Characterization of a Vertebrate Sperm Chemoattractant

Arizona State University, Scottsdale AZ

Investigators

Abstract

0130001 Chandler Critical steps in sperm-egg interaction during fertilization in animals include initiation of sperm motility, sperm chemotaxis toward the egg, sperm penetration of the extracellular matrix layers surrounding the egg, and binding and fusion of sperm with the egg plasma membrane. This proposal focuses on the important step of sperm chemotaxis, a process that has been widely studied in invertebrates such as the sea urchin but which has received little attention in vertebrates including mammals and humans. Dr. Chandler and Collaborators have now purified and sequenced allurin, a 21 kD protein obtained from the egg jelly layers of Xenopus laevis that represents the first vertebrate sperm chemoattractant to be isolated. Phylogenetic analysis of the allurin sequence suggests that this protein is related to the cysteine-rich secretory protein (CRISP) family of mammals, members of which are known to bind to sperm. They propose to molecularly clone the cDNA coding for allurin, to express recombinant allurin in bacteria, and to determine whether native and recombinant allurin both have chemoattractant activity for Xenopus sperm. Chemoattractant activity will be quantitated in vitro using a two chamber assay in which sperm cross a porous polycarbonate membrane barrier, and sperm will be observed microscopically by using a "choice" assay in which sperm swimming behaviors and trajectories are monitored by video microscopy as they approach agar wells filled with the chemoattractant protein. In addition, they plan to study the mechanism by which allurin acts on sperm with goals of 1) identifying and characterizing a sperm-surface receptor for allurin, and 2) determining what types of intracellular signals are elicited in sperm by allurin. Finally, they will determine the range of allurin expression in both reproductive and non-reproductive tissues. Protein expression will be assessed by immunocytochemistry and Western blotting while expression of mRNA coding for allurin will be detected by Northern blots and in situ hybridization. They anticipate that allurin-like proteins will be found in the reproductive tracts of both female and male amphibians.