Increase in macrophages in the testis of cathepsin a deficient mice suggests an important role for these cells in the interstitial space of this tissue

Cathepsin A (PPCA) is a lysosomal carboxypeptidase that functions as a protective protein for alpha-neuraminidase and beta-galactosidase in a multienzyme complex. In the present study, the testes of PPCA -/- mice from 2 to 10 months of age were compared with those of their wild type counterparts. While germ and Sertoli cells appeared comparable in appearance and distribution, the mean profile area of seminiferous tubules showed a significant decrease between wild type and PPCA -/- mice, suggesting changes to the seminiferous tubules and their contents. In addition, macrophages in the interstitial space (IS) of PPCA -/- mice were large, spherical, and filled with pale lysosomes, unlike those seen in wild type mice, and a quantitative analysis of their frequency per unit area of IS in PPCA -/- mice revealed a significant increase compared to that of wild type mice; this was also the case for their mean profile area. Absence of mitotic figures, cycling cells, or degenerating figures in the IS suggests that the major recruitment of macrophages appears to be from the circulation. In the IS, Leydig cells also showed an accumulation of large pale lysosomes in PPCA -/- mice, and their frequency also increased significantly as compared to wild type mice. In the electron microscope, a close association of Leydig cell microvilli with the surface of macrophages was pronounced in PPCA -/- mice. Since macrophages and Leydig cells interact by secreting various factors between each other, and considering the fact that Leydig cells show an accumulation of large pale lysosomes in PPCA -/- mice, it is suggested that macrophages accumulate as a result of abnormalities occurring in Leydig cells. Taken together, the data on increase in frequency of macrophages suggests important functions for these cells in both wild type and PPCA -/- mice.     

Intracellular distribution of lysosomal enzymes in the mouse pigment mutants pale ear and pallid

Summary The size distribution of lysosomes was determined in kidney proximal tubule cells of two mouse pigment mutants, pale ear and pallid, which have an increase in kidney lysosomal enzyme content caused by a decreased rate of secretion of lysosomal enzymes into urine. Both mutations have larger lysosomes when compared with normal mice. However, neither mutant contains the giant lysosomes (up to 11 micron diameter) common to the well-characterized beige mutant, which has a kidney secretory defect similar to the pale ear and pallid mutants. Subcellular distribution studies, performed by the osmotic shock technique, likewise suggested differences among the pigment mutants. A very high content of soluble enzyme, indicative of lysosomal fragility during homogenization, was found in extracts from the beige mutation. By comparison, the percent of soluble enzyme became progressively lower in extracts of the pallid and pale ear mutants and was lowest in extracts from normal mice. All 3 pigment mutants had normal concentrations of osmotically resistant membrane-bound lysosomal enzymes. This indicates that the excess, non-secreted, lysosomal enzyme in all three pigment mutants likely is present in classical lysosomal organelles rather than in other non-lysosomal subcellular membrane fractions. The results also illustrate that mammalian mutants which exhibit decreased lysosomal secretory rates can have strikingly different effects on morphology of lysosomes.Supported in Part by National Science Foundation Grant PCM 77-24804. E. K. N. was supported in part by United States Public Health Service Grant GM 007093-03.     

Further studies of the surface epithelium covering preovulatory rabbit follicles with special reference to lysosomal alterations

Summary The fine structure of the surface epithelium over preovulatory rabbit follicles was examined parallel with visualization of acid phosphatase at the electron microscopical level. Small enzyme positive vesicles were pinched off from the Golgi cisternae and similar vesicles fused and got incorporated into larger lysosomes of dense body type. Some lysosomes appeared in direct continuity with tubular enzyme positive structures. Other possible ways of increase of the lysosomal pool are indicated and discussed.As in previous studies a maximal accumulation of lysosomes was found in the apical epithelium at 8 h after an ovulatory dose of human chorionic gonadotrophin (HCG); thereafter a gradual loss of lysosomes ensued. Before the lysosomes disappeared from the surface epithelium they changed in character. They became more electron-lucent and revealed a fine-fibrillar matrix. Dense bodies deep in the cell interior appeared to communicate with each other and with the extracellular space below the surface epithelium. Openings were never seen towards the peritoneal cavity. The loss of lysosomal content from the apical surface epithelium before follicle rupture appeared in many respects similar to the histamine release process in mast cells.The findings support our working hypothesis that the surface epithelium over Graafian follicles is an essential source of proteolytic enzymes and that these may be released extracellularly and actively contribute to the dissolution of the follicular apex before rupture.This investigation was supported by grants from the Swedish Medical Research Council (Projects No. B74-12X-78-09C and B75-12X-78-10A). The technical assistance of Miss Ingalis Fransson and Miss Kerstin Nilsson is greatly appreciated     

