The overall objective of our studies is to determine the role of the novel CRES (cystatin-related epididymal-specific) protein in male reproductive function. The CRES protein exhibits a remarkably restricted pattern of expression and is highly regulated at the mRNA level by testicular factors. Of the 25 tissues examined, the male-specific CRES mRNA/protein has been localized to only the very proximal region of the caput epididymidis, the round/elongating spermatids of the testis, and to specific cells in the pituitary. The dramatic appearance and disappearance of the CRES protein in the testis and epididymis suggests that the protein may perform a specialized role during a very discrete phase of testicular and epididymal function. Furthermore, the homology of the CRES protein to the cystatin family of cysteine protease inhibitors, all of which are secretory binding proteins, suggests that it also may have protein binding activity. Indeed our preliminary studies suggest that CRES protein binds specifically to the head of the proximal caput epididymal spermatozoa in a very transient interaction. Therefore, our hypothesis is that the CRES protein may be a regulatory binding protein necessary for spermatogenic and sperm maturational events. Specifically, the CRES protein may bind to a sperm-associated protein, perhaps a protease. This binding may then result in the "activation" of the binding partner which subsequently may be critical for spermatogenic and sperm maturational events. To test our hypothesis we will: 1) extend our CRES protein localization studies by examining the cellular and intracellular localization of the CRES protein in the testis, epididymis, pituitary, and spermatozoa at the light and electron microscopic levels; 2) Isolate the rat CRES cDNA and prepare a rat CRES antibody for characterization of the rat CRES gene and protein; 3) begin CRES protein characterization studies by examining post-translational modifications of the CRES protein and cystatin activity; and 4) examine the interaction of CRES protein with mouse and rat sperm and initiate studies to identify the CRES protein binding partner. These studies will provide valuable information on a novel epididymal protein which may be important for sperm development and maturation.