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. © 1995 Wiley-Liss, Inc.     

The trafficking of prosaposin (SGP-1) and GM2AP to the lysosomes of TM4 Sertoli cells is mediated by sortilin and monomeric adaptor proteins

Prosaposin (SGP-1) and GM2 activator protein (GM2AP) are soluble sphingolipid activator proteins (SAPs) that are targeted to the lysosomal compartment of Sertoli cells to aid hydrolases in the breakdown of glycosphingolipids. To reach the lysosome, most soluble proteins must interact with the mannose 6-phosphate receptor (MPR). To be sorted from the Golgi, the MPR must bind to the Golgi associated, gamma-adaptin homologous, ARF binding proteins (GGAs), a group of monomeric adaptor proteins responsible for the recruitment of clathrin. It is well established, however, that the lysosomes of I-cell disease (ICD) patients have near normal levels of several lysosomal proteins, including prosaposin and GM2AP. ICD results from a mutation in the phosphotransferase that adds mannose 6-phosphate to hydrolases. Thus, prosaposin and GM2AP can traffic to lysosomes in a MPR independent manner. Previous work has demonstrated that an interaction with sphingomyelin in the Golgi membrane is necessary for the targeting of prosaposin by an unknown receptor. Using a TM4 Sertoli cell line, we tested the hypothesis that prosaposin and GM2AP are targeted to the lysosomal compartment via the sortilin receptor, which has been recently shown to have a GGA binding motif. Interestingly, dominant-negative GGAs, unable to bind clathrin to shuttle from the Golgi, prevented the trafficking of prosaposin and GM2AP to lysosomes. A dominant negative construct of sortilin lacking the GGA binding domain retained prosaposin and GM2AP in the Golgi. In conclusion, our results showed that the trafficking of prosaposin and GM2AP to the lysosome is dependent on sortilin.     

Rat clusterin isolated from primary Sertoli cell-enriched culture medium is sulfated glycoprotein-2 (SGP-2)

Clusterin, a glycoprotein originally isolated from ram rete testis fluid, is a dimer composed of monomers with non-identical NH2-terminal amino acid sequences. In view of its possible role in cell-cell interactions in the seminiferous epithelium, we sought to identify such a protein in the rat. Using the bioassay developed for the ovine protein, rat clusterin was purified to apparent homogeneity by HPLC from primary Sertoli cell-enriched culture media. This protein is also a heterodimer consisting of monomers of Mr 43,000 (alpha) and Mr 35,000 (beta). NH2-Terminal amino acid sequence analysis indicated that the alpha subunit has a sequence of NH2-SLMPLSHYGPLSFHNMFQPFFDMIHQAQQA and the beta subunit, NH2-EQEFSDNELQELSTQGSRYVNKEIQNAVQG. These two subunits show marked similarity with the corresponding subunits of ram clusterin isolated from rete testis fluid. Using an antibody against the alpha subunit of rat clusterin, a cDNA clone was isolated from a rat testicular lambda gt11 cDNA library. Analyses of the amino acid sequence derived from the isolated rat clusterin cDNA and of the NH2-terminal amino acid sequences indicate that rat clusterin is identical to a Sertoli cell glycoprotein previously designated sulfated glycoprotein-2.     

Role of epithelial cells of the male excurrent duct system of the rat in the endocytosis or secretion of sulfated glycoprotein-2 (clusterin)