Accumulation of indigestible substances reduces fusion competence of macrophage lysosomes

It is well known that mouse macrophages loaded with indigestible substances become highly vacuolated. However, why this vacuolization occurs and its effect on lysosome function and intracellular transport during endocytosis remain unknown. Here, macrophage vacuoles were formed by incubation with sucrose or a tripeptide of the D-isomer of alanine and were determined to be lysosomal in origin by staining with the lysosomal glycoproteins and lysosomal hydrolases. However, as indicated by confocal and electron microscopy, subsequent delivery of both fluid phase (lucifer yellow, horse-radish peroxidase) and receptor-bound ligands (IgG complexes) was significantly reduced, suggesting that indigestible material reduced the ability of the loaded lysosomes to fuse with endosomes containing newly internalized tracers. Nevertheless, ligands internalized by the vacuolated cells were degraded at almost the normal rate, indicating that degradation occurs in the absence of delivery to the loaded lysosomes. We have also found that this fusion inhibition occurs in human alveolar macrophages loaded with physiologic debris from smoking and asbestos. These results suggest that indigestible material within lysosomes, such as is present in residual bodies in vivo, may affect their fusion competence.     

Effect of weak bases on the intralysosomal pH in mouse peritoneal macrophages

The spectral characteristics of dextran, labeled with fluorescein, depend upon pH. We have loaded the lysosomes of mouse peritoneal macrophages with this fluorescence probe and used it to measure the intralysosomal pH under various conditions. The pH of the medium has no effect on the intralysosomal pH. Weakly basic substances in the medium cause a concentration-dependent increase in the intralysosomal pH. However, the concentration of base necessary to produce a significant change in the intralysosomal pH varies over a wide range for different bases. The active form of the base is the neutral, unprotonated form. Although most of these weak bases cause an increase in the volume of the lysosomes, increase in lysosomal volume itself causes only a minor perturbation of the intralysosomal pH. This was demonstrated in cells whose lysosomes were loaded with sucrose, and in cells vacuolated as a demonstrated in cells whose lysosomes were loaded with sucrose, and in cells vacuolated as a consequence of exposure to concanavalin A. The results of these studies are interpreted in terms of energy-dependent lysosomal acidification and leakage of protons out of the lysosomes in the form of protonated weak bases.     

Transport of human lysosomal neuraminidase to mature lysosomes requires protective protein/cathepsin A.

Human lysosomal N-acetyl-alpha-neuraminidase is deficient in two lysosomal storage disorders, sialidosis, caused by structural mutations in the neuraminidase gene, and galactosialidosis, in which a primary defect of protective protein/cathepsin A (PPCA) leads to a combined deficiency of neuraminidase and beta-D-galactosidase. These three glycoproteins can be isolated in a high molecular weight multi-enzyme complex, and the enzymatic activity of neuraminidase is contingent on its interaction with PPCA. To explain the unusual need of neuraminidase for an auxiliary protein, we examined, in transfected COS-1 cells, the effect of PPCA expression on post-translational modification, turnover and intracellular localization of neuraminidase. In pulse-chase studies, we show that the enzyme is synthesized as a 46 kDa glycoprotein, which is poorly phosphorylated, does not undergo major proteolytic processing and is secreted. Importantly, its half-life is not altered by the presence of PPCA. However, neuraminidase associates with the PPCA precursor shortly after synthesis, since the latter protein co-precipitates with neuraminidase using anti-neuraminidase antibodies. We further demonstrate by subcellular fractionation of transfected cells that neuraminidase segregates to mature lysosomes only when accompanied by wild-type PPCA, but not by transport-impaired PPCA mutants. These data suggest a novel role for PPCA in the activation of lysosomal neuraminidase, that of an intracellular transport protein.     

Ultrastructure of langur monkey epididymidis prior to and following vasectomy and vasovasostomy.

