Cathepsin A is expressed in a cell- and region-specific manner in the testis and epididymis and is not regulated by testicular or pituitary factors.

The epithelial cells of the testis are involved in the production, differentiation, and sustenance of sperm, and those of the epididymis play a major role in sperm maturation, protection, and storage. These tissues express various proteins that respond differently to androgens. Cathepsin A is a multifunctional lysosomal carboxypeptidase that also functions as a protective and an activator protein for neuraminidase and beta-galactosidase. In this study, cathepsin A was immunolocalized by light and electron microscopy using a polyclonal affinity-purified antibody on the testis and epididymis of normal, orchidectomized with or without testosterone supplementation, efferent duct-ligated, and hypophysectomized adult rats. In normal rats, cathepsin A expression was noted in lysosomes of Sertoli and Leydig cells but not in germ cells of the testis, as well as nonciliated cells of the efferent ducts. In the epididymis, a cell- and region-specific distribution of cathepsin A was noted. In experimentally treated animals, no changes were noted in the expression of cathepsin A. Immunolabeling of tissues examined at the electron microscopic level revealed that lysosomes were reactive. These data indicate cell- and region-specific expression of cathepsin A in cells of the testis and epididymis and also indicate that cathepsin A expression is not regulated by testicular or pituitary factors.

Role of sphingolipids in the transport of prosaposin to the lysosomes.

Prosaposin is the precursor of four lysosomal saposins that promote the degradation of glycosphingolipids (GSLs) by acidic hydrolases. GSLs contain a hydrophobic ceramide moiety, which acts as a membrane anchor, and a hydrophilic oligosaccharide chain that faces the lumen of the Golgi apparatus and extracellular spaces. By using fumonisin B1, PDMP and D609, we tested the hypothesis that sphingolipids mediate the transport of prosaposin to the lysosomes. Fumonisin B1 interferes with the synthesis of ceramide, PDMP blocks the formation of glucosylceramide and D609 blocks the formation of sphingomyelin. Fumonisin B1 produced a 59;-85% decrease in the density of gold particles in the lysosomes of CHO and NRK cells immunolabeled with anti-prosaposin antibody, and a 55% reduction in the lysosomes of CHO cells stably transfected with an expression vector containing a human prosaposin cDNA. To examine whether the mannose 6-phosphate receptor pathway was affected by this treatment, NRK and CHO cells treated or not with fumonisin B1 were labeled with anti-cathepsin A antibody. The results showed no significant differences in labeling of the lysosomes, suggesting that the effect of fumonisin B1 was specific. When fumonisin B1 and D609 were added to the media of transfected CHO cells, a decrease in immunofluorescence with anti-prosaposin antibody was observed by confocal microscopy. PDMP did not cause any reduction in immunoreactivity, indicating that sphingolmyelin appears to be involved in this process. In conclusion, our data support the hypothesis that sphingolipids, possibly sphingomyelin, are involved in the transport of prosaposin to the lysosomes.

Alpha-mannosidases involved in N-glycan processing show cell specificity and distinct subcompartmentalization within the Golgi apparatus of cells in the testis and epididymis.

The Golgi apparatus is enriched in specific enzymes involved in the maturation of carbohydrates of glycoproteins. Among them, alpha-mannosidases IA, IB and II are type II transmembrane Golgi-resident enzymes that remove mannose residues at different stages of N-glycan maturation. alpha-Mannosidases IA and IB trim Man9GlcNAc2 to Man5GlcNAc2, while alpha-mannosidase II acts after GlcNAc transferase I to remove two mannose residues from GlcNAcMan5GlcNAc2 to form GlcNAcMan3GlcNAc2 prior to extension into complex N-glycans by Golgi glycosyltransferases. The objective of this study is to examine the expression as well as the subcellular localization of these Golgi enzymes in the various cells of the male rat reproductive system. Our results show distinct cell-and region-specific expression of the three mannosidases examined. In the testis, only alpha-mannosidase IA and II were detectable in the Golgi apparatus of Sertoli and Leydig cells, and while alpha-mannosidase IB was present in the Golgi apparatus of all germ cells, only the Golgi apparatus of steps 1-7 spermatids was reactive for alpha-mannosidase IA. In the epididymis, principal cells were unreactive for alpha-mannosidase II, but they expressed alpha-mannosidase IB in the initial segment and caput regions, and alpha-mannosidase IA in the corpus and cauda regions. Clear cells expressed alpha-mannosidase II in all epididymal regions, and alpha-mannosidase IB only in the caput and corpus regions. Ultrastructurally, alpha-mannosidase IB was localized mainly over cis saccules, alpha-mannosidase IA was distributed mainly over trans saccules, and alpha-mannosidase II was localized mainly over medial saccules of the Golgi stack. Thus, the cell-specific expression and distinct Golgi subcompartmental localization suggest that these three alpha-mannosidases play different roles during N-glycan maturation.

