Lab Protocols

Experiments for the Teaching Lab

Sea Urchin Fertilization and Early Development

Every developmental lab needs a sea urchin fertilization experiment. There's probably nothing that demonstrates the early stages of development more clearly than these translucent echinoderm embryos. The developmental stages of the sea urchin up through gastrula are nearly identical to starfish development . The This protocol does not deal with sea urchin husbandry itself, but starts off assuming mature males and gravid females. I learned the techniques shown here at the Darling Marine Center developmental biology workshop. What little prior experience I had with sea urchins came from Dr. Charles D'Asaro at the University of West Florida.

Special Supplies:

Figure 1

Figure 3

Figure 5

Figure 2

Figure 4

Procedure:

  1. Obtain gametes from sea urchins (Fig.1). Before you begin, clearly mark some Pasteur pipets with pink tape and others with blue (for female and male use, respectively). Have the 0.5 M KCl solution on hand. Fill up an appropriately sized beaker with sea water and add an inverted pipet tripod to hold the future female urchin if necessary (Fig. 2, used in step 1C). Have a dry petri dish ready for collecting male sperm.
    1. Select a sea urchin from the holding tank, cup it in your hands with paper towels, and give it a few firm shakes.
    2. While holding the urchin upside down (oral side up), insert the loaded siringe between teeth and the "gums," and gently expel a small amount of KCl solution (Fig. 3). Do this quickly five times, once in every of the five slots between the teeth (Movie 1). Cup the urchin again in paper towels and give a few firm shakes. By the time you're complete, the urchin spines will probably be flailing (as much as sea urchins can flail).
    3. Flip the urchin aboral side up and inspect for any spawning; females will usually have pigmented eggs, while males will expel a milky white liquid. If male, place upside down (aboral side down) in the dry petri dish (result shown in Fig. 4, though the urchin is upright). If female, place upside down on top of the beaker filled with sea water (Fig. 5) . The female spawning can be quite spectacular to see (Movie 2).
  2. Prepare gametes for fertilization. Although you may go through several urchins before you obtain good results, one good male and one good female are often enough to provide samples for the whole class. Once both sperm and eggs are obtained it's time to prepare the slides.
    1. (Optional wash step) After the eggs have mostly settled to the bottom, decant off the majority of the sea water and refill (with sea water).
    2. Use a pink pipet to vacuum up eggs from the bottom of the beaker, and add two drops to a clean slide.
    3. Rub the corners of a cover slip across the ball of clay to create clay feet at each of its corners. These clay feet will prevent the cover slip from smashingh the eggs.
    4. Apply the cover slip with clay feet...the eggs are ready (still unfertilized at this point).
    5. Prepare the sperm by adding a single drop of the concentrated sperm to about 60 mL of sea water. Mix them throughout with a clean pipet. The appearance should be faintly clouded. Obvious/excessive turbity indicates potentially to much sperm which may result in polyspermy.
    6. The sperm are ready.
  3. Fertilization. At this point, each student should have their own slide with unfertilized eggs, and the instructor should have a batch culture of dilute sperm.
    1. Have the students use a 10X objective to observe the unfertilized eggs and note their appearance. If prefer to use dark field (See "Simple Tools"), but light field will also work.
    2. While on the microscope stage, add about two drops of dilute sperm to one edge of the cover slip. The sperm will be drawn under the coverslip, gradually progressing across the eggs.
    3. If successful, the students will be able to observe a wave of fertilization before their eyes, indicated by the physical "blistering" of the fertilization membrane away from the egg plasma membrane to form a "halo" around the egg.
  4. Batch Fertilization.To observe sea urchin development over time (up through the pluteus larval stage), they should be fertilized as a batch in small beakers instead of on slides.
    1. Pipet a large amount of unfertilized eggs into a small beaker filled with about 60 mL of sea water (add enough to create a visible layer on the bottom).
    2. Add 4-6 drops of the dilute sperm solution to the beaker and gently swirl.
    3. After 2-3 minutes, aliquots (two drops) can be immediately observed under a compound light microscope to look for fertilization efficiency. If not many sperm are visible and effeciency is low, try adding several more drops of sperm, although this could mean a bad batch of eggs.
    4. If fertilization is satisfactory, let the eggs settle to the bottom (5-10 minutes) and decant off the majority of the sea water (this removes the left over sperm and is an important step). Refill the beaker with sea water...do this twice.
    5. Cover the beaker with a loose covering, such as aluminum foil, and store at room temperature.
    6. Samplings can be made at any time to observe the various stages of development, though the actual time of the events may vary based on the species. In general, one can expect to see the first cleavage within 1 and 1/2 hours. It's not necessary to use a coverslip each sampling, just don't use the 40X objective.
    7. Important: In order to maintain good cultures over several days the successful blastulae need to be separated from junk. The day after fertilization, ciliated blatulae will be floating about the water column with the junk lying at the bottom. Decant off and SAVE the upper 80% of the culture and toss the rest.
    8. Continue to sample/observe this culture as long as possible.