Evidence for the deficiency of β-glucosidase-activating factor in fibroblasts of patients with I-cell disease

Summary Reduced activity of -glucosidase was shown in the cultured skin fibroblasts of four patients with I-cell disease when the enzyme was tested without the use of detergents. In the presence of taurocholate and triton X100 -glucosidase activity was normal. This suggested a deficiency of a -glucosidase-activating factor in I-cell fibroblasts rather than of the enzyme itself. The deficiency of -glucosidase activity was corrected to some extent by mixing cell lysates, and more effectively by cocultivation and fusion of I-cell disease and Gaucher fibroblasts. These results present evidence for the presence of a -glucosidase-activating factor in normal and Gaucher fibroblasts. In fibroblasts of patients with I-cell disease this activator is probably deficient, as is the case for most lysosomal enzymes.     

Cultured neonatal rat oligodendrocytes are enriched in acid hydrolase activities

We measured the activity of several acid hydrolases in oligodendrocyte and mixed glial (predominantly astrocytic) cell cultures prepared from neonatal rat cerebra. When compared with the mixed glial cultures, the cultured oligodendrocytes exhibited higher levels for all the hydrolases when activities were normalized to protein content. When enzymic activities were examined as a function of DNA content, oligodendrocytic -l-fucosidase, -d-glucuronidase, arylsulfatase, and N-acetyl--d-glucosaminidase were higher than in mixed glial cultures, whereas the activities of -d-glucosidase, -d-galactosidase and acid phosphatase were not elevated. These differences could not be accounted for by the fetal bovine serum present in the culture medium. The enrichment in acid hydrolase specific activities in the oligodendrocytes may be associated with a rapid turnover of at least some of the extensive myelin-like membranes formed by these cultured cells. Alternatively, the enrichment of acid hydrolase activity in the oligodendrocytes may be associated with intracellular vesicles of lysosomal origin which may play a role in myelin-like membrane assembly. Exactly which of the above two processes, or possible combinations thereof, is responsible for the present finding is not known.     

Saposins and Their Interaction with Lipids

The lysosomal degradation of several sphingolipids requires the presence of four small glycoproteins called saposins, generated by proteolytic processing of a common precursor, prosaposin. Saposins share several structural properties, including six similarly located cysteines forming three disulfide bridges with the same cysteine pairings. Recently it has been noted that also other proteins have the same polypeptide motif characterized by the similar location of six cysteines. These saposin-like (SAPLIP) proteins are surfactant protein B (SP-B), Entamoeba histolytica poreforming peptide, NK-lysin, acid sphingomyelinase and acyloxyacyl hydrolase. The structural homology and the conserved disulfide bridges suggest for all SAPLIPs a common fold, called saposin fold. Up to now a precise fold, comprising five -helices, has been established only for NK-lysin. Despite their similar structure each saposin promotes the degradation of specific sphingolipids in lysosomes, e.g. Sap B that of sulfatides and Sap C that of glucosylceramides. The different activities of the saposins must reside within the module of the -helices and/or in additional specific regions of the molecule. It has been reported that saposins bind to lysosomal hydrolases and to several sphingolipids. Their structural and functional properties have been extensively reviewed and hypotheses regarding their molecular mechanisms of action have been proposed. Recent work of our group has evidenced a novel property of saposins: some of them undergo an acid-induced change in hydrophobicity that triggers their binding to phospholipid membranes. In this article we shortly review recent findings on the structure of saposins and on their interactions with lipids, with special attention to interactions with phospholipids. These findings offer a new approach for understanding the physiological role of saposins in lysosomes.     

