Rat spermatogenic cell beta-D-galactosidase: characterization, biosynthesis, and immunolocalization.

In this study we have demonstrated that the rat sperm acrosomal beta-d-galactosidase is expressed in late spermatocytes and spermatids (round, elongated/condensed) during spermatogenesis. The enzyme is an exoglycohydrolase which, along with other exoglycohydrolases and proteases, is thought to aid in penetration of the zona pellucida, the extracellular glycocalyx that surrounds the mammalian egg. The presence of the enzyme in spermatocytes was confirmed by multiple approaches using biochemical, biosynthetic, and immunohistochemical protocols. The germ cells (spermatocytes, round spermatids, and elongated/condensed spermatids), purified from rat testis, were found to contain beta-galactosidase and four other glycohydrolases (beta-d-glucuronidase, alpha-d-mannosidase, alpha-l-fucosidase, and beta-N-acetylglucosaminidase). With the exception of alpha-l-fucosidase, the other enzymes assayed demonstrated a two- to threefold higher activity per cell in spermatocytes than in round spermatids. Immunoblotting approaches of affinity-purified germ cell extracts demonstrated several molecular forms of beta-galactosidase in spermatocytes and round spermatids; one of these forms (62 kDa) was seen only in round spermatids. The biosynthetic approach demonstrated that the enzyme is synthesized in spermatocytes and round spermatids in culture in high-molecular-weight precursor forms (90/88 kDa) which undergo processing to lower molecular weight mature forms in a cell-specific manner. The net result is the formation of predominantly 64- and 62-kDa forms in spermatocytes and round spermatids, respectively. The conversion of precursor forms to mature forms in the diploid and haploid cells in culture is rapid with t(1/2) of 6.5 and 9.0 h, respectively. Immunohistochemical approaches revealed an immunopositive reaction in the Golgi membranes, Golgi-associated vesicles, and lysosome-like structures in the late spermatocytes and early round spermatids. The forming/formed acrosome in round and elongated spermatids was also immunoreactive.

Biosynthesis, processing, and subcellular localization of rat spermbeta-D-galactosidase.

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 beta-D-galactosidase. In this paper, we report the localization of this enzyme along with its biosynthesis and processing. An antibody against rat luminal fluid beta-D-galactosidase was used to immunolocalize the enzyme in the testis and in epididymal spermatozoa. We found that beta-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 beta-D-galactosidase, 90 and 88 kDa. During pulse-chase analysis, a 56-kDa form appeared. Treatment of beta-D-galactosidase immunoprecipitates from testicular spermatozoa with N-glycanase or Endo H revealed that both the 90- and 88-kDa forms become a 70-kDa polypeptide on SDS-PAGE. Since Endo H or N-glycanase treatment provided similar results, the presence of extensive N-linked high mannose/hybrid-type glycans on these proteins is indicated. Treatment of the 56-kDa form of beta-D-galactosidase with Endo H or N-glycanase resulted in the appearance of 52- and 50-kDa forms, respectively. This result suggests that the 56-kDa form contains N-linked high mannose/hybrid as well as complex oligosaccharides. During epididymal maturation, the 90-kDa form of beta-D-galactosidase persists in caput epididymal spermatozoa and is gradually converted to a major 74-kDa form in cauda spermatozoa. In addition to the 90- to 74-kDa forms, cauda spermatozoa show a 56- to 52-kDa form on Western immunoblots. Since only the high-molecular weight forms of beta-D-galactosidase are present on immunoblots of isolated sperm heads, we suggest that they are acrosomal in origin and that the 56-kDa form, which is processed to 52 kDa in cauda spermatozoa, is associated with the cytoplasmic droplet.

Identification and androgen regulation of egasyn in the mouse epididymis.

