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D Djakiew

Publications and source records attributed to D Djakiew.

At least 37 records · Page 2Linked to original sources
PubMed1993

A 24,500 Da protein derived from rat germ cells is associated with sertoli cell secretory function.

A function and identify of a 24,500 Da protein derived from round spermatids of the rat testis was investigated with a specific polyclonal antiserum raised against RSP-24.5. The proteins released from cultured round spermatids significantly stimulated the secretion of de novo synthesized protein from cultured immature rat Sertoli cells 2.4-fold above control levels. Immunoprecipitation of RSP-24.5 from round spermatid protein further enhanced the stimulation of Sertoli cell protein secretion up to 3.1-fold above control levels, indicating that RSP-24.5 plays a role in the down regulation of Sertoli cell secretion. The antiserum recognized the 24,500 Da protein in Western blots of round spermatid protein, pachytene spermatoctye protein, Sertoli cell lysate and peritubular myoid cell lysate. A 40 amino acid sequence of a cyanogen bromide cleaved internal fragment of RSP-24.5 showed 80.5% homology to a phosphatidylethanolamine binding protein. These results suggest that phosphatidylethanolamine binding protein participates in the negative regulation of Sertoli cell secretory function during spermatogenesis.

Amino Acid Sequence↗
PubMed1993

Chemotaxis and chemokinesis of human prostate tumor cell lines in response to human prostate stromal cell secretory proteins containing a nerve growth factor-like protein.

The migration of three human prostate tumor epithelial cell lines (TSU-pr1, PC-3, DU-145) in response to secreted protein from a human prostate stromal cell line was investigated by using the modified blind-well Boyden chamber assay. Migrated cells were quantified by spectrophotometrically measuring the concentration of crystal violet stain extracted from their nuclei. Cell number was correlated linearly with the concentration of extracted crystal violet stain. All three tumor cell lines showed intrinsic migratory ability in the absence of chemoattractants, such that approximately 1-7% of plated cells migrated across the filter of the Boyden chambers during a 5-h incubation period. Prostate tumor cell migration was significantly enhanced (3-13-fold) in response to stromal cell secretory protein in a dose-dependent manner, whereas bovine serum albumin had no effect on stimulating tumor cell migration. Immunoprecipitation of the stromal cell secreted protein with a nerve growth factor antibody partially and significantly reduced its stimulatory activity for tumor cell migration. A Zigmond-Hirsch matrix assay of tumor cell migration in response to various concentration gradients of stromal cell secreted protein demonstrated both chemotaxis and chemokinesis by all three cell lines. These results are consistent with the stromal cell secretory protein stimulation of chemokinetic tumor cell migration through the capsule of the prostate. Outside of the prostate gland metastasis of tumor cells may occur by chemotaxis to preferential sites containing chemoattractants similar to or related to maintenance factors that can substitute for components of stromal cell secretory protein.

Carrier Proteins↗
PubMed1993

Stromal-epithelial paracrine interactions in the neoplastic rat and human prostate.