Sialidase-mediated depletion of GM2 ganglioside in Tay-Sachs neuroglia cells.

Tay-Sachs disease is a severe, inherited disease of the nervous system caused by accumulation of the brain lipid GM2 ganglioside. Mouse models of Tay-Sachs disease have revealed a metabolic bypass of the genetic defect based on the more potent activity of the enzyme sialidase towards GM2. To determine whether increasing the level of sialidase would produce a similar effect in human Tay-Sachs cells, we introduced a human sialidase cDNA into neuroglia cells derived from a Tay-Sachs fetus and demonstrated a dramatic reduction in the accumulated GM2. This outcome confirmed the reversibility of GM2 accumulation and opens the way to pharmacological induction or activation of sialidase for the treatment of human Tay-Sachs disease.

Cloning of the cDNA and gene encoding mouse lysosomal sialidase and correction of sialidase deficiency in human sialidosis and mouse SM/J fibroblasts.

Lysosomal sialidase occurs in a multienzyme complex that also contains beta-galactosidase and cathepsin A. We previously cloned the human lysosomal sialidase cDNA and characterized mutations in human sialidosis patients. Here, we report the cloning and expression of the mouse lysosomal sialidase cDNA and gene. The 1.77 kb cDNA encodes an open reading frame of 408 amino acids which shows high homology to the human lysosomal sialidase (80%), the rat cytosolic sialidase (65%) and viral and bacterial sialidases (50-55%). The sialidase gene is approximately 4 kb long and contains six exons. The five introns range in size from 96 to 1200 bp. Northern blot analysis revealed high expression of multiple sialidase transcripts in kidney and epididymis, moderate levels in brain and spinal cord, and low levels in adrenal, heart, liver, lung and spleen. Transient expression of the cDNA clone in sialidase-deficient SM/J mouse fibroblasts and human sialidosis fibroblasts restored normal levels of sialidase activities in both cell types. Immunocytochemically expressed sialidase co-localized with a lysosomal marker, LAMP2, confirming its lysosomal nature. Since sialidase activity requires its association with beta-galactosidase and cathepsin A, the expression of mouse sialidase within human sialidosis cells underlines the structural similarity between mouse and human enzymes and suggests that the mechanism for complex formation and function is highly conserved.

Apoptotic cell death in mouse models of GM2 gangliosidosis and observations on human Tay-Sachs and Sandhoff diseases.

Tay-Sachs and Sandhoff diseases are autosomal recessive neurodegenerative diseases resulting from the inability to catabolize GM2 ganglioside by beta-hexosaminidase A (Hex A) due to mutations of the alpha subunit (Tay-Sachs disease) or beta subunit (Sandhoff disease) of Hex A. Hex B (beta beta homodimer) is also defective in Sandhoff disease. We previously developed mouse models of both diseases and showed that Hexa-/- (Tay-Sachs) mice remain asymptomatic to at least 1 year of age while Hexb-/- (Sandhoff) mice succumb to a profound neurodegenerative disease by 4-6 months of age. Here we find that neuron death in Hexb-/- mice is associated with apoptosis occurring throughout the CNS, while Hexa-/- mice were minimally involved at the same age. Studies of autopsy samples of brain and spinal cord from human Tay-Sachs and Sandhoff diseases revealed apoptosis in both instances, in keeping with the severe expression of both diseases. We suggest that neuron death is caused by unscheduled apoptosis, implicating accumulated GM2 ganglioside or a derivative in triggering of the apoptotic cascade.

