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(703) ACQUISITION AND UBIQUITINATION OF ARYLSULFATASE A (AsA) DURING EPIDIDYMAL PASSAGE. Baska, Kathleen 1, Thompson, Winston3, Sutovsky, Miriam 1, Sutovsky, Peter1, 2, 1 Department of Animal Science, Columbia, MO3 Department of Obstetrics and Gynecology and Cooperative Reproductive Science Research Center, Atlanta, GA2 Department of Obstetrics and Gynecology, Columbia, MO ABSTRACT- Ubiquitin is a chaperone-protein marking other abnormal proteins or those proteins designated for proteolytic degradation. We hypothesize that during epididymal passage polyubiquitination leads to surface marking of defective spermatozoa, which could serve as an epididymal sperm quality control mechanism. Our goal is to identify the components of the ubiquitin system in epididymal fluid and to identify sperm surface proteins ubiquitinated in defective spermatozoa. Our work suggests that Arylsulfatase A (AsA), a protein secreted by the epididymis and binding to the surface of epididymal spermatozoa, is in fact one of the ubiquitinated substrates within defective epididymal sperm. Immunocytochemistry detected AsA immunoreactivity on both the sperm head acrosome and the principal piece of the sperm tail in bovine spermatozoa. Western blot analysis confirmed the presence of AsA in motile sperm fractions and whole sperm samples, and in lesser quantities in the immotile sperm fraction containing most of the ubiquitinated spermatozoa. AsA was also found to be present in the epididymal fluid collected from the caput and cauda epididymis. This indicates that in addition to intrinsic AsA expression during spermatogenesis in the testis, epididymal spermatozoa take up AsA on their surface during epididymal passage. Western blotting also detected the presence of the ubiquitin-carrier E2 and presumed E2-ubiquitin intermediates suggesting the presence of an active ubiquitination pathway in bovine epididymal fluid. These data contribute to a better understanding of the process of sperm maturation during epididymal passage. Supported by USDA and F21C-UM-C awards to PS. KEY WORDS: sperm, arylsulfatase a, epididymis, ubiquitin |
