Glycosidases in Cultured Rat Epididymal Cells: Enzyme Activity, Synthesis and Secretion

Abstract

During transit through the epididymis, spermatozoa acquire fertilizing the cell surface exhibits an altered glycoprotein pattern. Epididymal cells and their secretions contribute to these sperm-surface changes. To examine this process, epithelial cells from rat caput and cauda epididymidis were cultured and examined for the synthesis, processing and secretion of two glycoprotein-modifying enzymes, beta-galactosidase and beta-glucuronidase. Cells were cultured four days, incubated with D-2-[3H] mannose and L-[35S] methionine, and placed in isotope-free media. Levels of both cellular and secreted beta-galactosidase and beta-glucuronidase were determined by immunoprecipitation of cell homogenates or medium, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and scintillation counting of bands. During a 1-h pulse, both caput and cauda cells synthesize two precursor forms of beta-galactosidase (Mr = 84,000 and 87,000), which are processed to the mature (Mr = 63,000) enzyme during a 24-h chase. Caput cells release a high molecular weight (HMW) form (Mr = 90-100,000) and mature beta-galactosidase into the media, but not the Mr = 84-87,000 precursor. On the other hand, cauda cells release mostly mature beta-galactosidase. Ratios of radiolabeled mannose/methionine demonstrate a 7-fold greater mannose content in the cellular precursor of beta-galactosidase than in total protein. Another glycosidase, beta-glucuronidase, is synthesized as a Mr = 78,000-precursor which is processed to the mature Mr = 72,000 form. Medium in which caput and cauda cells were cultured contains both mature enzyme and a Mr = 94,000 form, but no 78,000-precursor form. Ratios of radiolabeled mannose/methionine in the cellular precursor of beta-glucuronidase are 2-fold greater than ratios in the total glycoprotein. Secretion is the major pathway of turnover for several epididymal glycosidases, since more than 50% of the total is secreted/day. These results indicate that cultured epithelial cells from the epididymis synthesize glycosidases and that processing and release differ, depending on the enzyme and the epididymal segment from which the epithelial cells were isolated.

Effect of swainsonine on rat epididymal glycosidases

“…Glycosidases have been identified in the epididymis (Conchie et ai, 1959) and in isolated rat epididymal epithelial cells (Kemp & Killian, 1978;Chapman & Killian, 1984;Skudlarek & Orgebin-Crist, 1986). The rat epididymis contains specific activity concentrations of glycosidases up to 90-fold greater than in other tissues (Skudlarek & Swank, 1981).…”

“…However, we have shown that both ß-glucuronidase and ß-galactosidase are synthesized in higher molecular weight precursor forms by cultured rat epididymal epithelial cells, processed to mature enzyme, and released into the extracellular medium (Skudlarek & Orgebin-Crist, 1986). These epididymal glycosidases may be involved in the modification of the sperm surface glycoproteins during epididymal transit.…”

“…For example, in-vivo feeding with swainso¬ nine increases a-D-mannosidase concentration in rat liver, brain and kidney, in pig liver and brain, but not in pig kidney (Tulsiani & Touster, 1983a;Tulsiani et ai, 1984;Novikoff, et ai, 1985). The epididymis is a rich source of glycosidases (Conchie et ai, 1959;Kemp & Killian, 1978;Skudlarek & Swank, 1981;Chapman & Killian, 1984;Skudlarek & Orgebin-Crist, 1986) and epididymal epithelial cells in culture release a-mannosidase, ß-glucuronidase and ß-galactosidase (Skudlarek & Orgebin-Crist, 1986). The present study examines the effect of swainsonine on these 3 epididymal glycosidases.…”

A 5-Kilobase Pair Promoter Fragment of the Murine Epididymal Retinoic Acid-binding Protein Gene Drives the Tissue-specific, Cell-specific, and Androgen-regulated Expression of a Foreign Gene in the Epididymis of Transgenic Mice

“…The CAT reporter gene was preferred to the lacZ reporter gene due to the high endogenous ␤-galactosidase activity present in the epididymis (26,27), which cannot be suppressed by the usual modification of…”

Mannose-6-Phosphate Receptors as a Molecular Indicator of Maturation of Epididymal Sperm

“…The epididymis is particularly rich in acid hydrolases, together with a developed and active lysosomal apparatu s [64,74]. In mice for instance, the highest levels of a-mannosidas e and b-hexosamini dase are expressed in epididymis [31].…”