Low density lipoprotein receptor-related protein-1 expression in the testis: regulated expression in Sertoli cells.

The low density lipoprotein receptor-related protein (LRP-1) is a multiligand receptor capable of mediating endocytosis of a wide array of ligands that relate to both lipoprotein metabolism and proteinase regulation. Many of its ligands are proteinases, proteinase-inhibitor complexes, and lipoproteins known to be contained within the luminal fluid of the seminiferous tubules or in the interstitial spaces of the testis. Immunocytochemical analysis was performed to characterize the pattern of expression of LRP-1 in cells of the rat testis. Immunoperoxidase staining localized LRP-1 to the cytoplasm of Sertoli cells. The distribution and intensity of the Sertoli cell staining was found to vary according to the stages of the cycle of the seminiferous epithelium. Staining was weak in the basal cytoplasm of Sertoli cells during stages II-VIII and strong and granular in the supranuclear cytoplasm during stages XII-XIV and stage I of the cycle. Immunogold labeling showed gold particles associated with the basal and adluminal plasma membranes, with endocytic vesicles, and with endosome membranes. Labeling was also observed on the plasma membrane and membranes of the endocytic apparatus in macrophages and Leydig cells in the interstitial space. Infusion of 125I-Labeled LRP-1 antibody into seminiferous tubules followed by radioautography showed silver grains overlaying the ad-luminal plasma membrane of Sertoli cells at time 0 and in endocytic vesicles and endosomes in the supranuclear region of Sertoli cells 10-minutes postinjection. When the 125I-Labeled LRP-1 antibody was injected into the interstitial space, silver grains overlayed the basal plasma membrane and coated endocytic pits of Sertoli cells at time 0 and, at 10 minutes, the grains labeled endosomes located in the basal pole of Sertoli cells. 125I-Labeled LRP-1 antibody also labeled the plasma membrane and the endocytic apparatus of macrophages and Leydig cells. The absence of immunogold labeling and radioautographic silver grains within lysosomes of Sertoli cells, Leydig cells, and macrophages suggests that internalized LRP-1 is recycled back to the cell surface. The presence of LRP-1 in the endocytic compartment of these cells is consistent with its functioning in the clearance of proteases involved in seminiferous tubule remodeling and/ or the uptake of cholesterol-bound lipoproteins necessary for the biosynthesis of testosterone. In conclusion, the results of these studies demonstrated for the first time the presence of LRP-1 receptor in the endocytic compartments of Sertoli cells and interstitial cells of the rat testis.

Cloning, expression and chromosomal mapping of human lysosomal sialidase and characterization of mutations in sialidosis.

Sialidase (neuraminidase, EC 3.2.1.18) catalyses the hydrolysis of terminal sialic acid residues of glyconjugates. Sialidase has been well studied in viruses and bacteria where it destroys the sialic acid-containing receptors at the surface of host cells, and mobilizes bacterial nutrients. In mammals, three types of sialidases, lysosomal, plasma membrane and cytosolic, have been described. For lysosomal sialidase in humans, the primary genetic deficiency results in an autosomal recessive disease, sialidosis, associated with tissue accumulation and urinary excretion of sialylated oligosaccharides and glycolipids. Sialidosis includes two main clinical variants: late-onset, sialidosis type I, characterized by bilateral macular cherry-red spots and myoclonus, and infantile-onset, sialidosis type II, characterized by skeletal dysplasia, mental retardation and hepatosplenomegaly. We report the identification of human lysosomal sialidase cDNA, its cloning, sequencing and expression. Examination of six sialidosis patients revealed three mutations, one frameshift insertion and two missense. We mapped the lysosomal sialidase gene to human chromosome 6 (6p21.3), which is consistent with the previous chromosomal assignment of this gene in proximity to the HLA locus.

