PARENT SESSION
Gonadal Function

LOCALIZING THE SITE OF SYNTHESIS AND CLONING THE GENE FOR A PROTEOLYTIC ENZYME FOUND IN TURKEY SEMINAL PLASMA.

Holsberger, Denise¹, Rice, Charles², Korn, Nancy¹, Thurston, Ronald¹, ^{1 2}

ABSTRACT- Our laboratory previously discovered a seminal plasma protease (TSPE) that is unique to turkeys. The exact role(s) of TSPE in semen is unclear, but it may contribute to factors essential for spermatozoal viability and longevity. Understanding the function of TSPE may lead to improvements in turkey semen preservation. TSPE was isolated from seminal plasma using Benzamidine-Sepharose 6B and Concanavalin A-Agarose affinity chromatography. Fractions containing TSPE were detected by amidase activity using the synthetic substrate N-benzoyl-DL-arginine-p-nitroanilide. Purification resulted in two protein subunits with approximate molecular weights of 40 and 66 kDa, as determined by SDS-PAGE. Further analysis of the TSPE subunits using thymol-sulfuric acid glycoprotein detection and casein zymogen gel electrophoresis revealed that the 40 kDa subunit is glycosylated and has proteolytic activity. Purified TSPE was used to produce two monoclonal antibodies, B3F6 and A4C6, which are specific for the 40 and 60 kDa subunits respectively. The testis, epididymal region, and liver tested negative for TSPE using immunohistochemistry. Blood serum was also negative for the TSPE protein. In contrast, the epithelium of the ductus deferens was strongly positive for TSPE while the epithelium of the ductus efferentes showed a lesser degree of binding of the aforementioned monoclonal antibodies. A cDNA library was constructed using mRNA isolated from turkey ductus deferens. The constructs will be screened using the monoclonal antibodies A4C6 and B3F6 in order to clone, sequence, and characterize the gene for TSPE. (Supported by USDA-CSREES #99-35203-8161 and NIH-R15-ES10556-01).

KEY WORDS: Meleagris gallopavo, seminal plasma, protease