Immunocytochemistry of lysosomal hydrolases and their precursor forms in normal and mutant human cells

Summary The acid hydrolases -glucosidase, -galactosidase,N-acetyl--d-hexosaminidase, -glucocerebrosidase and cathepsin D were studied immunocytochemically in normal and mutant human cells using monoclonal and affinity-purified polyclonal antibodies. For light microscopy, Rhodamine or Fluorescein-labelled conjugates were used, and for electron microscopy protein A-gold conjugates were employed. With the double labelling procedure, it was found that in normal fibroblasts every lysosome contained all the enzymes studied. The method described also enabled us to demonstrate the presence or absence of mutant enzyme protein in fibroblasts derived from patients with a genetic lysosomal enzyme deficiency.Immunoreactive acid hydrolases or their precursor forms were found in the rough endoplasmic reticulum, the cisternae of the Golgi complex, Golgi associated vesicles and lysosomes. This is in agreement with the present concept that the Golgi complex plays an essential role in the processing and targeting of lysosomal enzymes.     

Lysosomal α-galactosidase in endothelial cell cultures established from a fabry hemizygous and normal umbilical veins

Summary An endothelial cell line has been established from the umbilical vein obtained after abortion of a male fetus suffering from Fabry disease. This X-linked inborn error of glycosphingolipid catabolism results from deficiency of the lysosomal hydrolase -galactosidase. A the clinical manifestations of the disease are mainly caused by glycosphingolipid depositions in the endothelium of all vessels. The hemizygous cell line and eight endothelial cell lines originating from the umblical cords of normal newborns were grown for more than 10 passages. They had a short generation time that allowed us to get sufficient cells for qualitative and quantive investigations of -galactosidase. The enzyme in normal endothelial cells had a similar thermostability and isoelectric focusing pattern as that in fibroblasts, but the activity was essentially higher in endothelial cells. The hemizygous endothelial cells were deficient in -galactosidase A. It is concluded that endothelial cell lines are an important alternative to fibroblasts for in vitro studies of the lysosomal storage diseases.     

Structural analysis of the carbohydrate moieties of α-l-fucosidase from human liver

Acid -l-fucosidase (EC 3.2.1.51) was obtained from human liver and purified to homogeneity. The enzyme consists of four subunits; each of these has a molecular mass of 50 kD_a and bears oneN-linked carbohydrate chain. The structures of these chains were studied at the glycopeptide level by methylation analysis and 500-MHz¹H-NMR spectroscopy. Oligomannoside-type chains andN-acetyllactosamine-type chains are present in an approximate ratio of 31. While the oligomannoside-type chains show some heterogeneity in size (Man_5–8GlcNAc₂), theN-acetyllactosaminetype chains are exclusively bi-(2–6)-sialyl, bi-antennary in their structure.These observations on the carbohydrate moieties of -l-fucosidase substantiate our hypothesis [Overdijket al. (1986) Glycoconjugate J 3:339–50] with respect to the relationship between the oligosaccharide structure of lysosomal enzymes and their residual intracellular activity in I-cell disease. For the series of enzymes examined so far, namely, -N-acetylhexosaminidase, -l-fucosidase and -galactosidase, the relative amount ofN-acetyllactosamine-type carbohydrate increases, while the residual intracellular activity in I-cell disease tissue decreases in this order. The system which is responsible for preferentially retaining hydrolases with (non-phosphorylated) oligomannoside-type chains both in I-cells and in normal cells has yet to be identified.     

