1. Abstract Lithium has been shown to disturb development in sea urchin embryos, but its effects on fertilization have remained unexplored. To address this, sea urchin eggs (Arabacia punculata) were incubated for 10 minutes in varying concentrations of Lithium Chloride (LiCl); 0.01M, 0.1M, 0.3M, and 0.5M. After the incubation period, fertilization was attempted. Increasing concentrations of the LiCl showed a decrease in fertilization rates. Control, untreated eggs showed a 100% fertilization, whereas fertilization among LiCl treated eggs ranged from 50% (0.01M LiCl) to 7.41% (0.50M LiCl). LiCl treated eggs also showed a decrease in the number of 4-celled embryos, with an increased incidence of amorphous cells (0% amorphous in Control embryos versus 55.0% in 0.10M LiCl). Trypan Blue Staining showed that LiCl had no effect on the viability of the eggs, since all samples showed 100% stain exclusion. These data suggest that LiCl disturbs the fertilization process in sea urchin gametes, however, the continued viability of the cells may indicate that these effects are localized to the jelly coat and/or the osmolarity by affecting the sodium gradient within the cells such that the plasma membrane remains intact. Introduction Lithium is one of the most commonly prescribed drugs for the treatment of bipolar disorder, is a leading anode material for batteries, and a coolant in nuclear reactors. It is also used on spacecraft to remove carbon dioxide from the air (Encyclopedia Britannica). Lithium has been shown to have a major effect on the development of many different organisms, including sea urchins, Xenopus, and zebrafish, and even fruits such as tomatoes, by acting on Lithium sensitive enzymes such as GSK- 3β (Klein and Melton, 1996). Lithium Chloride (LiCl) has been shown to progressively block the cell cycle of sea urchin embryos by inhibiting the phosphoinositide signal pathway, an important regulator of embryonic cell division (Becchetti and Whitaker, 1997). LiCl has also been shown to alter nuclear β-catenin, an event which coincides with an increase in mesoderm and endoderm (Logan, et al., 1999). LiCl has also been suggested to act through the inhibition of glycogen synthase kinase- 3β (GSK-3β), a regulator in cell fate determination in many organisms (Klein and Melton, 1996). Although the effects of LiCl on embryonic development seem extensive, its effects on fertilization have remained virtually unexplored. Inject Urchins with 4cc 0.5M KCl Collect gametesIncubate oocytes in LiCl Lithium Chloride Interferes with Fertilization in Arabacia punculata Elizabeth M. Ziegler York College of Pennsylvania Control 0.01M 0.1M 0.3M 0.5M Discussion Lithium Chloride caused a decrease in fertilization rates as concentration increased. This may be due to interference with the jelly coat of the egg, which attracts the sperm to the egg for fertilization. LiCl showed a dose-dependent effect on the development embryos, as well as a teratogenic effect (Becchetti and Whitaker, 1997), an event supported by these data. The LiCl could also have affected the membrane potential of the oocytes leading to a change in the osmosis, making the oocytes hypotonic, and the integrity of the membrane, making it more permeable to ASW attributing to a change in osmosis or the pressure within the oocytes. As in previous studies LiCl incubation interfered with cell division in fertilized embryo, preventing embryos from cleaving to the 4-cell state. This has been attributed to interference with the phosphoinositide pathway, a block that occurs during metaphase and cytokinesis (Becchetti and Whitaker, 1997), and with the inhibition of GSK-3  (Logan, et al., 1999). Observations made in previous studies are not comparable because many were specifically looking at the activity of an enzyme or pathway of interest and did not report the effects on developmental rates. The continued viability of the oocytes, in concert with the decrease in fertilization may be due to effects on the jelly coat or on the permeability of the cell which could have lead to changes in the osmotic pressure due to changes in the concentration of ASW or perhaps sodium within the cell while leaving the cell membrane intact. Future Research How is cell viability maintained, while fertilization is inhibited and cells become amorphous? Literature Cited Becchetti, A., and Whitaker, M. 1997. Lithium blocks cell cycle transitions in the first cell cycles of sea urchin embryos, an effect rescued by myo -inositol. Development 124:1099-1107. Logan, C., Miller, J., Ferkowicz, M., and McClay, D. 1999 Nuclear β-catenin is required to specify vegetal cell fates in the sea urchin embryo. Development 126: 345-357. Klein, P. and Melton, D. 1996. A molecular mechanism for the effect of lithium on development. Developmental Biology 93: 8455-8459. Encyclopedia Britannica. Lithium. Available from: http://www.britannica.com/eb/article?eu=49668 Accessed 2002, August 30. Picture Cited Jean-Marie Cavanihac. 2000. http://www.microscopy- uk.org.uk/mag/artjul00/urchin1.html ThinkQuest team J0111704. 2001March 14. Available from: http://library.thinkquest.org/J0111704/picturegallery/pic14/pic14. html Acknowledgments Dr. Ricker, PhD Department Chair Results Increasing concentrations of the LiCl solution resulted in decreased fertilization rates (100% for the control group, 50% in 0.01M, 10% in 0.1M, 24.14% in 0.3M, and 7.41% in 0.5M). LiCl also showed a decrease in the number of 4-celled embryos (readings were taken on all embryos present: 90.91% in the control group, 66.67% in 0.01M, 15.38% in 0.1M, and 0.0% in 0.3M and 0.5M) and an increased number of amorphous cells (0% in the control group, 9.38% in 0.01M, 55.0% in 0.1M, 24.14% in 0.3M, and 37.04% in 0.5M). Trypan Blue staining showed that the LiCl had no effect on the viability of the oocytes (all samples showed 100% cell viability before fertilization). Objectives 1.) To examine the effects of LiCl on fertilization of sea urchin (Arabacia punculata) oocytes. 2.) To monitor the effects of LiCl on subsequent embryo development and compare them to previous studies. 3.) To examine the effects of LiCl on cell viability of sea urchin oocytes. Materials and Methods Artificial seawater was used during the collection, incubation, and fertilization of gametes. All experiments were performed at 24˚C. Lithium Chloride Solution 21.20g LiCl was dissolved in 100mL of ASW to obtain a 5.0M stock solution. This was then diluted down to achieve molarities of 0.01, 0.10, 0.30, and 0.50. Gamete Handling and Fertilization Gamete shed was induced by injecting KCl into the soft tissue around the mouth. Eggs were collected by placing the female aboral side down on top of a 250mL beaker filled with ASW, while sperm was pipetted off the male sea urchin and placed in a dry Petri dish. Equal portions of eggs were placed in beakers containing either ASW or LiCl solutions. Fertilization was achieved by diluting 3 drops of sperm in ASW and then by placing 3 drops of that solution in the egg containing beakers, after the eggs had been incubated for 10 min. Fertilize the gametes Figure 2. Mean percentage of fertilized and 4-cell embryos. Error bars indicate the standard error of the mean (SEM). A one-way ANOVA Test determined that the differences in SEM were significant. (ThinkQuest 2001) (Cavanihac 2000) Figure 1. Flow chart of key steps in methods.