Exercise 13.1 - Sea Urchin Embryology

LEVEL I

Materials

Procedure

  1. Place a commercially prepared slide of the sea urchin embryo development on the stage of the microscope and locate representative stages in the development of the organism. Draw and label the following stages:

  2. Indicate on your drawings the development of micromeres, mesomeres, and macromeres. Also identify the formation of primary mesenchyme and secondary mesenchyme during the formation of the gastrula.

  3. Collect a living sea urchin and place it "upside-down" in the top of a small beaker. The mouth should be exposed. With a clean, new needle, inject 1.0 ml of 0.5 M KCl into the tissue just adjacent to the oral opening. The fluid should enter the coelomic cavity, not the gut of the animal.

  4. Within a short period of time (several minutes) the sea urchin will discharge its gametes. The gametes will appear as either a yellow collection of eggs, or a white collection of sperm.
    For the eggs, immediately invert the sea urchin so that the eggs are shed directly into a small amount of salt water in the bottom of a new, clean beaker. It is important that the bottom of the sea urchin be in contact with the sea water and that the salt water be kept cool (15° C is ideal). The shed eggs are stable for several hours if kept cool.
    For sperm, the animal should be inverted over a dry beaker, that is with no salt water. Sperm are activated when diluted in sea water. If collected in the dry state and stored in a refrigerator, they will survive for several hours. When diluted, they will last only a matter of minutes.

  5. Place a small sample of the eggs in sea water onto a microscope slide and place the slide on the stage of a microscope. Focus on the eggs.

  6. With a clean pipette, collect a small drop of the sperm and place it into about 1 ml of salt water in a test tube. Gently stir to mix and activate the sperm, and then transfer a drop of the diluted sperm to the waiting eggs on the microscope stage.

    Do not contaminate the remaining eggs by contact with the sperm.

  7. Immediately place a coverslip on the suspension of sperm and eggs and observe. Once fertilization occurs, the edges of the coverslip can be sealed with parafin to prevent drying.
    For long term observation, a diluted sample of eggs should be fertilized and kept at 0° C. Periodically, wet mounts can be made of the developing embryos to examine the progress.

  8. Compare the development of the living embryos to the prepared slide stages. 17

    Notes

    The development of the sea urchin embryo occurs rather rapidly. Fertilization will probably occur before you can get the coverslip on the slide. Within about 1-2 minutes the fertilization membrane will lift off, and the first division should occur within 2-3 hours, depending on the temperature. The embryo will hatch in just over 24 hours and will complete its entire development within 72 hours.

    If close examination of the fertilization process is desired, place the eggs on a slide with a support to keep the coverslip just off the eggs (a second coverslip will do), add a coverslip and focus on the eggs with darkfield illumination. Place a drop of sperm just at the edge of the coverslip while observing the eggs through the microscope. With darkfield illumination the sperm will be visible and minute changes in the cortical region of the egg will be observed as a sperm penetrates the outer membrane.

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    Cell Biology Laboratory Manual
    Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
    St. Peter, MN 56082 -- cellab@gac.edu