Binding receptors for α-l-fucosidase in human B-lymphoid cell lines

An established mechanism for directing newly made acid hydrolases to lysosomes involves acquisition of mannose 6-phosphate residues by the carbohydrate portion of acid hydrolases followed by binding to specific membrane-bound transport receptors and delivery to lysosomes. Two distinct phosphomannosyl receptors (CI-MPR and CD-MPR) have been identified. Alternative mechanisms for trafficking acid hydrolases exist. This report examines means for the possible receptor-mediated intracellular transport of -l-fucosidase in lymphoid cells. The binding of -l-fucosidase to intact cells and to total cell membrane preparations, in conjunction with immunoassays of solubilized membrane preparations, revealed the presence of CI-MPR and CD-MPR on human lymphoid and fibroblast cell lines. The mean level of CD-MPR in nine lymphoid cell lines was 7.2-fold greater than CI-MPR. The mean level of CI-MPR in two fibroblast lines was 3.8-fold greater than CD-MPR. The mean content of CI-MPR was 19.5-fold greater in the fibroblasts than in the lymphoid cells. The CD-MPR content of fibroblasts and lymphoid cells was nearly equivalent. Among these cell lines were a fibroblast and a lymphoid line from the same individual. These results indicate that human B-lymphoid cells are deficient in CI-MPR and suggest that modulation of expression of CI-MPR and CD-MPR in lymphoid cells differs from that in fibroblasts, including cell lines with identical genomes. No specific receptor capable of binding -l-fucosidase independent of mannose 6-phosphate was demonstrable, despite published results that support the existence of a mannose 6-phosphate independent trafficking mechanism in lymphoid cells for this enzyme.     

Transglycosylation reactions by exoglycosidases from the termite Macrotermes subhyalinus

The ability of four exoglycosidases (-galactosidase, -glucosidase, -glucosidase and invertase) from the termite Macrotermes subhyalinus to catalyse tranglycosylation reactions was tested using lactose, cellobiose, maltose and sucrose as glycosyl donors and 2-phenylethanol as glycosyl acceptor. The experimental conditions were optimized in relation to the time course of the reaction, pH and concentrations of glycosyl donor and acceptor. Whereas the hydrolytic activity was largely predominant over the transferase activity with -galactosidase and -glucosidase, the transglycosylation activity represented 68% with -glucosidase. In addition, as demonstrated by the transglycosylation product formed, the hydrolysis of sucrose was catalysed by -glucosidase and not by invertase. On the basis of this work, -glucosidase from M. subhyalinus appears to be a valuable tool for the preparation of neoglycoconjugates.     

Oxygen free radicals induced release of lysosomal enzymes in vitro

Summary The effect of oxygen free radicals, generated by xanthine and xanthine oxidase, was studied on the release of lysosomal hydrolase from rat liver lysosomes in vitro. A lysosomal enriched subcellular fraction was prepared, using differential centrifugation technique, from the homogenate of rat liver. The biochemical purity of the lysosomal fraction was established by using the markers of different cellular organelles. Oxygen free radicals were generated in vitro by the addition of xanthine and xanthine oxidase. The release of lysosomal hydrolase (-glucuronidase) from the lysosomal fraction was measured. There was a 3 to 4 fold increase in the release of -glucuronidase activity in the presence of xanthine and xanthine oxidase when compared to that in the absence of xanthine and xanthine oxidase. In the presence of superoxide dismutase (SOD), a scavenger of oxygen free radicals, the xanthine and xanthine oxidase system was unable to induce the release of -glucuronidase activity from the lysosomes. Sonication (2 bursts for 15 sec each) and Lubrol (2 mg/10 mg lysosomal protein) treatment, which are known to cause membrane disruption, also induced the release of -glucuronidase from lysosomal fraction. This release of -glucuronidase by sonication and lubrol treatment was not prevented by SOD. These data indicate that lysosomal disruption is a consequence of oxygen free radicals, generated by xanthine and xanthine oxidase.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - EGTA Ethylene Glycol Bis-(-aminoethyl ether)N,N,-N,N-tetracetic acid - Tris Tris (hydroxymethyl) aminomethane - SOD Superoxide Dismutase     

Ultrastructural and immunocytochemical study of skin fibroblasts from normal and sialidosis patients