The expression and androgen regulation of egasyn, the endoplasmic reticulum-targeting protein of beta-D-glucuronidase, was examined in the mouse-epididymis. The proximal (caput) and distal (corpus & cauda) epididymal tissue extracts were prepared by homogenization and sonication in buffered Triton X-100 solution, and high speed centrifugation. The supernatant when resolved by 2D-PAGE under non-denaturing conditions and stained for esterase activity showed that the distal (but not proximal) epididymis of the normal mouse contain several specific forms of esterases. These forms include a series of four variants (pI 5.2-5.75) with high mobility (HM) and esterase activity, and three faintly staining variants (beginning at pI 6.0) with low mobility (LM). Several lines of evidence indicate that the specific esterases seen in the corpus/cauda epididymidis are egasyn-esterases. Firstly, these molecular forms were not seen in the distal epididymal extracts from the egasyn-deficient mouse. Secondly, the HM forms can be immunoprecipitated with anti-egasyn antibody, suggesting the presence of free egasyn. Finally, the LM forms disappeared after heat treatment (56 degrees C for 8 min), a condition known to dissociate egasyn:beta-D-glucuronidase complex. This result indicates that a small amount of egasyn is complexed with beta-D-glucuronidase. Immunoblotting (Western blot) studies (using anti-egasyn antibody) following resolution of egasyn released from the egasyn:beta-D-glucuronidase complex revealed a single band of an apparent molecular weight 64 kDa in the distal (but not proximal) epididymis, indicating that the mouse epididymal egasyn is identical or very similar to the liver egasyn. Castration of mice lead to the appearance of free and complexed egasyn forms in the proximal epididymis. Testosterone supplementation to the castrated mice resulted in the disappearance of the induced egasyn forms from the caput epididymidis. Taken together, these results indicate that the expression of egasyn in the epididymis is region-specific and is differentially regulated by androgens.

Role of luminal fluid glycosyltransferases and glycosidases in the modification of rat sperm plasma membrane glycoproteins during epididymal maturation.

It is generally accepted that mammalian spermatozoa undergo biochemical and morphological changes during epididymal transit, collectively termed epididymal maturation. Although many details of the biochemical modification are not fully understood, lectin binding studies from several laboratories strongly suggest that glycan moieties of sperm plasma membrane glycoproteins are extensively modified as spermatozoa transit from the proximal to the distal epididymis. In the present article, we summarize our studies with two sets of glycan modifying enzymes, namely glycosyltransferases (synthetic enzymes) and glycosidases (hydrolytic enzymes) in rat spermatozoa collected from different regions of the epididymis, and similar enzyme activities present in the epididymal luminal fluid. Our data show that the activities of these enzyme are high in the epididymal luminal fluid (> 80% of the total enzyme activities was in the plasma). Evidence presented in this report also demonstrates that: (1) at least one sperm surface glycoprotein (apparent molecular mass of 86 kDa) is fucosylated in vitro when caput spermatozoa are incubated with GDP [14C]fucose; and (2) a peanut agglutinin (PNA)-positive glycoprotein of 135-150 kDa present on plasma membrane of sperm from the caput (but not cauda) epididymidis is degalactosylated by digestion with purified luminal fluid beta-D-galactosidase. Taken together, these results strongly suggest a role for glycoprotein modifying enzymes in the modification of sperm surface glycoproteins during epididymal maturation.

Androgen regulation of molecular forms of beta-D-glucuronidase in the mouse epididymis: comparison with liver and kidney.

The expression and androgen regulation of beta-glucuronidase molecular forms were examined in mouse epididymis, liver, and kidney. Two-dimensional polyacrylamide gel electrophoresis performed under nondenaturing conditions showed that, compared to liver and kidney, which contain four microsomal (M1-M4) and a major lysosomal (L) form of beta-glucuronidase, the epididymis revealed regional differences and tissue specificity in the expression of the various molecular forms of the enzyme. Only the lysosomal form (pI 5.4-6.1) is present in the caput epididymidis while the corpus/cauda contains the lysosomal form, the free X form (pI 5.9-6.3) and the four microsomal forms (X form complexed with egasyn). Mutant mice that lack egasyn have no microsomal forms in the distal epididymis. In epididymal fluid, the lysosomal form is found throughout the epididymis, whereas the X form appears only in the corpus/cauda epididymidis. Sodium dodecyl Sulfate (SDS)-gel electrophoresis and western blot analysis of immunoprecipitated beta-glucuronidase revealed only one band corresponding to the L form (apparent molecular weight 74 kDa) in the caput epididymidis and two bands in the corpus/cauda (apparent molecular weights 73 and 75 kDa), corresponding to L and X forms, respectively. Castration of mice led to the suppression of the regional differences in the appearance of X and M forms in the epididymis. Testosterone supplementation to castrated mice restored the characteristic electrophoretic pattern of beta-glucuronidase in the caput epididymidis. In the liver and kidney, castration has no effect on the expression of the molecular forms, whereas androgen treatment induced the X form in the kidney. Histochemical localization of beta-glucuronidase confirmed the region specificity seen in the epididymis and in addition revealed cell specificity in the expression of beta-glucuronidase. These results indicate that beta-glucuronidase shows tissue specificity and, in the case of the epididymis, region and cell specificity. In addition, the enzyme in the different tissues responds differentially to androgens.

