The primary goal of this renewal proposal is to sufficiently characterize the proacrosin-acrosin system so that definitive information regarding its regulation can be obtained. Both biochemical and physiological approaches will be utilized. The biochemical characterization will first focus on the determination of the amino acid sequence of boar proacrosin and in defining the topography of the active site of acrosin. These latter studies will be accomplished by using affinity labeling reagents to determine which specific amino acids are involved in acrosin's enzymatic activity.
The second aim will be to characterize the precursors to proacrosin found in testis and epididymal boar sperm. This will be done to obtain a thorough understanding of nascent proacrosin so that the system can be quantitated and the regulation of its conversion elucidated. These studies will involve the precursor purification and characterization as well as the in vitro translation of boar testicular proacrosin mRNA. The physiological characterization will focus on the regulation of the proacrosin- acrosin system in live sperm. This will be accomplished by determined the effectiveness that specific inhibitors and antibodies to proacrosin and acrosin have on the sperm's ability to penetrate oocytes. These results will be quantitated by a kinetic enzyme analysis and specific forms that are affected will be identified by a highly sensitive gelatin-SDS-PAGE zymograph. For possible clinical and contraceptive purposes one of the test systems will utilize human sperm and then analyze their penetration of zona-free hamster eggs. A more in depth approach will focus on the well-characterized boar sperm proacrosin-acrosin system and the boar sperm's ability to undergo in vitro capacitation and in vitro fertilization of zona intact porcine ova. This study will result in a more complete understanding of the molecular events required for fertilization and could ultimately lead to possible means of controlling it for either contraceptive or fertility enhancement purposes. In addition, a more thorough comprehension of the regulatory mechanisms of this proteinase system will produce increased understanding of the possible regulation of other proteolytic enzyme systems which control many important biological processes.