The localization of sulfated glycoprotein-2 (clusterin; SGP-2) was investigated in the rete testis, efferent ducts, and epididymis of the rat using light (LM) and electron (EM) microscope immunocytochemistry. At the LM level, the epithelial cells of the rete testis and efferent ducts demonstrated an intense immunoperoxidase reaction over their apical and supranuclear regions, and sperm in the lumen of the efferent ducts were unreactive. In the EM, gold particles were found exclusively over the endocytic apparatus of these cells. In the proximal area of the epididymal initial segment, an insignificant immunostaining of epithelial cells and sperm was observed. However, the distal area of the initial segment showed a moderate staining over the epithelial principal cells and sperm, while in the intermediate zone of the epididymis a stronger reaction was observed over these cells. The strongest immunoperoxidase reaction was noted in the caput epididymidis, where it formed a distinct mottled pattern. Thus, while some principal cells were intensely stained, others were moderately or weakly stained; a few were completely unreactive. In the corpus and cauda epididymidis, the staining pattern was similar but not as intense. In the EM, only the secretory apparatus of these cells was found to be immunolabeled with gold particles. Sperm in the lumen of these different regions were also labeled. The epithelial clear cells were unreactive throughout the epididymis. Northern blot analysis substantiated these results and showed the presence of highest levels of SGP-2 mRNA in the caput epididymidis, especially in its proximal area, whereas increasingly lower levels were found in the corpus and cauda epididymidis. In summary, these results suggest that testicular SGP-2 dissociates from the sperm during passage through the rete testis and efferent ducts, where it is endocytosed by the epithelial cells lining these regions. In the epididymis, it is replaced by an epididymal SGP-2 that is secreted by the epithelial principal cells of the epididymis. Furthermore, in the epididymis, the principal cells appear to be in different functional states with respect to the secretion of epididymal SGP-2 within a given region of the duct as well as along the epididymal duct.     

Pachytene spermatocyte protein(s) stimulate sertoli cells grown in bicameral chambers: Dose-dependent secretion of ceruloplasmin,sulfated glycoprotein-1, sulfated glycoprotein-2, and transferrin

Summary Interactions between pachytene spermatocytes and Sertoli cells were investigated using the bicameral culture chamber system. Pachytene spermatocytes were isolated from adult rats with a purity in excess of 90% by centrifugal elutriation. The pachytene spermatocytes were cultured in a defined media and pachytene spermatocyte protein prepared from the conditioned media by dialysis and lyophilization. This pachytene spermatocyte protein was reconstituted at various concentrations and incubated with confluent epithelial sheets of immature Sertoli cells cultured in bicameral chambers. Pachytene spermatocyte protein stimulated secretion of total [³⁵S]methionine-labeled protein from Sertoli cells in a dose-dependent manner predominantly in an apical direction. This stimulatory effect of pachytene spermatocyte protein was domain specific from the apical surface of Sertoli cells, and seemed specific for secretion because total intracellular protein did not increase under the influence of pachytene spermatocyte protein. Pachytene spermatocyte protein and follicle-stimulating hormone additively stimulated Sertoli cell secretion. The physicochemical characteristics of the stimulatory pachytene spermatocyte protein are indicative of heat stability, whereas the stimulatory pachytene spermatocyte protein exhibit acid, dithiothreitol and trypsin sensitivity, and partial urea sensitivity. Furthermore, Sertoli cell secretion of ceruloplasmin, sulfated glycoprotein-1, sulfated glycoprotein-2, and transferrin in response to various concentrations of pachytene spermatocyte protein were determined by immunoprecipitate of these [³⁵S]methionine-labeled proteins with polyclonal antibodies. Maximal stimulation of ceruloplasmin and sulfated glycoprotein-1 secretion from Sertoli cells was observed at a dose of 50 μg/ml pachytene spermatocyte protein, whereas maximal stimulation of sulfated glycoprotein-2 and transferrin secretion from Sertoli cells was observed at a dose of 100 μg/ml of pachytene spermatocyte protein. These results suggest that pachytene spermatocytes modulate Sertoli cell secretory function of at least four proteins in the regulation of spermatogenesis. Supported by grant #DCB-8915930 (D. D.) from the National Science Foundation, Washington, DC.     

