Effect of UV Treatment on Early Development of Sea Urchin Embryos

互联网2013-09-05

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Objective:
To test the effects of UV radiation at 254nm on Sea Urchin embryo development after radiation at the 2-cell stage.

Background:
Sea Urchins exhibit radial holoblastic cleavage. The first and second cleavages are perpendicular to one another

and go through the animal and vegetal poles. The subsequent cleavages separate the cells into mesomeres,

macromeres, and micromeres. Sea Urchins undergo formation of the blastula and gastrulation before developing into a

mature embryo.

UV light is harmful to many organisms just as it is harmful to humans. Many eggs of marine animals including sea

urchins have a high concentration of mycosporine amino acids that protect them from UV damage. Mycosporine amino

acids are obtained through the diet of the adult and are stored in the egg. However, there is fear that the amphibians are

declining because of the increased UV-B reaching the earth's surface. UV-A and UV-B reach the earth's surface. UV-B is

increasingly hitting the earth's surface because of the depleting ozone layer. One of the more common effects of UV-B is

adjacent thymidines condensing into cyclobutane pyrimidine dimers (CBPDs) which impede DNA replication and

transcription. UV-C (200-280nm) is the most harmful type of UV to organisms, however, due to oxygen in the atmosphere

absorbing the UV rays, UV-C never reaches the Earth's surface. The effects of ozone depletion are likely to be most

devastating to organisms that are exposed to sunlight during their developmental stages. The sea urchin embryos are

transparent and poorly protected against UV. Sea urchins are exposed to UV radiation during all stages of their lifecycle.

Sea Urchins like many organisms have DNA repair enzymes, photolyases. UV radiation causes direct damage to proteins

and DNA by creating the formation of pyrimidine dimers and crosslinks between DNA and protein. Consequences of

these mutations are delayed cell divisions, DNA damage, developmental delays, and abnormalities. UV treatment can

cause immediate effects by damaging microtubules and long term effect by damaging RNA and DNA.

Experimental Design:

gametes in artificial seawater ( ASW ) 3 times

2) Add 1ml of diluted sperm to the gametes in ASW ( Fertilize gamates )

3) Wait until more than 75% of the embryos undergo first cleavage (check for cleavage under compound microscope periodically) See Figure 1.

4) Split the embryos into 4 open dishes labeled; 1min UV , 2 min UV, 5 min UV, Control

5) Place the open dish with embryos labeled 1 min UV 3 1/2 inches under the hand held UV lamp and turn on the short wave lamp for 1 minute while wearing protective goggles

6) Repeat for the two dishes labeled, 2 min UV, 5 min UV for times indicated

7) Wait for the second cleavage to occur, take pictures with digital camera while under the microscope of the control embryos and the three experimental groups of embryos.

8) Wait for the control embryos to hatch

9) Come in the next morning look under microscope and take pictures of all 4 groups of embryos to compare stages of development and percentage of normally developed cells

Figure 1. Sea Urchin embryos during first cleavage when radiated with UV-C.