Materials Needed
- Sea urchins
- 0.5 M KCl injections (for gamete extraction)
- sea water solution
- 10 mM PABA in sea water
- calcium-free sea water
- hypertonic sea water (sea water + 30 g/L NaCl)
- nylon filtering apparatus
- cylinders to hold filters
Procedure
1. Obtain adult sea urchins, species, Lytechinus variegates. 2. Harvest the gametes from these sea urchins using 0.5 M KCl injections (2mL.). Using a needle, piece the ventral side of the embryo, injecting small aliquots of KCl into each of the five symmetrical regions of the sea urchin. Gently agitate the sea urchin. 3. Observe the expulsion of the gametes to determine whether they are eggs or sperm. Collect eggs in a beaker filled with ASW and sperm in a dry petri dish. 4. Allow the sea urchin eggs to settle to the bottom of the beaker, remove the supernatant and wash the eggs with ASW containing PABA in order to prevent the hardening of the fertilization envelope. 5. Allow the sea urchin eggs to settle again, and repeat this washing process two more times. 6. Once the eggs have settled to the bottom of the beaker, pour off the PABA and replace with ASW. 7. In ASW, introduce the sperm to the eggs. 8. Observe the embryos. Note when the first and second cell divisions occur. 9. Transfer the four-celled embryos into hypertonic ASW solution in order to cause the cells to shrink away from one another. 10. Transfer approximately one fourth of the embryos into a test tube containing ASW labeled "control." 11. Pass the remaining embryos through a nylon mesh in order to separate the blastomeres. Check the resulting batch to insure that the majority of the embryos have been separated. 12. Collect a third of the remaining embryos (one fourth of the total embryos) in a test tube containing ASW labeled "first twinning." Place the remaining embryos back into the original beaker. 13. Observe these embryos to determine when they have again reached the four cell stage. 14. Repeat the twinning process (Steps 9 and 11). 15. Collect one half of these embryos in a test tube containing ASW labeled "second twinning." 16. Allow the remaining embryos to divide to the four celled stage. 17. Repeat the twinning process (Steps 9 and 11). 18. Collect these remaining embryos in a test tube of ASW labeled "third twinning." 19. Observe and photograph all of the embryos at the "zero hour" after twinning. 20. Allow the embryos to develop for twenty four hours. Observe and photograph. 21. Note any developmental differences that may have resulted from the twinning process.