Low density lipoprotein receptor-related protein-2 expression in efferent duct and epididymal epithelia: evidence in rats for its in vivo role in endocytosis of apolipoprotein J/clusterin.

Apolipoprotein J/clusterin/sulfated glycoprotein-2 (apo J) disassociates from spermatozoa and is endocytosed by epithelial cells lining the efferent ducts and epididymis. The low density lipoprotein receptor-related protein-2/megalin (LRP-2) has been shown to bind to apo J and mediates its endocytosis and lysosomal degradation in cultured cells. In this study, immunocytological techniques were used to localize LRP-2 in rat efferent ducts and epididymis and to determine whether its expression correlated with those epithelial cells involved in apo J endocytosis. Pronounced LRP-2 immunochemical staining was observed on the apical surfaces of epithelial cells lining the efferent ducts and in the intermediate zone, proximal caput, and corpus and cauda regions of the epididymis. Single immunogold labeling at the electron microscopic level showed LRP-2 to be present within coated pits, endocytic vesicles, and early endosomes of the nonciliated cells of the efferent ducts and the principal cells of the epididymis. In efferent ducts, double immunogold labeling showed both LRP-2 and apo J to be present in endocytic compartments including coated pits, endocytic vesicles, and early endosomes of nonciliated cells. However, while apo J was detected in late endosomes and lysosomes of nonciliated cells, LRP-2 was not. Apical tubules, possibly emerging from late endosomes, contained labeling for LRP-2 but not for apo J. Ciliated cells lying adjacent to nonciliated cells displayed no labeling for either LRP-2 or apo J. These results are consistent with the possibility that LRP-2 serves as an endocytic receptor for apo J in vivo and that after endocytosis the LRP-2 is recycled back to the cell surface while apo J is delivered to the lysosomes for degradation. To provide additional evidence implicating LRP-2 in apo J endocytosis, a receptor-associated protein (RAP), an antagonist of apo J binding to LRP-2, was injected into the efferent duct lumen. Subsequent immunocytological analysis of the efferent duct showed that the RAP treatment abolished the endocytosis of apo J by the nonciliated cells. Taken together, these data indicate that LRP-2 is a likely mediator of apo J endocytosis by the nonciliated efferent duct cells.

The Saccharomyces cerevisiae processing alpha 1,2-mannosidase is localized in the endoplasmic reticulum, independently of known retrieval motifs.

The yeast-specific alpha 1,2-mannosidase, Mns1p, converts Man,GlcNAc2 to a single isomer of Man8GlcNAc2 during N-linked oligosaccharide processing in Saccharomyces cerevisiae. Mns1p is a 68 kDa type II integral membrane glycoprotein with a very short amino terminal cytoplasmic tail of only two amino acids and a large carboxy-terminal catalytic region that is homologous to class 1 alpha 1,2-mannosidases from mammalian and other species. We have used immunofluorescence and immunoelectron microscopy to demonstrate that Mns1p is localized in the endoplasmic reticulum in Saccharomyces cerevisiae. As Mns1p contains none of the known endoplasmic reticulum retrieval motifs (HDEL, KK or RR), these results suggest that Mns1p is localized in the endoplasmic reticulum by a different retentin mechanism.

Expression and tissue distribution of rat sulfated glycoprotein-1 (prosaposin).