Rat sperm plasma membrane mannosidase: localization and evidence for proteolytic processing during epididymal maturation.

Previous studies from this laboratory have identified a novel alpha-D-mannosidase on the sperm plasma membranes of several species, including man, which may have a role in fertilization. The polyclonal antibody raised against an isoform of the enzyme purified from rat epididymal fluid was found to cross-react with the alpha-D-mannosidase activity present in the detergent-solubilized spermatozoa and sperm plasma membranes. In the present study, we have used affinity-purified as well as monospecific anti-mannosidase IgG to demonstrate that the sperm mannosidase is an integral plasma membrane component of the rat sperm and is localized on the periacrosomal region of the sperm head. In addition, we demonstrate proteolytic processing of the membrane-bound alpha-D-mannosidase during maturation of spermatozoa. The membrane fractions prepared from testis, and spermatozoa from the caput, corpus, and cauda regions of the epididymis, were solubilized in SDS and resolved by SDS-PAGE. The resolved polypeptides, when subjected to Western blot analysis using affinity-purified anti-mannosidase IgG as the primary antibody, revealed the presence of three specific immunoreactive bands (apparent M(r), 135, 125, and 115 kDa) in the membranes from testis, caput, and corpus spermatozoa. However, the cauda sperm plasma membranes showed only one immunoreactive band of apparent M(r) 115 kDa. The disappearance of the 135-and 125-kDa forms and the appearance of a sharp 115-kDa band on cauda spermatozoa suggests a precursor-product relationship between various molecular forms of the enzyme. Trypsin treatment of testicular and caput sperm membranes largely converted the precursor forms to the mature (115-kDa) form. The in vitro proteolysis resulted in an elevated level of the alpha-D-mannosidase activity in the caput (but not cauda) sperm plasma membrane. Inclusion of trypsin inhibitors (benzamidine and aprotinin) largely prevented the conversion of precursor form to the mature form. These data are consistent with the observed increase in the levels of sperm enzyme activity as spermatozoa move from the caput to the cauda region and suggest that the increase is due to the conversion of enzymatically inactive/less active high molecular weight precursor forms (135 and 125 kDa) into enzymatically active mature form (115 kDa) during sperm maturation.

Purification and characterization of two forms of beta-D-galactosidase from rat epididymal luminal fluid: evidence for their role in the modification of sperm plasma membrane glycoprotein(s).

Previous studies from this laboratory have identified rat epididymal luminal fluid acid beta-D-galactosidase activity which also optimally hydrolyses a glycoprotein substrate at neutral pH [Skudlarek, Tulsiani and Orgebin-Crist (1992) Biochem. J. 286, 907-914]. We have now separated the luminal fluid beta-D-galactosidase into two molecular forms by ion-exchange chromatography on a column of DE-52. The separated enzyme activities were purified to an apparent homogeneity by molecular-sieve chromatography followed by affinity chromatography on a column of immobilized p-nitrophenyl beta-D-thiogalactopyranoside. The purified forms, when resolved by SDS/PAGE under reducing conditions, showed apparent molecular masses of 84 and 97 kDa. Kinetic studies, including a pH-dependent substrate preference and pH-dependent association/dissociation, disclosed no differences between these two forms. The two forms had identical N-terminal amino acid sequences. However, the 97 kDa form contained much more total carbohydrate and sialic acid than the 84 kDa form. The carbohydrate moieties in the two forms were assessed by comparing their size on SDS/PAGE before and after treatment with endo-enzymes. The removal of N-linked glycans by treatment with N-glycanase or endoglycosidase F generated de-N-glycosylated polypeptides of an apparent molecular mass of 70 kDa, and indicated that the two forms contained varying amounts of asparagine (N)-linked high mannose/hybrid-type and biantennary complex-type oligosaccharides. This result and the fact that the two molecular forms had identical N-terminal amino acid sequences indicated that the two forms probably have identical or very similar polypeptides. The potential role of the enzyme in modification of sperm plasma membrane (PM) glycoproteins was examined by resolving caput sperm PM proteins (before and after treatment in vitro of the membranes with the purified beta-D-galactosidase) on SDS/PAGE, followed by staining with peanut agglutinin (PNA), a lectin which preferentially binds to Gal beta 1,3GalNAc-linkages found in O-linked glycoproteins. The evidence presented in this report has indicated that a PNA-positive glycoprotein of an apparent molecular mass of 135-150 kDa present on the caput (but not cauda) sperm PM is degalactosylated by the digestion in vitro of the membranes with purified luminal fluid beta-D-galactosidase. This result suggests a possible role for the epididymal luminal fluid beta-D-galactosidases.