The objectives of this study were to analyze morphologically, morphometrically and immunocytochemically the lysosomal compartment of normal fibroblasts and of fibroblasts with neuraminidase deficiency. The immunocytochemical analyses consisted of quantifying the distribution of saposins and -galactosidase in the lysosomes of these cells to test the hypothesis that neuraminisdase deficiency is associated with an impairment in the transport of these proteins to the lysosomal compartment. To test this idea, cultured skin fibroblasts of patients with or without sialidosis were prepared for electron microscopy and probed with antibodies against lysosomal -galactosidase and lysosomal saposins. The lysosomes of the affected cells had an abnormal accumulation of incompletely digested membranes which was associated with a significant lowering in the density of antigenic sites per lysosome. However, due to a significant increase in the number of lysosomes per affected cell, the total number of antigenic sites in control and neuraminidase deficient cells was similar. This presumably compensatory effect indicates that although the rate of production of -galactosidase and saposins remains unchanged, the transport of these molecules to the lysosomes is somehow affected. Our data also indicate that in the fibroblasts, lysosomes require a normal concentration of the three enzymes to maintain neuraminidase activity and sphingolipid degradation.     

Distribution of Fucoidan Hydrolases and Some Glycosidases among Marine Invertebrates

Thirty-three species of marine invertebrates from the Sea of Japan were analyzed for contents of fucoidan hydrolases and some glycosidases. Fucoidan hydrolase activity was assessed by examining the effect of animal tissue extracts on fucoidans from the two brown seaweeds Laminaria cichorioides and Fucus evanescens, which have different structural characteristics. The activity of glycosidases (-glucosidase, -galactosidase, -fucosidase, and -mannosidase) was determined using p-nitrophenyl derivatives of sugars as substrates. It was found that glycosidases and fucoidan hydrolases of different specificities are fairly widely distributed among marine invertebrates. Mollusks and some species of echinoderms and arthropods showed the highest enzymatic activity. This research will enable us to choose organisms for the separation and study of fucoidan hydrolases and glycosidases, which may be useful in determining the structure of fucoidans.     

Properties of α-l-iduronidase in cultured skin fibroblasts from α-l-iduronidase-deficient patients

Summary On DEAE cellulose column chromatography, -l-iduronidase in cultured skin fibroblasts was resolved into two distinct components, forms A and B. They had similar K_m values for 4-methylumbelliferyl--l-iduronide, but differed in pH optima and thermal stability. Form B was more heat-stable than form A.Residual -l-iduronidase activity in Hurler fibroblasts was heat-stable, while that in Scheie fibroblasts was heat-labile, and moreover, that in Hurler-Scheie compound fibroblasts lay intermediate between Hurler and Scheie syndromes. These findings demonstrated that Hurler syndrome, Scheie syndrome and Hurler-Scheie compound were enzymatically distinguishable.     

Human β-galactosidase and α-neuraminidase deficient mucolipidosis: Genetic complementation analysis of the neuraminidase deficiency

Summary Human -galactosidase and -neuraminidase deficient mucolipidosis [ML(gal^-neur^-)] is an inherited lysosomal enzymopathy which recently was designated as a sialidosis. We analyzed the neuraminidase deficiency of this disorder with genetic complementation analyses using a heterokaryon enrichment procedure. The genetic defects of two apparent variants of this disorder complemented the defects of the neuraminidase deficiency diseases, sialidosis I and mucolipidosis I, resulting in the restoration of neuraminidase activity in heterokaryons. The neuraminidase deficiency, therefore, may not be the primary defect in ML(gal^-neur^-) and is not an appropriate test for determining carrier status. The clinical and biochemical characteristics of this disorder suggest that a post-translational or processing event for these enzymes may be defective. The defect, however, is different from I-cell disease and pseudo-Hurler polydystrophy, two disorders of post-translational lysosomal enzyme biosynthesis, since complementation studies demonstrated recovery of intracellular -galactosidase and -neuraminidase levels in heterokaryons. The lack of human -galactosidase expression in man-mouse somatic cell hybrids formed from fibroblasts of the infantile onset type disorder suggests that the defect is not corrected by the mouse genome. The ML(gal^-neur^-) disorder therefore appears to be a distinct subtype of the inherited neuraminidase deficiencies in which the defect may occur in a post-translational or regulatory step which coordinately affects the expression of lysosomal -galactosidase and -neuraminidase.     

