Transcriptional regulation of the cystatin-related epididymal spermatogenic (Cres) gene in the reproductive tract and the identification of novel Cres-like genes

An essential event in male reproduction is formation of functionally mature spermatozoa. Testicular spermatozoa are non-functional and require critical post-testicular modifications in the long convoluted tubule known as the epididymis. Our laboratory previously identified a gene potentially involved in sperm maturation we called cystatin-related epididymal spermatogenic {Cres) which was restricted in its expression to the initial segment epididymidis. Subsequently, it was determined that CRES protein is secreted into the initial segment epididymal lumen and is present in sperm acrosomes and anterior pituitary gonadotrophs. CRES and recently identified CRES-Iike proteins appear to represent a unique subgroup in the cystatin superfamily of cysteine protease inhibitors based on the highly reproductive-specific expression and their divergence in key sequences found in classical cystatins. Recent studies in our laboratory suggest that CRES acts as a cross-class inhibitor of the serine protease prohormone convertase 2 (PC2), a protease involved in prohormone processing. Therefore, CRES may regulate specific proteolytic processing in reproductive tissues.

One aspect of understanding sperm maturation is to identify the molecular mechanisms which control the reproductive-specific expression of genes important in reproductive processes. The goal of these transcriptional regulation studies was to identify transcription factors and their cognate motifs that are critical for the mRNA expression of the Cres gene. Analysis of DNA-protein interactions demonstrates that CC/V\T/enhancer binding protein p transcription factor is required for basal levels of Cres gene expression in the epididymis and the anterior pituitary gonadotrophs. To begin to identify the Cres 5'-flanking sequences necessary for Crestspecific expression, we generated transgenic mice that contained approximately 1.6 kb of C/-es5'-flanking sequence driving the chloramphenicol acetyltransferase (CAT) gene. These studies demonstrated that 1.6 kb of Cres 5'-flanking sequence specifically drives CAT expression to the round spermatids of the testis but not in any other tissues including the epididymis and pituitary. Therefore, this promoter sequence can recapitulate the pattern of Cres-specific expression in the testis.

The reproductive-specific expression of Cres gene and two other genes similar to Cres, cystatin T and testatin, suggests there may be a group of Creslike genes that function primarily in the reproductive tract, much like the growing number of ADAM (A Disintegrin And Metalloprotease) proteins in the reproductive tract. By screening nucleotide databases, we identified two new members of the Cres subgroup of family 2 cystatins, called Cres2 and Cres3 and demonstrated they exhibit reproductive-specific expression. Cres2 mRNA expression is epididymal-specific, whereas the Cres3 mRNA is present in the testis, epididymis, and ovary.

Taken together, these studies demonstrate that epididymal and pituitary transcription of the Cres gene requires the C/EBPp transcription factor for basal levels of expression; however, testicular expression is not dependent on C/EBPs. Interestingly, Cres mRNA expression can be recapitulated only in the testis with 1.6 kb of the Cres promoter, suggesting the elements that control epididymal and pituitary expression of the Cres gene are either not contained in the 1.6 kb 5'- flanking region or perhaps repressors elements within this fragment suppress expression. In addition, we have identified two new members of the Cres subgroup of family 2 cystatins that show restricted expression to reproductive tissues, suggesting these genes have diverged not only in function but also in their transcriptional regulation as compared to the ubiquitously expressed cystatins. Therefore, multiple Cres-\ke genes may have evolved to have specific and overlapping roles in proprotein processing imperative for reproductive processes.