastula stage can be observed in 18-24 hours, early gastrula in 30-36 hours, and early
pluteus in 72-80 hours. Every 24 hours, freshen the water in the dish by carefully
drawing off at least half the water and replacing it with fresh seawater.
Keep a careful record of when each of the recognizable development steps occurs.
Include detailed drawings.
Time/Phase visible
Drawing/observations
Post-lab Questions:
1. What would happen if the sperm could not get into the egg?
2. How do organisms develop from the size of a sperm and egg to an adult? What
directs these changes?
3. Why is there such a difference in the size of the egg and the sperm?
4. Water currents in the ocean are much stronger than any sperm. How do sperm
and egg find each other?
5. Why do sea urchins have external fertilization? [Discuss the life style of the
developing sea urchin embryo in the water column compared to a very different
ecological niche of the adults on the ocean floor.] What are the adaptive
advantages of external fertilization? What are the adaptive advantages of internal
fertilization?
6. What would happen if one cell was removed or damaged at the 2 cell, 4 cell, 8
cell stage?
7. What would happen if the two cells were separated after the first cleavage and
allowed to develop on their own?
8. Why do cells with multiple chromosomes need the complex steps of mitosis to
divide in comparison to the steps of binary fission in bacteria?
9. Carefully draw a sea urchin embryo in the gastrula phase. Label all visible
tissues layers. Are sea urchins diploblastic or triploblastic?