The ultrastructural changes in langur monkey epididymis prior to and following vasectomy or vasovasostomy were studied. The epididymal epithelium of the intact langur monkey was found to consist mainly of principal cells and basal cells and frequently apical or mitochondria rich cells were found. Besides these cells intraepithelial lymphocytes were also a consistent feature of the epididymal epithelium. Principal cells identified by means of the tuft of the stereocilia on their apical surface, bear well developed Golgi bodies, endoplasmic reticulum, vesicles, vacuoles and multivesicular bodies. This suggests their active involvement in absorption and secretion. Basal cells present at the base of the lamina bear a few cellular organelles and strong interdigitations with the adjacent cells. Apical or mitochondria rich cells were characterized by clusters of mitochondria in the apical region of the cell and few microvilli on their apical surface. Lymphocytes with a large nucleus and a pale rim of cytoplasm were also found at the base of the epithelium. Secretory and absorptive functions of principal cells of the epididymal epithelium were found to be increased after vasectomy, as indicated by bulging of the apical portion of the principal cells and membrane bound structure in the lumen. An extensive increase in the number of lysosomes, vesicles and vacuoles was also observed. An increase in the number of macrophages with spermatozoa remnants in the lumen of epididymis suggests that the principal mechanism for spermatozoa disposal following vasectomy is intraluminal endocytosis by macrophages. Changes following vasectomy persisted in vasovasostomized animals even after 12 months of recanalization, which may contribute to the failure of functional reanastomosis.     

Neuraminidase 1 is a negative regulator of lysosomal exocytosis

Lysosomal exocytosis is a Ca2+-regulated mechanism that involves proteins responsible for cytoskeletal attachment and fusion of lysosomes with the plasma membrane. However, whether luminal lysosomal enzymes contribute to this process remains unknown. Here we show that neuraminidase NEU1 negatively regulates lysosomal exocytosis in hematopoietic cells by processing the sialic acids on the lysosomal membrane protein LAMP-1. In macrophages from NEU1-deficient mice, a model of the disease sialidosis, and in patients' fibroblasts, oversialylated LAMP-1 enhances lysosomal exocytosis. Silencing of LAMP-1 reverts this phenotype by interfering with the docking of lysosomes at the plasma membrane. In neu1-/- mice the excessive exocytosis of serine proteases in the bone niche leads to inactivation of extracellular serpins, premature degradation of VCAM-1, and loss of bone marrow retention. Our findings uncover an unexpected mechanism influencing lysosomal exocytosis and argue that exacerbations of this process form the basis for certain genetic diseases.     

Morphology of the bovine epididymis

The epididymis of the bull was divided into six regions, and morphological differences between regions were studied. The epithelium of all regions contained four cell types: principal and basal epithelial cells, and intraepithelial lymphocytes and macrophages. The epithelium of regions II-V also contained a few apical cells. Principal cells of all regions possessed an endocytotic apparatus including stereocilia underlain by canaliculi, coated vesicles, and subapical vacuoles (up to 1 micron in diameter); however, large vacuoles with a flocculent content and multivesicular bodies (up to 5 microns in diameter) were most numerous in regions II, III, and IV. The unique features of principal cells of region I were the presence of well-developed Golgi bodies, few lipid droplets, and whorls of smooth endoplasmic reticulum in the supranuclear cytoplasm. Numerous mitochondria, distended cisternae of rough endoplasmic reticulum, and dense granules characterized the infranuclear cytoplasm of the principal cells of regions II-VI; however, these features were more developed in region V. Apical cells were characterized by the apical location of the nucleus, many mitochondria in the apical cytoplasm, and few microvilli at the luminal border. Basal cells with few cytoplasmic lipid droplets were present throughout the length of the epididymis but appeared more numerous in region V. Intraepithelial lymphocytes were present at all levels of the epithelium but were never seen in the lumen. Intraepithelial macrophages containing heterogeneous granules, eccentric nuclei, and pseudopods were invariably seen near the basal area of the epithelium in all regions. These observations are discussed in an effort to define the role of each cell type in the epididymal epithelium.     