Localization of sulfated glycoprotein-2 (clusterin) on spermatozoa and in the reproductive tract of the male rat

Sulfated glycoprotein-2 (SGP-2) is one of the major proteins secreted by rat Sertoli cells and epididymal cells in culture. The disulfide-linked dimeric protein secreted by Sertoli cells and found in seminiferous tubule fluid is composed of monomers of Mr 47 000 and 34 000 whereas the epididymal protein exhibits monomers of Mr 40 000 and 29 000. When both forms were chemically or enzymatically deglycosylated, they yielded proteins of similar molecular weight. No modification of the higher molecular weight testicular form by epididymal cells or fluids could be detected in incubation media. SGP-2 mRNA was localized in epididymal epithelium by in situ hybridization. Northern blot analysis indicated the testicular and epididymal mRNAs were of similar size. These findings suggest that the two forms of the protein occur because of tissue-specific post-translational modifications. The detergent-extracted protein from washed testicular spermatozoa is of the higher molecular weight form while epididymal sperm carry the lower molecular weight form. Immunohistochemical evidence suggests that the testicular form is removed prior to the initial segment of the epididymis and the epididymal form is applied in the proximal caput epididymidis. SGP-2 was immunolocalized to the sperm membrane at the ultrastructural level and was distinctly different from the immunolocalization of outer dense fiber proteins and fibrous sheath proteins.     

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.     

Differential expression of extracellular matrix components in rat Sertoli cells.

We studied expression of laminin, fibronectin, and Type IV collagen in the testis by means of immunofluorescence and immunoblot analysis and also examined gene expression of fibronectin using the ribonuclease protection assay. By immunofluorescence on sections from 20-day-old rats, laminin, fibronectin, and Type IV collagen were found in the basement membrane of the seminiferous tubules and in the interstitial regions of the testis. No localization of any extracellular matrix components was found inside the sectioned cells. However, when Sertoli cells were cultured on glass coverslips, laminin and Type IV collagen were both found inside the cells, suggesting new synthesis. In cultured peritubular cells, Type IV collagen, laminin, and fibronectin were found within the cells. When examined by immunoblot analysis, freshly isolated Sertoli and peritubular cells from 20-day-old rats did not demonstrate production of laminin or fibronectin. After 5 days in culture, peritubular cells produced both laminin and fibronectin, whereas cultured Sertoli cells produced only laminin. In contrast, freshly isolated and cultured Sertoli and peritubular cells all produced Type IV collagen. Moreover, the ribonuclease protection assay indicated that the bulk of fibronectin gene expression occurs within the first 10 days of postnatal development, with lower maintenance levels occurring thereafter. These results indicate that in the testis the highest levels of expression of laminin and fibronectin occur during development and in primary cell culture, whereas expression of Type IV collagen is higher at later stages.     

Biosynthesis and molecular cloning of sulfated glycoprotein 2 secreted by rat Sertoli cells

Sulfated glycoprotein 2 (SGP-2) is the major protein secreted by rat Sertoli cells. Pulse-chase labeling shows that SGP-2 is synthesized as a cotranslationally glycosylated 64-kDa precursor that is modified to a negatively charged 73-kDa form before intracellular cleavage to the mature 47- and 34-kDa subunits. A plasmid cDNA library was constructed from immunopurified mRNA, and a recombinant clone containing the entire protein coding sequence of SGP-2 was isolated. The 1857-nucleotide cDNA consists of a 297-nucleotide 5' noncoding segment, a 1341-nucleotide coding segment, and a 219-nucleotide 3' noncoding sequence. The 5' noncoding region contains five ATG codons followed by four short open reading frames. The derived SGP-2 sequence has a molecular weight of 51,379 and contains six potential N-glycosylation sites. Proteolytic processing sites for the preproprotein were determined by amino-terminal sequencing of the isolated SGP-2 subunits. Northern blots show a wide tissue distribution for the 2.0-kb SGP-2 message, and computer sequence analysis indicates a significant relationship between SGP-2 and human apolipoprotein A-I.     