Beta-D-galactosidase of rat spermatozoa: subcellular distribution, substrate specificity, and molecular changes during epididymal maturation.

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.

Purification and characterization of rat epididymal-fluid alpha-D-mannosidase: similarities to sperm plasma-membrane alpha-D-mannosidase.

We have previously reported the occurrence and partial characterization of a novel alpha-D-mannosidase activity on rat sperm plasma membranes [Tulsiani, Skudlarek and Orgebin-Crist (1989) J. Cell Biol. 109, 1257-1267]. Here, we report the presence of a similar alpha-D-mannosidase activity in a soluble form in rat epididymal fluid. The soluble enzyme was purified nearly 500-fold with 9-12% recovery to a state approaching homogeneity using: (1) (NH4)2SO4 precipitation; (2) affinity chromatography on immobilized mannan and D-mannosamine; (3) ion-exchange (DE-52) column chromatography; (4) molecular-sieve chromatography. The enzyme was eluted from the final column (Sephacryl S-400) at an apparent molecular mass of 460 kDa. When resolved by SDS/PAGE (under denaturing conditions), the enzyme showed a major protein band (115 kDa) and few very minor bands. The polyclonal antibody raised against the major protein band was found to cross-react with the alpha-D-mannosidase activity present in epididymal fluid (soluble) and detergent-solubilized spermatozoa from the rat and mouse. This result suggested that the soluble and membrane-bound enzyme activities shared a common antigenic site(s). The antibody was used to characterize further the alpha-D-mannosidase activity(ies) present in the rat epididymal fluid and rat sperm plasma membranes. Data from these studies show that the two forms are similar in (a) subunit molecular mass, (b) substrate specificity and (c) inhibitory effect of several sugars. These similarities suggest that the soluble and membrane-bound alpha-D-mannosidase activities are isoforms. Immunoprecipitation studies after solubilization of the testis and epididymal particulate fraction from sexually immature rats show that the testis (but not the epididymis) contains the immunoreactive alpha-D-mannosidase activity. This result and the fact that spermatozoa from the rat rete testis show alpha-D-mannosidase activity indicate that the sperm enzyme is synthesized in the testis during spermatogenesis.

Glycosylation of rat sperm plasma membrane during epididymal maturation.