Homotypic paracrine interactions in the rat and human prostate have been investigated using prostatic stromal cells and neoplastic epithelial cells (PA-III, rat; TSU-pr1, human). Secretory proteins prepared from each cell type were used to determine the dose dependent regulation of growth (DNA synthesis) of the corresponding homotypic responder cell, as determined by 3H-thymidine incorporation. PA-III secretory protein stimulated rat stromal cell proliferation by 1.8-fold. This stimulatory activity of PA-III protein on stromal cell proliferation was partially reduced (approximately 35%) by treatment with nerve growth factor (NGF) antibody, whereas neither acidic fibroblast growth factor (aFGF) antibody nor basic fibroblast growth factor (bFGF) antibody immunoneutralized the stimulatory activity of PA-III cell protein. In the corresponding opposite interaction, rat stromal cell protein modulated PA-III growth in a biphasic manner. At lower concentrations of stromal cell protein (1.25 micrograms/ml) PA-III cell growth was stimulated by 1.6-fold, whereas at higher concentrations of protein (100 micrograms/ml) PA-III cell growth was inhibited to 60%. Treatment of the stromal cell protein (1.25 micrograms/ml and 100 micrograms/ml) with NGF antibody reduced PA-III cell relative growth to approximately 30% and 5%, respectively. bFGF antibody treatment of stromal cell protein at 1.25 micrograms/ml did not influence relative growth, whereas bFGF antibody treatment of 100 micrograms/ml stromal cell protein reduced relative growth by an additional 40%. Treatment of the stromal cell protein (1.25 micrograms/ml and 100 micrograms/ml) with aFGF antibodies reduced relative growth from that observed at these two protein concentrations by approximately 50% in both cases. Human epithelial TSU-pr1 protein stimulated human stromal cell proliferation approximately 1.7-fold. Treatment of TSU-pr1 protein with NGF antibody resulted in stimulation of human stromal cell proliferation (4-fold). In the corresponding opposite interaction, human stromal cell secretory protein stimulated TSU-pr1 epithelial cell proliferation in a dose-dependent manner up to a maximum of 2.6-fold. This stimulation of TSU-pr1 proliferation by stromal cell secretory protein was reduced to 20% of maximal levels by treatment with antibody against NGF, whereas antibodies against bFGF and aFGF did not significantly influence the stimulatory effect of stromal cell secretory protein mediated proliferation of TSU-pr1 cells. These results suggest that prostatic stromal cells and neoplastic epithelial cells secrete several paracrine factors. One of these factors is nerve growth factor-like, and appears to have a major non-neurotrophic influence on the paracrine regulation of prostatic growth.

3T3 Cells↗
PubMed1993

A 29,000 M(r) protein derived from round spermatids regulates Sertoli cell secretion.

Within the last decade it has become accepted that germ cells can modulate Sertoli cell function in a paracrine interactive manner during the regulation of spermatogenesis. In this context, we undertook to identify a specific factor in round spermatid conditioned media that could stimulate Sertoli cell secretory function. Rat round spermatids isolated by centrifugal elutriation were cultured and the concentrated conditioned media were fractionated by Sephacryl S-200 gel filtration column chromatography. The biological activity of the fractionated round spermatid protein was assessed as stimulation of total protein and transferrin secretion from Sertoli cells that had been isolated from 18-day-old immature rat testes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the gel-filtration fractions showed two predominant proteins of 29,000 and 24,500 molecular weight which coexisted in the fractions containing the greatest biological activity. These two proteins were transferred to a nitrocellulose membrane and excised to raise polyclonal antibodies. Western blot analysis of the 29,000 M(r) protein demonstrated that it specifically occurred in round spermatid conditioned media, whereas no immunoreactive band was observed in either the conditioned media or cell lysates of other testicular cell types such as primary spermatocytes, Sertoli cells and peritubular myoid cells. Following subcellular fractionation of round spermatids by differential centrifugation, the 29,000 M(r) protein was detected by Western blots specifically in the cytosolic fraction of round spermatids, and was absent from the nuclear, mitochondrial, lysosomal and microsomal fractions. The antibody did recognize a few higher molecular bands in the cytosolic fraction which may represent precursor forms of the 29,000 M(r) protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Reduced expression of the low affinity nerve growth factor receptor in benign and malignant human prostate tissue and loss of expression in four human metastatic prostate tumor cell lines.

In the human prostate, a low affinity (p75) nerve growth factor (NGF) receptor (NGF-R) localizes to the epithelia while a NGF-like protein localizes to the stroma. This NGF-like ligand, derived from prostate stromal cell cultures, has been shown to participate in paracrine mediated growth of a human tumor epithelial cell line (TSU-prl) in vitro. In order to investigate the role of the NGF-R in neoplastic growth we have examined the expression of the NGF-R in normal prostate tissues, benign prostatic hyperplasia tissues, adenocarcinoma tissues, and four metastatic tumor cell lines of the human prostate. In primary epithelial cell cultures of normal human prostate the p75 NGF-R was localized by immunocytochemistry to cytoplasmic vesicles. Furthermore, Western blot analysis of the NGF-R in subcellular fractions of normal prostate tissue identified an M(r) 75,000 immunoreactive protein in the microsomal fraction under nonreducing conditions of sodium dodecyl sulfatepolyacrylamide gel electrophoresis. However, microsomal preparations of five prostatic adenocarcinoma and five benign prostatic hyperplasia specimens showed varying immunoreactivity among samples, all of which expressed less of the p75 NGF-R than the normal tissue. Interestingly, microsomal preparations of the human prostatic epithelial cell lines, TSU-prl, DU-145, PC-3, and LNCaP did not show NGF-R expression by immunoblot analysis. Hence, expression of the p75 NGF-R in normal prostate tissue, partial loss of NGF-R expression in benign and malignant prostate tissue, and complete loss of NGF-R expression in the four metastatic tumor cell lines, suggests an inverse association of p75 NGF-R expression with the neoplastic progression of the human prostate.