Sulfated glycoprotein-1 (SGP-1/prosaposin) exists as a sulfated secreted protein or as a lysosomal precursor of four smaller saposin molecules. The protein exhibits ubiquitous expression, evolutionary conservation, and diverse tissue inducibility. The lysosomal form of SGP-1 plays a role in the hydrolysis of glycolipids and sphingomyelin. The function of the secreted form of SGP-1 is still unclear. However, it could act as a glycolipid transfer protein, since several gangliosides (a series) were found to bind with high affinity to prosaposin. To identify cell types that produce SGP-1 mRNA, we constructed an SGP-1 cDNA and used for screening of different rat tissues by Northern blot analysis. To localize the translation product of SGP-1 transcripts, we immunostained the same tissues with an anti-SGP-1 antibody. The SGP-1 cDNA construct was generated by amplifying a rat testicular Zap cDNA library by PCR (polymerase chain reaction) with two synthetic oligonucleotide primers. A positive signal of 1.7 KB was isolated, subcloned into the pGEM-7Zf (+). Sequence analysis showed a near-identical nucleotide and amino acid similarity to a previous rat SGP-1 cDNA. The majority of the heterogeneites were conservative substitutions. Northern blot analysis demonstrated that all examined rat tissue and organs have SGP-1 mRNA. Immunocytochemistry identified two staining patterns in the cytoplasm of positive cells: (a) a granular reaction characteristic of lysosomes in the supranuclear and basal regions of epithelial cells and in the perinuclear region of neurons; and (b) a homogeneous reaction in the cytoplasm of Sertoli cells, Type II pneumocytes, macrophages, and epithelial cells lining the choroid plexus. The latter staining pattern could be characteristic of cells that exhibit a secretory routing of SGP- 1. The production of SGP-1 by a variety of specialized cells lining fluid compartments suggests that its secreted form has a role in the transport of lipids in biological fluids, possibly by the formation of soluble complexes with glycolipids. Similarly, the lysosomal form of SGP-1/prosaposin and their derived saposins also solubilizes certain glycolipids to promote their degradation by specific hydrolases.

Trafficking of sulfated glycoprotein-1 (prosaposin) to lysosomes or to the extracellular space in rat Sertoli cells.

Sulfated glycoprotein-1 (prosaposin) exists in 2 forms: a 65kDa form targeted to lysosomes and a 70kDa form secreted extracellularly. In order to understand the sorting and targeting mechanisms of the two forms of SGP-1, we have compared their maturation, processing, and secretion in rat Sertoli cells in vivo. Metabolic labeling experiments in vivo demonstrated that the 65kDa form is synthesized first, then post-translationally modified to the 70kDa form of SGP-1. Subcellular fractionation of testicular homogenate was used to obtain Golgi fractions containing up to 50-fold enrichment in galactosyltransferase. Permeabilization of enriched Golgi fractions with saponin released the 70kDa form, but did not affect the 65kDa protein. While excess free mannose 6-phosphate did not release lysosomal SGP-1, it released the 35kDa cathepsin L from Golgi membranes. Using quantitative electron-microscopic immunocytochemistry, the lysosomal contents of SGP-1 were shown to increase significantly after the administration of tunicamycin in vivo. Therefore, the trafficking of the 65kDa form of SGP-1 to the lysosomes appears to be independent of the M6P-receptor pathway. The 70kDa form of SGP-1 was found to aggregate within perforated Golgi fractions in a process which depends on low pH and calcium ions. We conclude that the targeting of the 65kDa form of SGP-1 to the lysosomes involves an early association with Golgi membrane that is independent of mannose 6-phosphate receptors.

Dramatically different phenotypes in mouse models of human Tay-Sachs and Sandhoff diseases.

We have generated mouse models of human Tay-Sachs and Sandhoff diseases by targeted disruption of the Hexa (alpha subunit) or Hexb (beta subunit) genes, respectively, encoding lysosomal beta-hexosaminidase A (structure, alpha) and B (structure, beta beta). Both mutant mice accumulate GM2 ganglioside in brain, much more so in Hexb -/- mice, and the latter also accumulate glycolipid GA2. Hexa -/- mice suffer no obvious behavioral or neurological deficit, while Hexb -/- mice develop a fatal neurodegenerative disease, with spasticity, muscle weakness, rigidity, tremor and ataxia. The Hexb -/- but not the Hexa -/- mice have massive depletion of spinal cord axons as an apparent consequence of neuronal storage of GM2. We propose that Hexa -/- mice escape disease through partial catabolism of accumulated GM2 via GA2 (asialo-GM2) through the combined action of sialidase and beta-hexosaminidase B.