Spermatozoa acquire fertilizing ability during passage through the epididymis. Modification of oligosaccharide moieties on sperm surface glycoproteins are some of the biochemical changes believed to be important in the production of functionally mature spermatozoa during passage through the epididymis. In an attempt to understand the mechanism underlying these modifications, we quantified four glycosyltransferase activities (the enzymes that catalyze the transfer of sugar residues from nucleotide sugar donor to the sugar chains on glycoproteins and glycolipids) of spermatozoa and fluid from various regions of the epididymis. Our results are as follows. (1) Only 10-20% of the total glycosyltransferase activities (sialyltransferase, fucosyltransferase, galactosyltransferase, and N-acetyl glucosaminyltransferase) sedimented with the spermatozoa; the remaining 80-90% of the four enzymes were present in soluble form in the epididymal fluid. (2) When the four transferase activities were expressed per 10(6) spermatozoa, only sialyltransferase and fucosyltransferase activities showed maturation-dependent changes. The former enzyme was significantly higher on the proximal caput spermatozoa and the latter on the distal caput spermatozoa. The higher levels of the two enzymes on caput spermatozoa could be due to their binding to the endogenous sugar acceptor molecules on the sperm surface, and subsequent release following sequential sialylation and fucosylation of the molecules in the proximal and distal caput spermatozoa, respectively. (3) When spermatozoa from the proximal and distal caput, corpus, and proximal and distal cauda were incubated with fucose-labeled nucleotide sugar (GDP[14C]fucose), higher levels of radioactivity were routinely incorporated into the spermatozoa from the distal caput. (4) The [14C]fucose-labeled spermatozoa or sperm plasma membranes, when solubilized, resolved on SDS-PAGE, and visualized by autoradiography, showed that the radioactivity had been incorporated into an endogenous acceptor of 86 kDa (major component) and several minor components. Treatment of the solubilized spermatozoa with N-glycanase suggested that the [14C]fucose is mainly present on N-linked oligosaccharide units. These studies demonstrate that some of the sperm surface components are fucosylated during sperm maturation. The potential significance of the in vitro fucosylation of sperm surface components in the production of functionally mature spermatozoa is discussed.

Rat epididymal luminal fluid acid beta-D-galactosidase optimally hydrolyses glycoprotein substrate at neutral pH.

Several glycosidases, purified and characterized from mammalian tissues, have been shown to be optimally active under acidic conditions when p-nitrophenyl (PNP) or 4-methylumbelliferyl glycosides are used as substrates. Although high levels of the glycosidases are present in the epididymal lumen, their physiological role remains uncertain. To be functional, the glycosidases are expected to be enzymatically active at or near the physiological pH of luminal fluid. In this report, we demonstrate that the rat epididymal luminal fluid beta-D-galactosidase, optimally active toward PNP beta-D-galactoside at pH 3.5, shows maximum activity towards a glycoprotein substrate ([Gal-3H]fetuin) at neutral pH. Several lines of evidence, including immunoprecipitation studies using antibody to the acid beta-D-galactosidase, and substrate competition studies, indicate that PNP galactosidase and [3H]Gal galactosidase activities are caused by a single enzyme, and that the two substrates are probably cleaved by the same catalytic site(s). Competition studies with various disaccharides indicate that this enzyme is capable of cleaving a variety of galactose linkages found in both O- and N-linked oligosaccharides. Molecular-sieve column chromatography of the beta-D-galactosidase of luminal fluid under several conditions of buffer and pH show that, whereas the enzyme eluted as a tetramer (apparent M(r) 320,000) under acidic conditions (pH 3.5-4.3), only dimers and monomers (apparent M(r) 180,000 and 92,000 respectively) were observed in neutral conditions (pH 6.8). This aggregation/dissociation phenomenon is reversible. These studies indicate that beta-D-galactosidase is present in the luminal fluid in dissociated forms, and is therefore optimally active towards glycoprotein substrates at physiological pH. The potential role of the enzyme in modification of sperm surface glycoproteins is discussed.

Biochemical alterations in the proacrosin-acrosin system during epididymal maturation of the rat spermatozoa.

Acrosin, an acrosomal serine protease, is believed to have a role in fertilization. The enzyme is synthesized in an enzymatically inactive precursor form, proacrosin, and is processed to enzymatically active form(s). In the studies presented here, maturation-associated changes in the proacrosin-acrosin system of rat spermatozoa are reported. Acid-solubilized components of spermatozoa from caput, corpus, and cauda epididymidis were resolved on gelatin-sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and the proteolytic bands visualized by enzymography. These studies reveal the presence of one form (52 kDa), two forms (52 and 41 kDa), and four forms (52, 41, 34, and 31 kDa) in the spermatozoa from caput, corpus, and cauda, respectively. The findings suggest that the enzymatically inactive high molecular weight component (proacrosin) present in the caput spermatozoa is partially converted to the low molecular weight components (acrosin) during epididymal transit. The sensitivity of these molecular forms to an inhibitor of acrosin, p-nitrophenyl p'-guanidino benzoate (NPGB), and the fact that all four forms cross-reacted with the antibody against guinea pig testis proacrosin, suggest that these molecular forms are proacrosin-acrosin components. To understand the mechanism of the changes in molecular forms, spermatozoa from caput, corpus, and cauda regions were subjected to in vitro activation, and the acid-solubilized components resolved on gelatin-SDS-polyacrylamide gel. A smaller component of 34 kDa was generated from both the caput and corpus spermatozoa. No changes in the molecular form(s) of cauda spermatozoa were observed, even after in vitro activation for 4 hours. Inclusion of NPGB during in vitro activation blocked generation of the new molecular form from the caput spermatozoa. These studies indicate that intra-acrosomal events during epididymal transit may be important in the production of functionally mature spermatozoa.

