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During spermatogenesis, spermatids synthesize constituent proteins present in mature spermatozoa; however, little information exists on the molecular processes involved. In previous studies, this laboratory reported the characterization of rat sperm β-d-galactosidase. In this paper, we report the localization of this enzyme along with its biosynthesis and processing. An antibody against rat luminal fluid β-d-galactosidase was used to immunolocalize the enzyme in the testis and in epididymal spermatozoa. We found that β-d-galactosidase is localized within the acrosomal cap of spermatids and in the acrosome and cytoplasmic droplet of epididymal spermatozoa. A combination of germ cell radiolabeling, immunoprecipitation, SDS-PAGE, and autoradiography revealed that spermatids produce two forms of β-d-galactosidase, 90 and 88 kDa. During pulse-chase analysis, a 56-kDa form appeared. Treatment of β-d-galactosidase immunoprecipitates from testicular spermatozoa with N-glycanase or Endo H revealed that bo...

In previous studies, we reported that rat epididymal fluid acid beta-D-galactosidase, which optimally cleaves a synthetic substrate (PNP beta-D-galactoside) at pH 3.5, shows maximum activity at pH 6.8 when a glycoprotein is used as a substrate [Skudlarek MD, Tulsiani DRP, Orgebin-Crist M-C. Biochem J 1992; 286: 907-914]. We now describe a similar pH-dependent substrate preference for rat sperm beta-D-galactosidase. We found that only 10-14% of total beta-D-galactosidase (and other glycosidase) activity was associated with spermatozoa. The remaining enzyme activities were present in soluble form in the luminal fluid. When the glycosidase levels were expressed per 10(6) sperm, all enzymes showed a progressive increase in spermatozoa from the caput to the corpus or proximal cauda followed by a sharp decline in spermatozoa from the distal cauda epididymidis. The observed decrease in beta-D-galactosidase activity could not be explained by the loss of cytoplasmic droplets (which have a low enzyme activity relative to spermatozoa) or the presence of inhibitors/activators of the enzyme activity in spermatozoa from the proximal or distal epididymis. However, we found that the changes in beta-D-galactosidase activity during sperm maturation in the epididymis were accompanied by changes in the molecular form(s) of the enzyme. Western blot analysis using an antibody to beta-D-galactosidase showed a progressive processing of the 82-kDa immunoreactive band in caput spermatozoa to an 80-kDa diffuse band in cauda spermatozoa. The sperm-associated beta-D-galactosidase form(s) does not appear to be due to adsorption and/or binding of the luminal fluid beta-D-galactosidase, which contained a 97-kDa form in fluid from the caput and two forms, of 97 kDa and 84 kDa, in corpus and cauda fluids. The observed difference in the molecular forms of the sperm and luminal fluid was found to be due to differential glycosylation, since de-N-glycosylation of various forms of beta-D-galactosidase generated a single immunoreactive form of 70 kDa. Subcellular localization studies and assay for the beta-D-galactosidase activity in the enriched plasma membrane and acrosomal membrane fractions suggested the likelihood that the activity of beta-D-galactosidase and other glycosidases is present in the acrosome and is readily released during sperm disruption. The evidence suggests that sperm beta-D-galactosidase may be functional within the acidic environment of the acrosome during sperm maturation as well as in the neutral environment of the oviduct after the zona-induced acrosome reaction.

Early studies have shown that spotted locoweed (Astragalus lentiginosus) has an adverse effect on male reproduction. Rams fed locoweed showed a reduced number of primary and secondary spermatocytes and spermatids in the testis, and of spermatozoa in the epididymis and vas deferens. In addition, the Sertoli cells and other epithelial cells were severely vacuolated. Swainsonine, an indolizidine alkaloid, has been identified as the sole or principal toxin in locoweed and perhaps in the plants of genus Swainsona. The toxin is an inhibitor of lysosomal alpha-D-mannosidase, cytosolic alpha-D-mannosidase, and Golgi mannosidase II. The in vitro and in vivo inhibition of Golgi mannosidase II induces the production of abnormal glycoproteins. Since epididymis-mediated modifications of sperm-surface glycoproteins are believed to be important for sperm-egg interactions, we initiated studies to determine effects of swainsonine on processing and catabolism of N-linked glycoproteins in male reproductive tissues. The results presented in this report indicate that feeding of the alkaloid led to accumulation of mannose-rich oligosaccharides (OS) in the testis and epididymis of rats. The major OS was purified from the reproductive tissues of swainsonine-fed rats, and its structure was deduced by comparison of the size of the OS before and after treatment with jack bean alpha-D-mannosidase, and by affinity column chromatography. In addition, the rat epididymal epithelial cells produced abnormal glycoproteins when cultured in the presence of the toxin. This result provides indirect evidence for the presence of a swainsonine-sensitive mannosidase II-like processing enzyme in the epididymal epithelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Previous studies from this laboratory and others have identified several enzymes on the surface of mammalian spermatozoa. Some of these enzymes, namely a galactosyltransferase and a novel alpha-D-mannosidase, are believed to play a ligand-like role in recognizing and binding to the complementary moiety(ies) present on zona pellucida glycoconjugates. However, little or no information is available about the occurrence of these enzymes in human spermatozoa. In the present report, we show that a very small amount of the total galactosyltransferase activity present in human semen is associated with spermatozoa. Moreover, our failure to find a significant amount of the enzyme on sperm plasma membranes suggests that the enzyme is not associated with the sperm surface. Therefore, it is unlikely that galactosyltransferase in humans has the same ligand-like role in zona binding that is demonstrated in mouse sperm. In contrast, nearly 5% of alpha-D-mannosidase activity was repeatedly found in the salt-washed plasma membrane fraction. The recovery and enrichment of the alpha-D-mannosidase was nearly one-half that observed for adenylate cyclase and nearly one-third that for phosphodiesterase I, the two sperm plasma membrane marker enzymes. The differential enrichment and recovery of the sperm surface alpha-D-mannosidase is consistant with our previous studies in rat spermatozoa, and suggests that alpha-D-mannosidase may be localized on morphologically distinct region(s) of the sperm plasma membranes. The properties of human sperm surface alpha-D-mannosidase are quite similar to those reported by us for rat sperm plasma membrane mannosidase, but quite different from human sperm acid alpha-D-mannosidase. In addition, whereas anti-rat epididymal alpha-D-mannosidase antibody (IgG-fraction) cross-reacted with the human sperm acid alpha-D-mannosidase, no cross-reactivity was observed with the sperm surface mannosidase. A small amount of fucosyltransferase (less than 1% of the enzyme originally present on spermatozoa) was found in the salt-washed plasma membrane, but the enrichment of the enzyme was only one-tenth of that observed for adenylate cyclase. The potential ligand-like role of human sperm surface alpha-D-mannosidase and other sperm surface enzymes during fertilization is discussed.

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.