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/μm² 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 aucts. 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. Together these results suggest that the labeling content of SGP-1 within lysosomes of nonciliated cells of the efferent ducts is not dependent on luminal or circulating androgens, nor is it dependent on a testicular factor entering the lumen of the ducts. It does appear, however, that SGP-1 synthesis and targeting to secondary lysosomes is dependent on a pituitary factor that may have a direct or an indirect effect on the nonciliated cells. © 1995 Wiley-Liss, Inc.     

Production of components of extracellular matrix by cultured rat Sertoli cells

Sertoli cells from rats aged 15, 20, and 25 days were cultured in plastic dishes and extracted with Triton X-100 (0.1 percent w/v) or sodium deoxycholate (2 percent w/v). Residues left after extraction were found to contain three proteins characteristic of extracellular matrix (fibronectin, collagen IV, and laminin). These proteins were identified by four methods: indirect immunofluorescence, co-migration with standard proteins on electrophoresis in polyacrylamide gels, immunoblotting (Western blots), and immunoprecipitation after incubating the Sertoli cells with [35S]methionine. In addition, fibronectin was identified by immunoelectron microscopy with a second antibody conjugated to colloidal gold. In the same cell residues, heparan sulfate was tentatively identified by the first of these methods. The cells used in these studies were shown, by electron microscopy, to be essentially pure cultures of Sertoli cells (greater than 95% pure). Since 100 percent of the cells examined showed positive and specific immunofluorescent staining with well-characterized antibodies to the four components of the extracellular matrix, and since studies with colloidal gold revealed the presence of fibronectin closely associated with and inside cells identified by electron microscopy as Sertoli cells, it must be concluded that Sertoli cells synthesize these four proteins and presumably heparan sulfate. Evidently, cultured Sertoli cells can synthesize and secrete some of the components of an extracellular matrix.     

Sulfated glycoprotein-1 (saposin precursor) in the reproductive tract of the male rat

Sulfated glycoprotein-1 is one of the major protein secretion products of rat Sertoli cells in culture. This 70,000 Mr protein shares substantial sequence similarity with human prosaposin, the precursor of lysosomal saposins. Saposins are known to enhance the activity of lipid modifying enzymes presumably by solubilizing the lipids. We report here the immunolocalization of sulfated glycoprotein-1 in the cells and fluid of the male reproductive tract. The protein is present in secondary lysosomes of Sertoli cells and also in the luminal fluid of seminiferous tubules and epididymis. The highest concentrations of the protein are in seminiferous tubule fluid and rete testis fluid, while relatively low amounts are found in cauda epididymal fluid and serum. Sulfated glycoprotein-1 is believed to be involved in degradation of lipids in residual bodies and may also assist in modification of membrane lipids during sperm maturation.     

Cyclic endocytic activity and kinetics of lysosomes in Sertoli cells of the rat: a morphometric analysis

Native ferritin was injected into the rete testis of rats, and seminiferous tubules infused with the tracer were collected 6 h later and prepared for electron microscopic analysis. As a result of internalization of the tracer by Sertoli cells, label was found within 12-66% of the secondary lysosomes, depending on the stage of the cycle of the seminiferous epithelium. The Zeiss MOP-3 instrument was used on selected electron microscope photographs to measure a number of morphometric parameters. Applying appropriate formulae and a computerized program, it was possible to determine the absolute numbers of labeled and unlabeled secondary lysosomes per Sertoli cell for each one of the 14 stages of the cycle. Knowing the duration of these stages, it was also possible to evaluate the turnover kinetics and life span of lysosomes for each stage of the cycle. The percentage of ferritin-labeled lysosomes, regarded as an index of the endocytic activity of Sertoli cells, remained low in stages II to VIII, increased abruptly during stage IX, stayed high during stages X to XIV, and decreased to a low level during stage I of the following cycle. Correspondingly, the turnover of secondary lysosomes was relatively slow and their life span relatively long during stages II through VIII, while the turnover of lysosomes was faster and their life span shorter during stages X through XIV-I of the cycle. During stage IX, there was a sharp drop in the number of lysosomes per Sertoli cell associated with a fast rate of disappearance and a remarkably short life span of less than 4 h for the lysosomes. These features, characteristic of stage IX, are explained by the rapid fusion of lysosomes with residual bodies, which are phagocytosed by Sertoli cells at this particular stage of the cycle. The accelerated endocytosis taking place during stages IX through XIV of the cycle may explain the reduction of the surface area of the adluminal plasma membrane of Sertoli cells as well as the reduction in volume of the tubular lumen observed during these stages. Thus, the demonstrated cyclic endocytic activity of Sertoli cells and several other cyclical events taking place within seminiferous tubules correlate well.     

