The Effects of Hydrogen Peroxide on the Fertilization of Sea Urchins Arbacia Punctulata

The Effects of Hydrogen Peroxide on the Fertilization of Sea Urchins Arbacia punctulata

Biology 188

Thursday 9:30 Lab

Brenda Seidel

13 April 2006

The Effects of Hydrogen Peroxide on Sea Urchins Arbacia punctulata

Biology 188

Thursday 9:30 Lab

Brenda Seidel

13 April 2006

Abstract

The objective of this experiment is to determine what concentration of hydrogen peroxide in a salt water environment would be best for sea urchin fertilization. Our hypothesis is that an environment with a concentration between 10-8 M and 10-10M of hydrogen peroxide will result in the highest successful fertilized sea urchin egg count. Sea urchin eggs and sperm were placed in separate test tubes with hydrogen peroxide concentrations of 10-1 to 10-12 M. Fifteen eggs were selected from each test tube and recorded to be fertilized, unfertilized, or abnormal. The results show that the highest count of successfully fertilized eggs was observed in the tube with a 10-12 M concentration of hydrogen peroxide. Additionally, it was observed that there was a low count of abnormal and unfertilized eggs in this tube. The data obtained in this experiment shows that an environment with a hydrogen peroxide concentration of 10-12 M is ideal for fertilization in sea urchins.

Introduction

In sea urchins, Arbacia punctulata, a high concentration of sperm and egg often result in polyspermy, the fertilization of an egg by more than one sperm. Polyspermy ultimately leads to uneven division and death (Coburn et al 1981). Offspring cannot result from eggs fertilized by more that one sperm (Aranows et al 1981). In sea urchin eggs, hydrogen peroxide is produced right after fertilization to combat this problem. A sea urchin dual oxidase, Udx1, in the egg generates the hydrogen peroxide to block polyspermy (Wong et al 2004, Wong et al 2005). In the absence of hydrogen peroxide, all eggs will undergo polyspermy and die. In Coburn et al's experiment, catalase was added to destroy hydrogen peroxide, and polyspermy occurred in one hundred percent of all fertilized eggs (1981).

However, hydrogen peroxide can also be harmful to sea urchin fertilization. A high concentration of hydrogen peroxide results in changes in sperm motility and a decrease in egg fertilization (Aranow et al 1981). Aranow et al observed that a concentration between 10-3 M and 10-6 M of hydrogen peroxide causes this to happen. Therefore, a lower concentration of hydrogen peroxide would be needed to be affective in aiding fertilization in sea urchins.

The objective of this experiment is to determine what concentration of hydrogen peroxide would be best for the fertilization of sea urchin eggs. This concentration will need to be high enough to prevent polyspermy but also low enough to not cause a decrease in sperm motility. Our hypothesis is that an environment with a concentration between 10-8 M and 10-10M of hydrogen peroxide will result in the highest successful fertilized sea urchin egg count. This hypothesis will be tested by observing sea urchin eggs and sperm in samples of sea water with hydrogen peroxide concentrations of 10-1 M to 10-12 M. A count of fertilized eggs, unfertilized eggs, and abnormal eggs will be taken and used to determine which concentration of this toxin is best for fertilization in sea urchins.

Materials and Methods

Sea urchins were received via overnight delivery nine days before the experiment was conducted. Thirty-six of these sea urchins were placed in a thirty gallon, 72 degree Fahrenheit tank that had a salinity of 30ppm. All seawater used in this experiment was artificially made using Instant Ocean and filtered water. Sea urchin eggs and sperm was collected seven days before the experiment and refrigerated. A sea urchin egg solution was prepared by adding 3ml of a 10% sea urchin egg solution and 100ml of seawater to a 250ml beaker. A sea urchin sperm solution was prepared by adding 10 micro liters of sperm to 100ml of seawater. These solutions were prepared right before the experiment. Twenty-six test tubes were labeled 1A to 13A and 1B to 13B. 2,700 micro liters of seawater were added each test tube. 300 micro liters of hydrogen peroxide were added to tubes 1A and 1B. Then, using a pipette, 300 micro liters of the solution in tubes 1A and 1B were taken out and added to tubes 2A and 2B respectively. Once again, 300 micro liters of the solution in tubes 2A and 2B were taken out and added to tubes 3A and 3B respectively. This process was done for each tube up to tubes 12A and 13B to make serial dilutions of hydrogen peroxide. Therefore, tubes 1A and 1B had a hydrogen peroxide concentration of 10-1 M, tubes 2A and 2B had 10-2 M, tubes 3A and 3B had 10-3 M, tubes 4A and 4B had 10-4 M, , tubes 5A and 5B had 10-5 M, tubes 6A and 6B had 10-6 M, tubes 7A and 7B had 10-7 M, tubes 8A and 8B had 10-8 M, tubes 9A and 9B had 10-9 M, tubes 10A and 10B had 10-10 M, tubes 11A and 11B had 10-11 M, and tubes 12A and 12B had 10-12 M. Hydrogen peroxide was not added to tubes 13A and 13B. These tubes were used as controls for this experiment. Then, 100 micro liters of the sea urchin sperm solution and 1000 micro liters of the sea urchin egg solution were added to each tube using a pipette. In the first trial, two drops of each tube was then transferred to a different depression slide with a pipette and placed under a light microscope. Each slide was observed and fifteen eggs were chosen at random to record data on. If a slide had less than fifteen eggs, all of the eggs on the slide were chosen. It was then determined whether each egg was fertilized, unfertilized, or abnormal and recorded. After the first trial, a second trial was done and the numbers obtained from both were averaged. Then, the data obtained for tubes 1A to 13A and 1B to 13B was averaged and used to determine the ideal hydrogen peroxide concentration for sea urchin fertilization

Results

The results from this experiment show that hydrogen peroxide substantially increases the number of successfully fertilizations. As shown in Table 1, only an average of 4.5 eggs was fertilized in the control tubes, 13. A notable increase in the number