Effects of ammonia on processing and secretion of precursor and mature lysosomal enzyme from macrophages of normal and pale ear mice: evidence for two distinct pathways

Lysosomal enzymes have been shown to be synthesized as microsomal precursors, which are processed to mature enzymes located in lysosomes. We examined the effect of ammonium chloride on the intracellular processing and secretion of two lysosomal enzymes, beta-glucuronidase and beta-galactosidase, in mouse macrophages. This lysosomotropic drug caused extensive secretion of both precursor and mature enzyme forms within a few hours, as documented by pulse radiolabeling and molecular weight analysis. The normal intracellular route for processing and secretion of precursor enzyme was altered in treated cells. A small percentage of each precursor was delivered to the lysosomal organelle slowly. Most precursor forms traversed the Golgi apparatus, underwent further processing of carbohydrate moieties, and were then secreted in a manner similar to secretory proteins. The lag time for secretion of newly synthesized beta-galactosidase precursor was notably longer than that for the beta-glucuronidase precursor. The source of the secreted mature enzyme was the lysosomal organelle. Macrophages from the pale ear mutant were markedly deficient in secretion of mature lysosomal enzyme but secreted precursor forms normally. These results suggest that ammonia-treated macrophages contain two distinct intracellular pathways for secretion of lysosomal enzymes and that a specific block in the release of lysosomal contents occurs in the pale ear mutant.     

Lysosomal cholesterol accumulation in macrophages leading to coronary atherosclerosis in CD38−/− mice

The disruption in transportation of oxLDL‐derived cholesterol and the subsequent lipid accumulation in macrophages are the hallmark events in atherogenesis. Our recent studies demonstrated that lysosomal Ca²⁺ messenger of nicotinic acid adenine dinucleotide phosphate (NAADP), an enzymatic product of CD38 ADP‐ribosylcyclase (CD38), promoted lipid endocytic trafficking in human fibroblast cells. The current studies are designed to examine the functional role of CD38/NAADP pathway in the regulation of lysosomal cholesterol efflux in atherosclerosis. Oil red O staining showed that oxLDL concentration‐dependently increased lipid buildup in bone marrow‐derived macrophages from both wild type and CD38^?/?, but to a significant higher extent with CD38 gene deletion. Bodipy 493/503 fluorescence staining found that the deposited lipid in macrophages was mainly enclosed in lysosomal organelles and largely enhanced with the blockade of CD38/NAADP pathway. Filipin staining and direct measurement of lysosome fraction further revealed that the free cholesterol constituted a major portion of the total cholesterol segregated in lysosomes. Moreover, in situ assay disclosed that both lysosomal lumen acidity and the acid lipase activity were reduced upon cholesterol buildup in lysosomes. In CD38^?/? mice, treatment with Western diet (12 weeks) produced atherosclerotic damage in coronary artery with striking lysosomal cholesterol sequestration in macrophages. These data provide the first experimental evidence that the proper function of CD38/NAADP pathway plays an essential role in promoting free cholesterol efflux from lysosomes and that a defection of this signalling leads to lysosomal cholesterol accumulation in macrophages and results in coronary atherosclerosis in CD38^?/? mice.     

β-hexosaminidase immunolocalization and α- and β-subunit gene expression in the rat testis and epididymis

β-hexosaminidase is an essential lysosomal enzyme whose absence in man results in a group of disorders, the G_M2 gangliosidoses. β-hexosaminidase activity is many times higher in the epididymis than in other tissues, is present in sperm, and is postulated to be required for mammalian fertilization. To better understand which cells are responsible for β-hexosaminidase expression and how it is regulated in the male reproductive system, we quantitated the mRNA expression of the α- and β-subunits of β-hexosaminidase and carried out immunocytochemical localization studies of the enzyme in the rat testis and epididymis. β-hexosaminidase α-subunit mRNA was abundant and differentially expressed in the adult rat testis and epididymis, at 13- and 2-fold brain levels, respectively. In contrast, β-subunit mRNA levels in the testis and epididymis were 0.3- and 5-fold brain levels. During testis development from 7–91 postnatal days of age, testis levels of α-subunit mRNA increased 10-fold and coincided with the appearance of spermatocytes and spermatids in the epithelium; in contrast, β-subunit mRNA was expressed at low levels throughout testis development. In isolated male germ cells, β-hexosaminidase α-subunit expression was most abundant in haploid round spermatids, whereas the β-subunit mRNA was not detected in germ cells. Within the epididymis both α- and β-subunit mRNA concentrations were highest in the corpus, with 1.5-fold and 9-fold initial segment values, respectively. Light microscopic immunocytochemistry revealed that β-hexosaminidase was localized to Sertoli cells and interstitial macrophages in the testis. In the epididymis, β-hexosaminidase staining was most intense in narrow cells in the initial segment, principal cells in the caput, and proximal corpus, and clear cells throughout the duct. Electron microscopic immunocytochemistry revealed that β-hexosaminidase was predominantly present in lysosomes in Sertoli and epididymal cells. The cellular and regional specificity of β-hexosaminidase immunolocalization suggest an important role for the enzyme in testicular and epididymal functions. Mol. Reprod. Dev. 46:227–242, 1997. © 1997 Wiley-Liss, Inc.     