Compartmental distribution of beta-hexosaminidase isoenzymes in I-cell fibroblasts.

A characteristic of the human lysosomal disorder I-cell disease is an abnormal excretion of most lysosomal hydrolases, including beta-N-acetyl-D-glucosaminidase (EC 3.2.1.30; beta-hexosaminidase) by cultured skin fibroblasts. Treatment of I-cell cultures with cycloheximide or tunicamycin demonstrated that (1) I-cell fibroblasts rapidly excrete all newly synthesized beta-hexosaminidase, (2) two qualitatively distinct pools of beta-hexosaminidase isoenzymes exist inside I-cell fibroblasts, one of which is a rapid-turnover excretory pool, and (3) the induction of an abnormal glycosylation of beta-hexosaminidase by tunicamycin in normal or I-cell fibroblast cultures does not affect subsequent excretion of the enzyme.     

Immunocytochemical localisation of some lysosomal hydrolases,their presence in luminal fluid and their directional secretion by human epididymal cells in culture

The way in which the human epididymis modifies spermatozoa during their sojourn in this structure might be clarified by knowledge of the nature of its secretions. We have examined the presence of several lysosomal hydrolases in human epididymal tissue and fluids, and their synthesis and secretion by monolayer cultures. Tissues were obtained from men undergoing orchidectomy for prostatic carcinoma. The enzymes cathepsin D and acid -glucosidase were localised in the lysosomes of epithelial cells from the corpus epididymidis, by an immunocytochemical technique. Cathepsin D was also found in epithelial cells of the efferent ducts within lysosomes, apical vesicles and multivesicular bodies. No immunolocalisation of acid glucosidase in the efferent ducts or on the microvilli of the corpus was demonstrable. Cathepsin D, -hexosaminidase (N-acetylglucosaminidase) and -glucosidase were measurable in the luminal fluid from the human corpus epididymidis; -hexosaminidase was secreted into the culture medium by confluent monolayers of epididymal and efferent duct cells. Immunoprecipitation of cell extracts and culture medium of these cultures incubated with ³⁵S-methionine revealed that the precursors of cathepsin D and -hexosaminidase were synthesized and secreted by such monolayers. Thus, active lytic enzymes are secreted by the human epididymis and could modify sperm membranes.     

Expression of α-d-mannosidase in man-hamster somatic cell hybrids

Two types of -d-mannosidase isozymes are present in human white blood cells, human diploid fibroblasts, and HeLa cells. One of these (the S isozyme) constitutes the major -d-mannosidase of the human cells, has a pH optimum of 4.4, and is associated with lysosomes. The other (the F isozyme) is most active at pH 6, is acid labile, and is located in the soluble portion of the cytoplasm. The expression of human lysosomal -d-mannosidase was examined in man-hamster hybrid clones, and was found to be concordant with that of phosphohexose isomerase in 54 of 55 primary clones. A locus specifying human lysosomal -d-mannosidase has therefore been assigned to chromosome 19.This work was supported by NIH Grants HD 04807-07 and HD 06285-04 and by a research grant (5 PO 1 HB 06276-04) to the Mental Retardation Research Center of the Children's Hospital Medical Center, Boston, Massachusetts, from the NIH.     

The myofibroblast markers α-SM actin and β-actin are differentially expressed in 2 and 3-D culture models of fibrotic and normal skin

