Immunolocalization of CRES (cystatin-related epididymal spermatogenic) protein in the acrosomes of mouse spermatozoa

The CRES (cystatin-related epididymal spermatogenic) protein is a member of the cystatin superfamily of cysteine protease inhibitors and exhibits high ly restricted expression in the reproductive tract. We have previously show n that: CRES protein is present in elongating spermatids in the testis and is synthesized and secreted by the proximal caput epididymal epithelium. Th e presence of CRES protein in developing germ cells and in the luminal flui d surrounding maturing spermatozoa prompted us to examine whether CRES prot ein is associated with spermatozoa. In the studies presented, indirect immu nofluorescence, immunogold electron microscopy, and Western blot analysis d emonstrated that CRES protein is localized in sperm acrosomes and is releas ed during the acrosome reaction. Interestingly, while the 19- and 14-kDa CR ES proteins were present in testicular and proximal caput epididymal sperma tozoa, the 14-kDa CRES protein was the predominant form present in mid-capu t to cauda epididymal spermatozoa. Furthermore, following the ionophore-ind uced acrosome reaction, CRES protein localization was similar to that of pr oacrosin/acrosin in that it was detected in the soluble fraction as well as associated with the acrosome-reacted spermatozoa. The presence of CRES pro tein in the sperm acrosome, a site of high hydrolytic and proteolytic activ ity, suggests that CRES may play a role in the regulation of intraacrosomal protein processing or may be involved in fertilization.