Altered secretion and accumulation of kidney glycosphingolipids by mouse pigmentation mutants with lysosomal dysfunctions

The kidney and urine glycosphingolipids of five pigmentation mutants which are known to have altered secretion of kidney lysosomal enzymes were examined. Among 34 pigmentation mutants which have been studied (Novak, E. K., Wieland, F., Jahreis, G. P., and Swank, R. T. (1980) Biochem. Genet. 18, 549-561) eight are known to have a 1.5- to 2.5-fold increase in kidney beta-glucuronidase in testosterone-treated females. These mutants appear to have defects in lysosomal processing, and because the mutations are at separate loci, each mutant probably affects different steps in assembly and/or exocytosis of lysosomes and related subcellular organelles. To test whether the neutral glycosphingolipids, galabiglycosylceramides, and globotriglycosylceramides thought to be associated with kidney lysosomes (McCluer, R. H., Williams, M. A., Gross, S. K., and Meisler, M. H. (1981) J. Biol. Chem. 256, 13112-13120) also exhibit abnormal secretion in the mutants with lysosomal enzyme abnormalities, the mutants beige-J, pale ear, light ear, pallid, and ruby eye-2-J were studied. The kidney and urine neutral glycosphingolipids from males of each mutant and C57BL/6J control mice were analyzed by high performance liquid chromatography. Beige-J, light ear, and pale ear showed marked increases in total kidney glycolipids; globotriglycosylceramides accounted for the bulk of the increase. Ruby eye-2-J showed less marked but significantly increased quantities of one galabiglycosylceramide and the globotriglycosylceramides in kidney. Pallid showed no significant increase in total kidney glycolipids but the globotriglycosylceramides appeared slightly elevated. In terms of the decrease in total urinary glycosphingolipids, the mutants fell into 2 categories. Beige-J, light ear, and pale ear were severely affected, whereas ruby eye-2-J and pallid were affected to a much lesser extent. Within the most severely affected group the excretion of the globotriglycosylceramides was more severely affected than that of the galabiglycosylceramides. The galabiglycosylceramides and globotriglycosylceramides appear to be specific markers of lysosomal membranes, but the independent behavior of these two classes of lipids during testosterone induction in normal mice and the differential effects on their secretion by different mutants indicate that they do not always exist in a characteristic ratio in a single type of subcellular organelle. All of the mutants accumulate organelles in their kidney proximal tubules which have distinct morphological characteristics as seen by electron microscopy.     

The effect of the immunization of animals with a live plague vaccine on the lysosome count in organ-specific macrophages

Vital fluorochromatic lysosomes of peritoneal, liver, splenic and lung macrophages of white mice, white rates, and guinea-pigs are studied. Reliable differences in a quantity of lysosomal granules of macrophages from various tissues as well as differences between macrophages from the same tissues of different experimental animals are found. At 21 days after animal immunization with live plague vaccine EV the most significant changes in the number of lysosomal granules are revealed in white mice. The number of lysosomes in guinea pigs increased in 1 and 7 days after vaccination, in 14 days their number became normal.     

Vacuolization of mucolipidosis type II mouse exocrine gland cells represents accumulation of autolysosomes