To characterize the differences between fibrotic myofibroblasts and normal fibroblasts, we studied two differentiation markers: -smooth muscle (SM) actin, a specific marker of myofibroblast differentiation, and -actin, which is overexpressed in the fibrotic tissue. Experiments were performed on fibroblasts isolated from normal pig skin and on subcutaneous myofibroblasts isolated from pig radiation-induced fibrosis. Three culture models were used: cells in monolayers, equivalent dermis, consisting of fibroblasts embedded into a matrix composed of type I collagen, and in vitro reconstituted skin, in which the matrix and containing life fibroblasts were overlaid with keratinocytes. Samples were studied using immunofluorescence and western-blotting. In monolayers cultures, both fibrosis and normal cells expressed -SM actin. Furthermore, similar amounts of -actin protein were found. In these conditions, the resulting alterations in the phenotypes of cells made comparison of cultured fibrotic and normal cells irrelevant. Under the two 3-D culture models, normal fibroblasts no longer expressed -SM actin. They expressed -actin at the basal level. Moreover, the fibrotic myofibroblasts in both 3-D models retained their differentiation features, expressing -SM actin and overexpressing -actin. We found that this normalization was mainly related to the genomic programmation acquired by the cells in the tissue. Cellular motility and microenvironment were also involved, whereas cellular proliferation was not a major factor. Consequently, both three-dimensional models allowed the study of radiation-induced fibrosis in vitro, provided good extrapolations to in vivo conditions and avoided certain of culture artefacts.     

Dual localization of acid hydrolases in endoplasmic reticulum and in lysosomes

Summary Dissimilar enzyme locations obtained on occasion by the post- and simultaneous-coupling techniques employing the substrate naphthol AS-BI -glucosiduronic acid were attributed to the inadequate incorporation of substrate into lysosomal membranes in the post-coupling technique on the one hand, as well as to the inhibition of cytoplasmic enzyme by diazotate in the simultaneous coupling technique on the other hand. The use of a fixative solvent mixture prior to the enzyme staining reaction appeared to labilize lysosomal membranes, to improve fixation and to eliminate fiber artefacts. In male mice which have been androgenized by the injection of gonadotrophin, kidney homogenates, subsequently prepared, exhibited an immediate increase in the specific activity of microsomal -glucuronidase while lysosomal -glucuronidase was unchanged for the first 36 hours.This event at 36 hours corresponded with enhanced cytoplasmic but not lysosomal staining. Diffuse reactions in enzyme morphology are discussed as well as the origin of lysosomal -glucuronidase in mouse kidney and the dual localization of hydrolases in endoplasmic reticulum and lysosomes.     

Lysosomal α-D-mannosidase

-mannosidosis in the human is an autosomal recessive lysosomal storage disease caused by a deficiency of lysosomal -D-mannosidasea actvity. Lysosomal -D-mannosidase is involved in the catabolism of N-linked glycoproteins through the sequential degradation of high-mannose, hybrid and complex oligosaccharides. This review is focused on human, mouse, bovine and feline genes coding for lysosomal -D-mannosidase. In particular the exon-intron structure of the genes, their promoters, and the identification of mutations causing the disease have been examined. The construction, by homologous recombination, of a mouse model of -mannosidosis is reported.     

Die sauren Hydrolasen des Nebenhodenepithels von Ratten nach Kastration und Kryptorchismus