Molecular role of sulfated glycoprotein-1 (SGP-1/prosaposin) in Sertoli cells.

Sulfated Glycoprotein-1 (SGP-1) is a major polypeptide secreted by rat Sertoli cells. Sequence analysis revealed a 70% sequence similarity with human prosaposin and a 80% similarity with mouse prosaposin. Both human and mouse prosaposin are 65-70 kDa proteins cleaved in the lysosomes into four 10-15 kDa proteins designated saposins A, B, C and D. Lysosomal saposins function as enzymatic activators that promote the hydrolysis of certain glycolipids. SGP-1 (70 kDa) was first considered as being exclusively secreted to the extracellular space. However, recent immunocytochemical studies using an anti SGP-1 antibody demonstrated the presence of this protein in Sertoli cell lysosomes. In addition Sertoli cell lysosomes isolated by cellular fractionation were found to contain a 65 kDa form of SGP-1 or lysosomal prosaposin, as well as, the 15 kDa saposins. Morphological and immunocytochemical evidences also indicated that both prosaposin and saposins may reach Sertoli cell phagosomes by lysosomal fusion. These phagosomes contain cytoplasmic residual bodies detached from spermatids during spermiation. Thus, prosaposin and their derived saposins must play a role in the hydrolysis of membrane glycolipids present in phagocytosed residual bodies. On the other hand, the function of the secreted form of SGP-1 is still unclear. However, SGP-1 was seen to interact with the plasma membrane of developing spermatids. Due to its capacity to bind certain types of gangliosides, SGP-1 appears to act as glycolipid transfer between Sertoli cells and the developing spermatids.

Targeting of endogenous sulfated glycoprotein-1 and -2 to lysosomes within nonciliated cells of the efferent ducts during postnatal development of the rat.

Sulfated glycoprotein (SGP)-1 and -2, secretory products of Sertoli cells, are secreted into the lumen of seminiferous tubules where they bind to late spermatids. Once released, the spermatozoa traverse the efferent ducts where these proteins detach from their surface and are endocytosed by the nonciliated cells. In adult animals, SGP-1 and SGP-2 are also synthesized by nonciliated cells and targeted from the Golgi apparatus to lysosomes. The purpose of the present study was to determine the pattern of expression of SGP-1 and SGP-2 within nonciliated cells during postnatal development. The efferent ducts of animals at different postnatal ages were prepared for an electron microscopic immunocytochemical quantitative analysis as well as for Northern blot analysis. The data expressed as labeling content (no. gold particles/micron 2 and taking into account the volume of the endocytic organelles and the cell) revealed that anti-SGP-1 labeling in endosomes of nonciliated cells was minimal at 15, 21, and 29 days of age. On the other hand, the lysosomal labeling content showed a significant increase by day 29 compared to 15 and 21-day-old animals indicating that an endogenous form of SGP-1 was being synthesized by nonciliated cells and targeted to lysosomes. By day 39 a significant increase in endosomal labeling occurred; this was attributed to the endocytosis of Sertoli-derived SGP-1 which coincided with the entry of spermatozoa into the lumen of these ducts at this age. Lysosomal labeling showed further significant increases at days 39, 49, and then again at day 90. Northern blot analysis detected SGP-1 mRNA transcripts at all postnatal ages examined. While decreases or increases in transcripts could not be determined due to the greater amount of tissue present with increasing age, these data taken together support the idea of an endogenous form of SGP-1 being synthesized by nonciliated cells and targeted to lysosomes during postnatal development. In the case of SGP-2, endosomal labeling was minimal at 15, 21, and 29 days of age but was significantly increased by day 39, with similar values at all subsequent ages. The high value at day 39 was attributed to the endocytosis of SGP-2 which coincided with the entry of spermatozoa into the lumen at this age. Lysosomal labeling, on the other hand, was low at days 15 and 21 but peaked at day 29 at a time when endosomal labeling was minimal. These results suggested the synthesis of an endogenous form of SGP-2 which was being targeted to lysosomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential expression of cathepsins B and D in testis and epididymis of adult rats.