Asparagine-linked glycoprotein biosynthesis in rat epididymis. Presence of a mannosidase II-like enzyme.

Previous studies from this laboratory using p-nitrophenyl alpha-D-mannoside (p-NPM) as substrate provided no evidence for the presence of mannosidase II in the rat epididymis [Skudlarek & Orgebin-Crist (1988) J. Reprod. Fertil. 84, 611-617]. However, rat epididymal epithelial cells cultured in the presence of swainsonine, an inhibitor of mannosidase II, produce abnormally processed N-linked glycoproteins containing hybrid-type oligosaccharides instead of complex-type [Tulsiani, Skudlarek & Orgebin-Crist (1990) Biol. Reprod. 43, 130-138], a result providing indirect evidence for the presence of mannosidase II-like enzyme in rat epididymis. In the studies described here, we present evidence for the occurrence of this processing enzyme in rat epididymal Golgi membranes. This enzyme is an integral Golgi membrane component. Like liver mannosidase II, the epididymal enzyme cleaves alpha 1,3- and alpha 1,6-linked mannosyl residues from GlcNAcMan5GlcNAc. However, unlike liver mannosidase II, the epididymal enzyme shows no activity towards the synthetic substrate, p-NPM. The epididymal mannosidase cross-reacts with liver anti-(mannosidase II) antibody, a result suggesting that the two enzymes share a common antigenic site(s). Immunoblotting studies following resolution of liver and epididymal Golgi membranes on SDS/PAGE show that, whereas the liver mannosidase II was resolved as a doublet of Mr 120,000 and 122,000, only the Mr 120,000 band was observed in the epididymal Golgi membranes. Immunoblotting of the Golgi-rich fractions, resolved under non-denaturing conditions, showed different patterns of charge and/or size isomers from the two tissues. These studies demonstrate tissue-specific differences in processing enzymes with similar function.

Swainsonine induces the production of hybrid glycoproteins and accumulation of oligosaccharides in male reproductive tissues of the rat.

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)

Human sperm plasma membranes possess alpha-D-mannosidase activity but no galactosyltransferase activity.

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.

Novel alpha-D-mannosidase of rat sperm plasma membranes: characterization and potential role in sperm-egg interactions.

During the course of a study of glycoprotein processing mannosidases in the rat epididymis, we have made an intriguing discovery regarding the presence of a novel alpha-D-mannosidase on the rat sperm plasma membranes. Unlike the sperm acrosomal "acid" mannosidase which has a pH optimum of 4.4, the newly discovered alpha-D-mannosidase has a pH optimum of 6.2, and 6.5 when assayed in sperm plasma membranes and intact spermatozoa, respectively. In addition, the two enzymes show different substrate specificity. The acrosomal alpha-D-mannosidase is active mainly towards synthetic substrate, p-nitrophenyl alpha-D-mannopyranoside, whereas the sperm plasma membrane alpha-D-mannosidase shows activity mainly towards mannose-containing oligosaccharides. Evidence is presented which suggest that the sperm plasma membrane alpha-D-mannosidase is different from several processing mannosidases previously characterized from the rat liver. The newly discovered alpha-D-mannosidase appears to be an intrinsic plasma membrane component, since washing of the purified membranes with buffered 0.4 M NaCl did not release the enzyme in soluble form. The enzyme requires nonionic detergent (Triton X-100) for complete solubilization. The enzyme is activated by Co2+ and Mn2+. However, Cu2+ and Zn2+ are potent inhibitors of the sperm plasma membrane alpha-D-mannosidase. At a concentration of 0.1 mM, these divalent cations caused nearly complete inactivation of the sperm enzyme. In addition methyl-alpha-D-mannoside, methyl-alpha-D-glucoside, mannose, 2-deoxy-D-glucose, and D-mannosamine are inhibitors of the sperm surface alpha-D-mannosidase. The physiological role of the newly discovered enzyme is not yet known. Several published reports in three species, including the rat, suggest that the sperm surface alpha-D-mannosidase may have a role in binding to mannose-containing saccharides presumably present on the zona pellucida.

