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Localization of a Proteolytic Enzyme Within the Efferent and Deferent Duct Epithelial Cells of the Turkey (Meleagris gallopavo) Using Immunohistochemistry¹

^a Department of Animal and Veterinary Sciences, Clemson University, Clemson, South Carolina 29634 ^b Clemson Institute of Environmental Toxicology, Clemson University, Pendleton, South Carolina 29670

Turkey seminal plasma contains a serine protease found to be distinct from the spermatozoal acrosin. However, the origin and biological roles of this enzyme are unknown. Our experimental objective was to identify the cellular source of this protease within the male reproductive tract. The enzyme was isolated from seminal plasma using benzamidine-Sepharose 6B chromatography. A synthetic substrate, N-benzoyl-DL-arginine p-nitroanilide, was used to detect fractions containing the enzyme. The affinity chromatography technique yielded a 150-fold increase in amidase activity. Analysis of the protease by SDS-PAGE revealed two protein bands with relative molecular masses of 37 000 and 61 000. Proteolytic activity was detected within the smaller band as evidenced by casein digestion. Further analysis of the purified protein revealed that the smaller protein band was glycosylated. To determine the cellular source of the protease, a panel of mouse monoclonal antibodies was then developed against the purified protease, and used in immunohistochemistry. Frozen tissue sections from the liver, testis, epididymal region, and deferent duct were fixed in 4% (w/v) paraformaldehyde, permeabilized with 0.2% (v/v) (octylphenoxy)polyethoxyethanol followed by routine immunohistochemistry procedures. Monoclonal antibodies did not bind to tissue sections from either the liver or testis, or to blood plasma proteins. Both the distal portion of the efferent duct and the deferent duct were immunoreactive. We concluded that the protease found in turkey seminal plasma is concentrated to the distal efferent duct and the deferent duct epithelial cells.

First decision: 7 November 2001.

¹ This work was supported by the U.S. Department of Agriculture, CSREES 99-35203-8161 and by the National Institutes of Health, R15-ES10556-01. Technical Contribution 4735 of the South Carolina Agriculture and Forestry Research System.

² Correspondence: Ronald Thurston, Department of Animal and Veterinary Sciences, Clemson University, Box 340361, Clemson, SC 29634. FAX: 864 656 1033; ront{at}clemson.edu