We previously reported that mice deficient in UDP-GlcNAc:lysosomal enzyme GlcNAc-1-phosphotransferase (mucolipidosis type II or Gnptab -/- mice), the enzyme that initiates the addition of the mannose 6-phosphate lysosomal sorting signal on acid hydrolases, exhibited extensive vacuolization of their exocrine gland cells, while the liver, brain, and muscle appeared grossly unaffected. Similar pathological findings were observed in several exocrine glands of patients with mucolipidosis II. To understand the basis for this cell type-specific abnormality, we analyzed these tissues in Gnptab -/- mice using a combined immunoelectron microscopy and biochemical approach. We demonstrate that the vacuoles in the exocrine glands are enlarged autolysosomes containing undigested cytoplasmic material that accumulate secondary to deficient lysosomal function. Surprisingly, the acid hydrolase levels in these tissues ranged from normal to modestly decreased, in contrast to skin fibroblasts, which accumulate enlarged lysosomes and/or autolysosomes also but exhibit very low levels of acid hydrolases. We propose that the lysosomal defect in the exocrine cells is caused by the combination of increased secretion of the acid hydrolases via the constitutive pathway along with their entrapment in secretory granules. Taken together, our results provide new insights into the mechanisms of the tissue-specific abnormalities seen in mucolipidosis type II.     

Effect of alterations in the size of the vacuolar compartment on pinocytosis in J774.2 macrophages

J774.2 macrophages cultured in medium containing 10 mg/ml sucrose accumulate the sugar by pinocytosis and become highly vacuolated, due to the sugar's osmotic effect within the vacuolar compartment. When such cells are incubated in medium containing 0.5 mg/ml invertase, the enzyme reaches the sucrose vacuoles by pinocytosis, then cleaves the sugar to more permeant monosaccharides. Within 4 hours, the vacuoles shrink to smaller, phase-dense organelles (Cohn and Ehrenreich, 1969, J. Exp. Med., 129:201). We have used this reversible expansion of the lysosomal compartment to address two questions: (1) Does the increased size of the lysosomal compartment affect pinocytic accumulation of solute, and (2) what is the fate of the vacuolar membrane and its soluble content during invertase-induced vacuole shrinkage? Using lucifer yellow (LY) as a probe for pinocytic fluid influx and efflux, we found that vacuolated cells accumulated 30–50% less LY than controls and returned to higher rates of pinocytosis after invertase-induced vacuole shrinkage. A similar reduction in LY accumulation was achieved after feeding cells latex beads to increase the size of the lysosomal compartment. Thus, treatments that increased the size of the lysosomal compartment reduced solute accumulation via pinocytosis. A dramatic shrinkage of LY-containing sucrose vacuoles followed pinocytosis of invertase. Despite this reduction in size of the LY-containing vacuoles, the overall rate of LY efflux did not increase significantly during invertase-induced vacuole collapse. Electron microscopy revealed that during shrinkage, the excess vacuolar membrane was compressed into whorled membranous organelles (residual bodies), with fluid markers (colloidal gold and, by inference, LY) trapped inside. The trapping of LY inside lysosomes as J774.2 macrophages returned to their normal dimensions indicates that nearly all of the surplus membrane contents were removed from circulation as well.     

Differentiation of granuloma cells (epithelioid cells and multinucleated giant cells): a morphometric analysis. Investigations using the model of experimental autoimmune (anti-TBM) tubulo-interstitial nephritis

Morphometric analysis disclosed distinct differences between blood monocytes, tissue monocytes (i.e. immature macrophages), epithelioid cells and multinucleated giant cells as well as phagocytic macrophages (i.e. mature macrophages) in the granuloma model of autoimmune (anti-TBM) tubulo-interstitial nephritis. The numerical density of lysosomes decreased slightly in tissue monocytes compared with blood monocytes but showed a pronounced increase during the formation of epithelioid cells. The lysosomal compartments of epithelioid cells and multinucleated giant cells resembled each other very closely, but the giant cells obviously produced additional lysosomes of small diameter (80-120 nm). Phagocytic macrophages displayed a total numerical density of lysosomes similar to that of tissue monocytes but the mean diameter of the lysosomes was markedly greater. Thus the volume density of lysosomes was highest in phagocytic macrophages. The blood monocytes exhibited the smallest lysosomal compartment. In tissue monocytes, epithelioid cells, and multinucleated giant cells the volume densities of the lysosomes were greater than in blood monocytes and remained relatively constant because the increase in numerical density was counterbalanced by a decrease in mean granule diameter. We found only minor differences in mitochondrial volume densities among the five cell populations. The shape of the mitochondria, however, changed steadily from short rotational ellipsoids in the blood monocytes to rather elongated and slender bodies in the multinucleated giant cells. The results suggest that epithelioid cells and multinucleated giant cells are active cells which may contribute by their specific performances, to the immunologic microenvironment of the granuloma.     

