Mechanisms of Calcium Release and Egg Activation in Chaetopterus

Howard University, Washington DC

Investigators

Abstract

This research will identify intracellular mechanisms involved in the initiation of development after fertilization by determining the mechanisms responsible for Ca 2+ oscillations in fertilized eggs. Eggs of the marine invertebrate, Chaetopterus, will be used as a model to study ligand-gated Ca 2+ release mechanisms and to perform correlated in vivo and in vitro studies to identify which is/are responsible for Ca 2+ oscillations in fertilized and parthenogenetically- activated eggs. Synthesis of the endogenous ligands for these channels will also be studied. The results of these studies may have great significance for the control of fertility because they will contribute important information to our understanding of mechanisms by which sperm initiate the development of the egg. They will directly test hypotheses concerning the mechanisms by which Ca 2+ becomes available to activate eggs and other cells in response to extracellular signals. Since the organism to be studied is evolutionarily very distant from frogs and sea urchins, the results will provide important data as to the general applicability of mechanisms of egg activation. These eggs undergo Ca 2+ oscillations upon fertilization as do those of mammals and many, possibly most, other organisms with the exceptions of frogs and sea urchins. Therefore, these data are more likely to inform us of mechanisms of fertilization in other organisms, including mammals, than would data obtained from sea urchin and frog eggs. Lastly, since these Ca 2+ release mechanisms are also involved in other cells in response to other signals, these results will also have broad relevance to mechanisms by which other cells respond to extracellular signals. The hypotheses to be tested are: 1) that inositol 1,4,5-trisphosphate is responsible for Ca2+ oscillations and egg activation at fertilization, and 2) that other small ligands such as cyclic ADP-ribose or nicotinic acid adenine dinucleotide phosphate also contribute to these oscillations. The procedures to be used will include: 1) fluorescence spectrometry to assay Ca2+ release and sequestration in vitro, 2) fluorescence and confocal microscopy to examine the movements of Ca 2+ within eggs as a function of activation by sperm or parthenogenetic agents, 3) immunofluorescence microscopy to localize endogenous ligand-gated Ca2+ release channels in the eggs, 4) western blotting to identify the proteins that make up these channels, and 5) light microscopy as a means to determine the extent of egg activation and development. This study will also integrate research and education as well as broaden the participation of underrepresented minorities (African-Americans) in scientific research. The applicant institution is a historically Black university, and the supported graduate student will be African-American. This individual's training will be enhanced by exposure to the environment of the Marine Biological Laboratory, Woods Hole, MA.