Cathepsins are specific proteases in lysosomes that participate in the degradation of proteins, some of which are derived from endocytosis. In this study we examined the immunocytochemical localization of cathepsin B and D antibodies in cells of rat testis and epididymis, using light and electron microscopic immunocytochemistry. In testis, cathepsin D was immunolocalized over lysosomes of Sertoli cells and Leydig cells and on the acrosome of spermatids. Cathepsin B was found over lysosomes of macrophages. Non-ciliated cells of the efferent ducts revealed intense immunogold labeling over lysosomes with both anti-cathepsin B and D antibodies. In epididymis, cathepsins B and D showed marked variations in expression over the different epithelial cells and regional differences for a given cell type. Anti-cathepsin D antibodies showed intense labeling over lysosomes of principal cells in the corpus and proximal cauda. In contrast, anti-cathepsin B antibodies revealed intensely labeled lysosomes of principal cells of the distal initial segment, intermediate zone, and caput epididymidis, with weaker labeling in other regions. Clear cells of the proximal caput epididymidis revealed intensely labeled lysosomes for anti-cathepsin D antibodies. In the distal caput, clear cells showed a variable reaction pattern from intensely labeled to unreactive. Basal cells of teh intermediate zone and proximal caput region were intensely reactive for anti-cathepsin D antibodies. There was no staining over clear or basal cells with anti-cathepsin B antibodies. Taken together, these results demonstrate cell-specific and regional differences in the distribution of cathepsins B and D in cells of the male reproductive system. Such results suggest substrate specificity with regard to protein turnover within lysosomes of cells of testis and epididymis.

Hormonal regulation of sulfated glycoprotein-1 synthesis by nonciliated cells of the efferent ducts of adult rats.

The objective of this study was to define the factors regulating the endogenous production of sulfated glycoprotein-1 (SGP-1) in nonciliated cells of the efferent ducts. To this end we examined five different groups of animals undergoing the following experimental procedures: (1) hypophysectomized animals at 7, 14, and 28 days, (2) 7-day hypophysectomized rats receiving testosterone implants given at various time intervals thereafter, (3) castration at various time intervals up to 7 days, (4) 7-day castrated rats receiving testosterone implants at various time intervals thereafter, and (5) castrated rats given testosterone implants immediately after castration and sacrificed at different time intervals thereafter. Efferent ducts were fixed by perfusion with 4% paraformaldehyde and 0.5% glutaraldehyde in phosphate buffer for quantitative immunocytochemical analysis at the level of the electron microscope. For each experimental condition and their controls, the number of gold particles/micron2 within the endosomal and lysosomal compartments was calculated taking into account the changes in both the volume of the cell and organelles being quantified and expressed as labeling content. The results revealed that hypophysectomy (up to 4 weeks) caused a marked significant decrease in the SGP-1 labeling content of the endosomal and lysosomal compartments. The labeling content of the lysosomal compartment of efferent ducts from rats castrated for up to 1 week did not change significantly. However, there was a significant decrease in the labeling content of endosomes. This decrease is due to SGP-1, which is secreted by Sertoli cells, not being available for uptake in the efferent ducts. These results suggested that testosterone is not required for maintaining the high labeling content of SGP-1 within lysosomes of nonciliated cells, but that a pituitary factor appears to be needed. The administration of testosterone at different intervals to 7-day castrated animals resulted in a significant decrease of lysosomal SGP-1, suggesting that testosterone under these experimental conditions inhibits the production of a pituitary factor that maintains the high labeling content of SGP-1 within lysosomes of the nonciliated cells. Testosterone administered to 7-day hypophysectomized animals over a 24-hr period had no effect on the labeling content of SGP-1 within lysosomes. However, the administration of testosterone to animals immediately following castration showed no differences in the labeling content of SGP-1 within compared to controls.(ABSTRACT TRUNCATED AT 400 WORDS)

Role of sulfated glycoprotein-1 (SGP-1) in the disposal of residual bodies by Sertoli cells of the rat.