L-Glutamate in the extracellular space regulates endogenous D-aspartate homeostasis in rat pheochromocytoma MPT1 cells

In previous studies [FEBS Lett. 434 (1998) 231, Arch. Biochem. Biophys. 404 (2002) 92], we demonstrated for the first time that D-aspartate (D-Asp) is synthesized in cultured mammalian cell lines, such as pheochromocytoma 12 (PC12) and its subclone, MPT1. Our current focus is analysis of the dynamics of D-Asp homeostasis in these cells. In this communication, we show that L-glutamate (Glu) and L-Glu transporter substrates in the extracellular space regulate the homeostasis of endogenous D-Asp in MPT1 cells. D-Asp is apparently in dynamic homeostasis, whereby endogenous D-Asp is constantly released into the extracellular space by an undefined mechanism, and continuously and intensively taken up into cells by an L-Glu transporter. Under these conditions, L-Glu and its transporter substrates in the medium may competitively inhibit the uptake of D-Asp via the transporter, resulting in accumulation of the amino acid in the extracellular space. We additionally demonstrate that DL-TBOA, a well-established L-Glu transporter inhibitor, is taken up by the transporter during long time intervals, but not on a short time-scale.     

Biosynthesis and molecular cloning of sulfated glycoprotein 1 secreted by rat Sertoli cells: sequence similarity with the 70-kilodalton precursor to sulfatide/GM1 activator

Sulfated glycoprotein 1 (SGP-1) is one of the abundant proteins secreted by rat Sertoli cells. Pulse-chase labeling shows that SGP-1 is synthesized as a cotranslationally glycosylated 67-kilodalton (kDa) precursor which is posttranslationally modified to a 70-kDa form before secretion to the extracellular space. A plasmid cDNA library was constructed from immunopurified mRNA, and two overlapping clones coding for the entire protein coding sequence were isolated. The cDNAs represent 27 nucleotides of 5' noncoding sequence, 1554 nucleotides of coding sequence, and 594 nucleotides of 3' noncoding sequence. The derived SGP-1 sequence contains 554 amino acids and has a molecular weight of 61,123. Four potential N-glycosylation sites occur within the sequence. An internal region of SGP-1 shows 78% sequence identity with the 67 N-terminal amino acids described for human sulfatide/GM1 activator (SAP-1). Sequence comparisons suggest that SGP-1 is the precursor to sulfatide/GM1 activator; however, the secretion of the protein from Sertoli cells is distinct from the proteolytic processing and lysosomal compartmentalization which have been described for human fibroblasts. The presence of internal sequence similarity suggests that three additional binding sites may occur in SGP-1. Northern blots show similar levels of expression for the 2.6-kilobase SGP-1 mRNA in all tissues examined. The site of SGP-1 synthesis in testis was localized to Sertoli cells by immunofluorescence and in situ hybridization.     

