The role of the lipid soluble dolichols, long chain polyprenyls with a saturated Alpha-isoprene unit, in the synthesis of N-glycosidically linked glycoproteins during rat spermatogenesis will be investigated. We have identified four potentially key regulatory sites (enzymatic steps). These are the dolichol kinase and dolichyl phosphate phosphatase enzymes which directly control the concentration of dolichyl phosphate in cellular membranes. The levels of dolichyl phosphate then regulate the rate of N-glycosidically linked glycoprotein synthesis. The processing enzymes, both the Alpha-glucosidases and the Alpha-mannosidases, will also be examined. The Alpha-glucosidases may regulate the en bloc transfer of oligosaccharide from the oligosaccharide-lipid (dolichol) to peptide. The Alpha mannosidases determine the type of oligosaccharide produced, i.e., high mannose or complex type. The processing enzymes will be assayed using as substrate both the rho-nitro-phenyl glycoside and 14C-labeled oligosaccharides synthesized in vitro using labeled nucleotide sugars and testicular microsomes. We will characterize each of these enzymes in testicular tissue and optimize assay systems. Following characterization, we will follow these enzymes in the maturing rat from 14 to 60 days of age and correlate activities with the appearance of the various development stages of the spermatogenic cell line. These enzymatic activities will also be measured in purified populations of spermatogenic cells isolated using elutriation and percoll density gradient centrifugation. Since glycoproteins constitute much of both the acrosome and the antigenic components of the cell surface, this study should provide a better understanding of the factors constrolling both acrosome formation and the glycopotein composition of the cell surface. In addition, we will examine the molecular structure of testicular dolichols since some question exists regarding the nature of the Alpha-isoprene unit. Spectral analysis, NMR, IR and mass-spectroscopy, will be conducted on purified testicular dolichols. The distributions of these enzymes in the various cellular membrane systems will also be examined. The long-term goals of studies such as these include defining the enzymatic changes associated with spermatogenesis and the identification eventually of the signals regulating such processes.
| Nyquist, S E; Holt, S (1986) Rat testicular dolichols: distribution and accumulation with age. Biol Reprod 34:89-95 |
| Berkowitz, L; Nyquist, S E (1986) Dolichol kinase activity in the developing rat testis. Biol Reprod 34:518-26 |