Adenocarcinoma↗
PubMed1992

Density dependent polarized secretion of a prostatic epithelial cell line.

The polarized secretions (apical/basal) of newly synthesized total protein and proteases from prostatic epithelial sheets of PA-III cells grown in dual compartment chambers were investigated at various cell densities and culture conditions. PA-III cells grown in a serum free defined medium (SFDM) form morphologically polarized monolayers of epithelial cells. These cells secreted their 35S-methionine labeled total protein in a predominantly apical direction (apical/basal ratio, 4-8 fold), with a lesser proportion of protein secreted apically at lower cell densities of the PA-III cell monolayer. PA-III cells grown in 5% fetal calf serum (FCS) are morphologically squamous, comparable to the anaplastic phenotype, and exhibited an inversion of polarized total protein secretion (apical/basal ratio, 0.4-0.9 fold), with an increased proportion of total protein secreted in a basal direction at lower cell densities. Since the culture of PA-III cells in FCS may approximate the anaplastic phenotype we investigated the polarized secretion of proteases from these cells at various cell densities, and compared them with the secretory pattern of protease secretion from polarized PA-III cells cultured in SFDM. At lower cell densities of the PA-III cells grown in FCS the polarity of protease secretion was inverted such that metalloproteinases, tissue type plasminogen activator, and a 72 kD gelatinase were secreted in a predominantly basal direction, as well as urokinase and a gelatinase of 26 kD that were secreted more or less equally into the apical and basal compartments of the chambers. On the other hand, for cultures of PA-III cells grown in SFDM the aforementioned proteases exhibited predominantly an apically directed polarity of secretion. These results suggest that the anaplastic phenotype characterized by a loss of polarized structure may also be characterized by a functional loss or inversion of polarized secretion. The consequences of such a loss or inversion of polarized secretion would be to increase the localized concentrations of proteases along the basal domain of cells thereby facilitating degradation of the basement membrane and interstitial tissue in vivo.

Adenocarcinoma↗
PubMed1992

Distribution of nerve growth factor-like protein and nerve growth factor receptor in human benign prostatic hyperplasia and prostatic adenocarcinoma.

Recent observations from our laboratory have identified a nerve growth factor (NGF)-like protein in conditioned media of stromal cells and neoplastic epithelial cells of the human prostate which mediates paracrine interactive growth of both cell types in vitro. In order to investigate the location of this NGF-like protein in the human prostate in vivo, and whether a nerve growth factor receptor (NGF-R) could be identified, we have carried out immunocytochemical studies on frozen tissue sections of human benign prostatic hyperplasia (BPH), prostatic adenocarcinoma and normal prostatic tissue. The NGF-like protein localized predominantly to the stromal component of BPH, adenocarcinoma and normal (non-cancerous) prostatic tissue. Conversely, the NGF-R localized predominantly to the epithelial cells of these tissues. Renal tissue provided negative controls for both the NGF-like protein and the NGF-R. The testis provided positive controls for both the NGF-like protein and the NGF-R. These results provide corroborative evidence for a NGF-like protein produced by stromal cells which interacts with a NGF-R on the adjacent epithelial cells thereby mediating paracrine interactive growth regulation of the human prostate.

Adenocarcinoma↗
PubMed1991

Regulation of growth by a nerve growth factor-like protein which modulates paracrine interactions between a neoplastic epithelial cell line and stromal cells of the human prostate.