Biogenesis of lysosomes in marshall cells and in cells of the male reproductive system

The mechanism of plasma membrane trafficking and degradation is still poorly understood. This investigation deals with the biogenesis of lysosomes during endocytic flow in Marshall cells and in various cell types of the male reproductive system. Marshall cells were exposed to ammonium chloride (NH4Cl) and leupeptin after labeling with cationic ferritin. In some experiments, the treated cells were immunogold labeled with anti-prosaposin antibody. NH4Cl and leupeptin are lysosomotropic agents that affect the endosomal-lysosomal progression. Testes, efferent ducts and epididymis from mouse mutants with defects affecting plasma membrane degradation were also used to analyze this process. NH4Cl produced a retention of cationic ferritin in endosomes and hindered the endosomal/lysosomal progression. Leupeptin did not affect this process. NH4Cl decreased the labeling of prosaposin in endosomes and lysosomes, while leupeptin increased the labeling of prosaposin in lysosomes. The number of lysosomes per cytoplasmic area was higher in treated cells than in controls. These findings suggest that leupeptin affected lysosomes whereas NH4Cl affected both endosomes and lysosomes. The endosomal and lysosomal accumulation of prosaposin induced by the treatment with NH4Cl and leupeptin indicated that the site of entry of prosaposinwas both the lysosome and endosome. Electron microscopy (EM) of tissues from mouse mutants with defects affecting plasma membrane degradation substantiated these observations. The EM analysis revealed a selective accumulation of multivesicular bodies (MVBs) and the disappearance of lysosomes, in testicular fibroblasts, nonciliated cells of the efferent ducts and principal cells of the epididymis, suggesting that MVBs are precursors of lysosomes. In conclusion: (1) endosomes and MVBs are a required steps for degradation of membranes; (2) endosomes and MVBs are precursors of lysosomes; and (3) endosomes, MVBs, and lysosomes appear to be transient organelles.     

Increase in lysosomal numbers and activity in the rat uterine luminal and glandular epithelium during early pregnancy: a histochemical study

Lysosomal acid phosphatase was studied at both the light and electron microscope level in the rat uterine luminal and glandular epithelium, at oestrous, late dioestrous and day 6 of pregnancy. At the light-microscopic level lysosomal numbers were quantified and statistically analysed. A morphological study was also carried out on the lysosomes at the electron-microscopic level in the above-mentioned stages. Quantification of lysosomal numbers found day 6 of pregnancy to have a significantly higher lysosomal population in the luminal and glandular epithelium compared to non-pregnant states. At the electron-microscopic level the luminal epithelial lysosomes were frequently observed in an invaginated or vesiculated form whereas these characteristics were rarely observed during late dioestrous and were non-existent during oestrous. Generally, the lysosomes appeared more active in the luminal epithelium at day 6 of pregnancy compared to the non-pregnant state. The findings are discussed in reference to the role of the lysosome at the time of blastocyst implantation.     

LAMP proteins are required for fusion of lysosomes with phagosomes

Lysosome-associated membrane proteins 1 and 2 (LAMP-1 and LAMP-2) are delivered to phagosomes during the maturation process. We used cells from LAMP-deficient mice to analyze the role of these proteins in phagosome maturation. Macrophages from LAMP-1- or LAMP-2-deficient mice displayed normal fusion of lysosomes with phagosomes. Because ablation of both the lamp-1 and lamp-2 genes yields an embryonic-lethal phenotype, we were unable to study macrophages from double knockouts. Instead, we reconstituted phagocytosis in murine embryonic fibroblasts (MEFs) by transfection of FcgammaIIA receptors. Phagosomes formed by FcgammaIIA-transfected MEFs obtained from LAMP-1- or LAMP-2- deficient mice acquired lysosomal markers. Remarkably, although FcgammaIIA-transfected MEFs from double-deficient mice ingested particles normally, phagosomal maturation was arrested. LAMP-1 and LAMP-2 double-deficient phagosomes acquired Rab5 and accumulated phosphatidylinositol 3-phosphate, but failed to recruit Rab7 and did not fuse with lysosomes. We attribute the deficiency to impaired organellar motility along microtubules. Time-lapse cinematography revealed that late endosomes/lysosomes as well as phagosomes lacking LAMP-1 and LAMP-2 had reduced ability to move toward the microtubule-organizing center, likely precluding their interaction with each other.