Zusammenfassung Auf Grund von Verteilungsdifferenzen verschiedener Epithelzelltypen (Hauptzellen, helle Zellen vom Typ I–IV, Flaschenzellen, Basalzellen) und intraepithelialer Lymphocyten im Verlauf des Nebenhodenganges sowie von Zell-, Lokalisations- und Aktivitätsdifferenzen der sauren Hydrolasen Phosphatase, -d-N-Acetylglucosaminidase (NAG), -d-Glucuronidase, -d-Galactosidase (-Gal), -d-Galactosidase, -d-Mannosidase (-Man), unspezifische Esterasen und Dipeptidylpeptidase (DPP) I und II im Epithel kann der Nebenhoden in 10 Zonen untergliedert werden. Meist kommen die Hydrolasen in Lysosomen, möglicherweise aber auch im endoplasmatischen Reticulum und Golgi-Apparat sowie in Sekretgranula der Epithelzellen vor. Daher laufen offenbar außer intraepithelialen Verdauungsprozessen, deren Intensität von der betreffenden Zone und vom jeweiligen Zelltyp abhängt, auch Sekretionsvorgänge saurer Hydrolasen (NAG, -Gal, -Man, DPP II) ins Lumen ab. — Experimente (bilaterale und unilaterale operative und chemische Kastration mit und ohne Androgensubstitution, Kryptorchismus) zeigen, daß der Nebenhoden danach in proximodistaler Richtung reagiert; etwas schematisch läuft die Reaktion in 3 Phasen ab; außerdem entwikkeln sich vermutlich aus Hauptzellen sog. neue basale Zellen mit speziellem Lysosomenapparat, und ohne Substitution sinkt die Hydrolasenaktivität; die Abnahme selbst kann sich enzym-, zonen- und zellspezifisch verhalten. Nach bilateraler Kastration stirbt initial innerhalb oder außerhalb des Epithels bevorzugt in Zone 1 und 2 ein bestimmter Hauptzelltyp; nach Kryptorchismus erscheinen Spermatiden im Nebenhodengang, wandern ihn hinunter und inkorporieren die Lumenhydrolasen. — Diese und weitere Daten deuten darauf hin, daß vor allem die Serumandrogene, aber auch die Lumenandrogene durch lokal unterschiedliche sowie zell- und hydrolasenspezifische Effekte das Nebenhodenepithel regulieren. Vielleicht sind darüber hinaus nur bestimmte Hauptzellen androgensensitiv und werden durch Androgene beeinflußt; die restlichen könnten hingegen androgeninsensitiv sein und über andere, noch unbekannte Mechanismen gesteuert werden. Die Ganglumenhydrolasen werden selektiv von Nebenhodenepithelzellen sezerniert; eventuell sind dafür die hellen Zellen vom Typ I und II in Zone 5 und 6 verantwortlich.

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Acid hydrolases in the epididymal epithelium of rats after castration and cryptorchidism

Summary On the basis of distribution differences of various types of epithelial cells (principal cells, clear cells of type I–IV, flask cells, basal cells) and intraepithelial lymphocytes in the course of the epididymal duct as well as cell, localization and activity differences of the acid hydrolases phosphatase, -d-N-acetylglucosaminidase (NAG), -d-glucuronidase, -d-galactosidase (-Gal), -d-galactosidase, -d-manosidase (-Man), non-specific esterases and dipeptidylpeptidase (DPP) I and II in the epithelium the epididymis of mature rats can be subdivided into 10 zones. Mostly, these hydrolases are localized in lysosomes but may also be linked to the endoplasmic reticulum, Golgi apparatus and secretion granules of the epithelial cells. Therefore, beside intraepithelial digestive processes, the intensity of which varies in dependency on the zone and cell type, secretion of some acid hydrolases (NAG, -Gal, -Man, DPP II) into the lumen seems to be possible. —In a series of experiments (bilateral and unilateral surgical and chemical castration with and without androgen substitution, cryptorchidism) it can be shown that the epididymis reacts in a proximodistal direction; 3 phases of epithelial response may be distinguished; furthermore, so-called new basal cells with a special lysosomal apparatus develop presumably from principal cells, and without substitution the hydrolase activity decreases; the decrease itself depends on the investigated enzyme, cell type and zone. After bilateral castration the initial event is the death of one special type of principal cell inside or outside the epithelium preferentially in zone 1 and 2; after cryptorchidism spermatides enter the lumen, move the duct downwards and incorporate the luminal hydrolases. — These and additional data suggest a regulation of the epididymal epithelium by serum androgens but also luminal androgens via locally different as well as cell and hydrolase specific effects. Possibly, only one type of principal cell is androgen-sensitive and is influenced by androgens respectively; the others may be insensitive to androgens and are regulated by mechanisms presently not yet known. The luminal hydrolases are actually secreted by epithelial cells of the epididymis; presumably, the clear cells of type I and II in zone 5 and 6 are responsible for their secretion.

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