Sulfated glycoprotein-1 (SGP-1) is a polypeptide secreted by Sertoli cells in the rat. Sequence analysis revealed a 76% sequence similarity with human prosaposin produced by various cell types. Human prosaposin is a 70 kDa protein which is cleaved in the lysosomes into four 10-15 kDa polypeptides termed saposins A, B, C, and D. The function of lysosomal saposins is to either solubilize certain membrane glycolipids or to form complexes with lysosomal enzymes and/or their glycolipid substrate to facilitate their hydrolysis. The present investigation dealt with the delivery of SGP-1 into the phagosomes of Sertoli cells; these phagosomes contain the residual bodies which detach from the late spermatids at the time of spermiation. Immunogold labeling with anti-SGP-1 antibody was found over Sertoli cell lysosomes, but was absent from phagosomes formed after phagocytosis of spermatid residual bodies in the apical Sertoli cell cytoplasm in stages VIII and early IX of the cycle of the seminiferous epithelium. The phagosomes found later in the basal Sertoli cell cytoplasm in stages IX and X of the cycle became labeled with the antibody as the components of the residual bodies rapidly underwent lysis and disappeared from the Sertoli cells. Sertoli cell lysosomes isolated by cell fractionation (estimated purity of 80%) were found to contain a 65 kDa form of SGP-1 or prosaposin, as well as the 15 kDa polypeptides or saposins. Thus, it appears that this unique lysosomal form of SGP-1 reached the Sertoli cell phagosomes and that their derived polypeptides, the saposins, must play a role in the hydrolysis of membrane glycolipids found in phagocytosed residual bodies.

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 beta-galactosidase in the lysosomes of these cells to test the hypothesis that neuraminidase 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 beta-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 beta-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.

Sulfated glycoprotein-2 synthesized by nonciliated cells of the efferent ducts is targeted to the lysosomal compartment.

The epithelial nonciliated cells of the efferent ducts are specialized in internalizing many luminal substances. The nonciliated cells actively endocytose sulfated glycoprotein-2 (SGP-2), a major secretory protein of Sertoli cells and a homologue of human apolipoprotein J. This study was undertaken to investigate the internalization of Sertoli-derived SGP-2 and synthesis of an endogenous efferent duct form of SGP-2 by nonciliated cells targeted to their secondary lysosomes on animals whose efferent ducts were ligated and/or received injections of tunicamycin. The regulation of synthesis of the endogenous form of SGP-2 within nonciliated cells by hormones in general and testosterone in particular was also examined using hypophysectomized and castrated animals with or without subsequent testosterone replacement. Quantitative electron microscope immunocytochemistry was performed on groups of animals fixed with 4% paraformaldehyde and 0.5% glutaraldehyde in phosphate buffer for each experimental condition and their controls. In each case, the labeling density (number of gold particles/micron 2) within the endosomal (endosomes) and lysosomal (dense multivesicular bodies and secondary lysosomes) compartments was calculated. The results revealed that ligation of the efferent ducts resulted in a significant decrease in the labeling density of the endosomal and lysosomal compartments. However, a baseline of about 18% of controls was still observed in the lysosomal compartment 24 h after ligation. In this compartment similar values were noted 24 h after tunicamycin treatment in conjunction with or without ligation. These results suggest that an endogenous form of SGP-2 is synthesized by nonciliated cells and presumably targeted via small vesicles from the Golgi apparatus to the lysosomal compartment, but that the major portion of SGP-2 within this compartment is derived via endocytosis of testicular SGP-2. Hypophysectomy and castration also showed significant decreases in the labeling densities of these two compartments, but again a baseline level of labeling was noted in the lysosomal compartment. Subsequent testosterone administration to 7-day hypophysectomized or castrated animals had no effect on the labeling density of the lysosomal compartment, as values comparable to the effect of hypophysectomy or castration alone were noted. Taken together these results suggest that the nonciliated cells of the efferent ducts synthesize an endogenous form of SGP-2 that is targeted to the lysosomal compartment and which is not regulated by pituitary factors or testosterone.