Exposure in utero to di(n-butyl) phthalate alters the vimentin cytoskeleton of fetal rat Sertoli cells and disrupts Sertoli cell-gonocyte contact

Di(n-butyl) phthalate (DBP) is commonly used in personal care products and as a plasticizer to soften consumer plastic products. Male rats exposed to DBP in utero have malformations of the male reproductive tract and testicular atrophy characterized by degeneration of seminiferous epithelium and decreased sperm production. In the fetal testis, in utero exposure to DBP reportedly resulted in reduced testosterone levels, Leydig cell aggregates, and multinucleated gonocytes (MNG). We investigated whether exposure in utero to DBP affects rat fetal Sertoli cells and compromises interactions between Sertoli and germ cells in the developing testis. Histological examination showed that MNG occurred at low frequency in the normal fetal rat testis. Exposure in utero at the dose level of DBP above estimated environmental or occupational human exposure levels significantly increased the number of these abnormal germ cells. Postnatally, MNG exhibited aberrant mitoses and were detected at the basal lamina. MNG were not apoptotic in the fetal and postnatal rat testes, as indicated by TUNEL. Sertoli cells in DBP-exposed fetal testis had retracted apical processes, altered organization of the vimentin cytoskeleton, and abnormal cell-cell contacts with gonocytes. The effect of DBP on Sertoli cell morphology at the level of light microscopy was reversed after birth and cessation of exposure. Our data indicate that fetal Sertoli cells are targeted by exposure in utero to DBP and suggest that abnormal interactions between Sertoli and germ cells during fetal life play a role in the development of MNG.     

Characteristics of lysosomes in the rat placental cells

Six acid hydrolases, cytochrome oxidase, and alkaline phosphatase were demonstrated in 0.25 m sucrose homogenates of rat chorioallantoic placenta. The acid hydrolases were: acid phosphatase, β-glucuronidase, N-acetyl-β-glucosaminidase, β-galactosidase, and acid deoxyribonuclease, showing optimum activity near pH 4.5; cathepsin, with optimum activity near pH 3.6. The free acid hydrolases present in cytoplasmic extracts expressed 20–40% of their total activity. “Total” activity was defined as the enzyme activity observed in the presence of Triton X-100, while “free” activity denoted enzyme activity measured under similar assay conditions except in the presence of sucrose and absence of Triton X-100. The decreased activity or latency in the assays for the free activity of acid phosphatase, acid deoxyribonuclease, and cathepsin persisted after incubation at pH 5 and 37 ° up to an hour. In contrast, this latency did not persist after incubation of the β-glycosidases. Additionally, the free activity of all the designated enzymes of the cytoplasmic extract was in excess of the nonsedimentable activity observed.     

Germ cell binding to rat Sertoli cells in vitro

The interaction between male germ cells and Sertoli cells was studied in vitro by co-incubation experiments using isolated rat germ cells and primary cultures of Sertoli cells made germ cell-free by the differential sensitivity of germ cells to hypotonic shock. The germ cell/Sertoli cell interaction was examined morphologically with phase-contrast and scanning electron microscopy and then quantified by measuring radioactivity bound to Sertoli cell cultures after co-incubation with added [3H]leucine-labeled germ cells. Germ cell binding to Sertoli cell cultures was the result of specific adhesion between these two cell types, and several features of this specific adhesion were observed. First, germ cells adhered to Sertoli cell cultures under conditions during which spleen cells and red blood cells did not. Second, germ cells had a greater affinity for Sertoli cell cultures than they had for cultures of testicular peritubular cells or cerebellar astrocytes. Third, germ cells fixed with paraformaldehyde adhered to live Sertoli cultures while similarly fixed spleen cells adhered less tightly. Neither live nor paraformaldehyde-fixed germ cells adhered to fixed Sertoli cell cultures. Fourth, germ cell binding to Sertoli cell cultures was not immediate but increased steadily and approached a maximum at 4 h of co-incubation. Saturation of germ cell binding to Sertoli cell cultures occurred when more than 4200 germ cells were added per mm2 of Sertoli cell culture surface. Finally, germ cell binding to Sertoli cell cultures was eliminated when co-incubation was performed on ice. Based on these observations, we concluded that germ cell adhesion to Sertoli cells was specific, temperature-dependent, and required a viable Sertoli cell but not necessarily a viable germ cell. These results have important implications for understanding the complex interaction between Sertoli cells and germ cells within the seminiferous tubule and in the design of future experiments probing details of this interaction.