Effect of swainsonine on rat epididymal glycosidases.

Each epididymis of control and swainsonine-fed rats (5 micrograms/ml drinking water) was divided into 5 segments, and tissue, spermatozoa and sperm-free supernatants were prepared from each segment. When levels of 3 lysosomal glycosidases and total protein were determined, the proximal cauda contained the greatest concentration of glycosidase. The specific-activity profile for beta-glucuronidase and beta-galactosidase was similar in swainsonine-fed and control rats. However, the concentration of alpha-D-mannosidase in tissue of all segments was significantly greater in swainsonine-fed rats than in age-matched controls. Enzyme activity for alpha-D-mannosidase after swainsonine treatment was significantly greater in spermatozoa from the caput, than in spermatozoa from the corpus and the cauda epididymidis. Since the alpha-D-mannosidase activity was optimal at pH 4.5 and studies with highly specific antibody to lysosomal alpha-D-mannosidase immunoprecipitated all of the alpha-D-mannosidase present in detergent extracts of epididymal tissue, spermatozoa, and sperm-free supernatant, the enzyme studied is of lysosomal origin.

Synthesis and processing of lysosomal enzymes of mouse kidney after treatment with [3H]fucose.

[3H]Fucose, intravenously injected into androgen-treated mice, was incorporated at high rates into the immunopurified kidney lysosomal enzymes beta-glucuronidase and beta-galactosidase. Initially, label was incorporated into high molecular weight proenzyme forms. Processing of fucose-labeled proenzyme forms to lower molecular weight mature forms was very rapid, being detectable at 30 min, and complete by 90 min, compared with the several hours required for processing of lysosomal enzymes labeled with amino acids. This result is consistent with addition of fucose residues within the Golgi apparatus just before transfer of lysosomal proenzyme forms to the lysosome where maturation is thought to occur. The combination of the high rates of incorporation of [3H]fucose and the known metabolic stability of this precursor sugar suggests that the mouse kidney system is advantageous for studies of the synthesis, processing, and degradation of fucose-containing complex oligosaccharides of lysosomal enzymes and, by extension, of other kidney glycoproteins.

Secretion of proteins and glycoproteins by perifused rabbit corpus epididymal tubules: effect of castration.

Protein synthesis in epididymal tissue of intact and castrated rabbits was studied after incubation of epididymal minces with [35S]-cysteine or [35S]-methionine and protein separation by two-dimensional gel electrophoresis. Regional differences in the pattern of protein synthesized were observed. Castration did not change overall protein synthesis, but it reduced these regional differences. The presence of 5 alpha-DHT in the culture medium of the proximal corpus epididymidis perfused for 24 hr did not increase overall protein synthesis in tubules from intact or castrated rabbits and did not reinitiate synthesis of the proteins that had disappeared after castration. The kinetics of glycoprotein synthesis and secretion were studied by light and electron microscopy autoradiography at 0.5, 2, 6, and 24 hr after exposure to [3H]-mannose, [3H]-fucose, and [3H]-glucosamine. Changes in the distribution of mannose- and glucosamine-labeled material indicated that the decline in grain density over the epithelium from 30 min to 24 hr coincided with an increasing reaction over the stereocilia border from 30 min to 2 hr and in the lumen from 2 to 24 hr. The distribution of fucose-labeled material indicated that the grain reaction over the epithelium declined more rapidly than with the mannose label. When the glucosamine-labeled sperm mass was released from the tubules, the labeled material was lost after the first washing, indicating that the glucosamine-labeled glycoproteins did not bind firmly to corpus spermatozoa within 24 hr. After castration, both mannose- and fucose-labeled materials migrated to the cell apex more rapidly than in the intact animal, but they were not released as readily into the lumen. The culture of epididymal tubules from castrated males with 5 alpha-DHT for 24 hr did not promote the release of either mannose- or fucose-labeled material into the lumen. However, testosterone given in vivo for 2 weeks restored secretion of mannose-labeled material into the lumen.