Nerve growth factor-like substance(s) were identified in both conditioned media of a human prostatic tumor epithelial cell line (TSU-pr1) and a human prostatic stromal cell line (HPS) by Western blot analysis and bioassay of neurite outgrowth of PC12 cells. Nerve growth factor-beta (NGF) immunofluorescence was also localized to secretory vesicles in the cytoplasm of both the TSU-pr1 and HPS cells. Western blot of the TSU-pr1 and HPS cell-secreted protein identified an Mr 65,000 major protein which immunoreacted with murine NGF antibody. NGF Western blot of HPS cell-secreted protein also identified an Mr 42,000 minor band under reduced and nonreduced conditions and an Mr 61,000 minor band under reduced conditions. The secreted protein from the TSU-pr1 cells (50 micrograms/ml) and HPS (50 micrograms/ml), as well as murine NGF (50 ng/ml) or human recombinant NGF (50 ng/ml), stimulated neurite outgrowth from PC12 cells. This neurite outgrowth activity was partially inhibited by treatment with NGF antibody. Neither the serum containing growth medium nor bovine serum albumin (50 micrograms/ml) stimulated neurite outgrowth. The NGF-like secretory protein appeared to play a role in the paracrine regulation of prostatic growth between TSU-pr1 cells and HPS cells. The relative growth of TSU-pr1 cells, as indicated by [3H]thymidine incorporation, in response to HPS secretory protein was stimulated 2.8-fold in a dose-dependent manner. In the converse interaction, the relative growth of HPS cells in response to TSU-pr1 secretory protein was stimulated 1.8-fold in a dose-dependent manner. Immunoneutralization of TSU-pr1 and HPS secretory protein was performed with antibody against NGF, acidic fibroblast growth factor, and basic fibroblast growth factor. Removal of the NGF-like protein from the maximal stimulatory dose of TSU-pr1 secretory protein (100 micrograms/ml) with NGF antibody reduced HPS proliferation to 52% of maximal levels, and immunoneutralization of the NGF-like protein in the maximal stimulatory dose of HPS secretory protein (20 micrograms/ml) also reduced TSU-pr1 proliferation to 16% of maximal levels. Addition of normal rabbit serum or prior immunoprecipitation of either TSU-pr1 or HPS secretory protein with antibody against acidic fibroblast growth factor and basic fibroblast growth factor did not inhibit the proliferation of either cell type. These results suggest that TSU-pr1 tumor cells and HPS cells secrete NGF-like protein(s) which modulate their paracrine interactive growth in vitro.

Adrenal Gland Neoplasms↗
PubMed1991

Germ cell mitogenic activity is associated with nerve growth factor-like protein(s).

The mitogenicity of germ cell proteins released from round spermatids (RS) and pachytene spermatocytes (PS) was investigated. Germ cells were isolated by centrifugal elutriation from 90-day-old rat testes and incubated in a supplement enriched culture media that lacked exogenous proteins. The conditioned culture media of RS and PS were dialysed/concentrated and lyophilized to prepare RS protein (RSP) and PS protein (PSP). Mitogenic activity of RSP and PSP was determined by 3H-thymidine incorporation into Swiss 3T3 fibroblasts. RSP and PSP stimulated 3H-thymidine incorporation by fibroblasts in a dose-dependent manner. At a higher concentration of RSP (300 micrograms/ml), fibroblast proliferation was stimulated from 6- to 20-fold of control cultures, whereas PSP (300 micrograms/ml) stimulated fibroblast proliferation 2.5-fold of control cultures. Since RSP exhibited substantially greater mitogenic activity than PSP we further investigated the RSP mitogenic substance(s) by immunoneutralization with antibodies against several growth factors. The mitogenic activity of RSP was significantly reduced by treatment with nerve growth factor (NGF) antibody, while neither the treatment of RSP with acidic fibroblast growth factor (aFGF) antibody, nor basic fibroblast growth factor (bFGF) antibody significantly modified the mitogenic activity of RSP. Interestingly, murine NGF-beta, recombinant human NGF-beta, and bovine serum albumin (BSA) did not exhibit mitogenic activity on 3T3 fibroblasts. Nevertheless, the presence of a NGF-like protein in RS and PS was confirmed by indirect immunofluorescence staining with a murine NGF antibody. Subsequently, a Western blot analysis with the NGF antibody identified two immunoreactive bands of 41 +/- 2 kDa and 51 +/- 1 kDa in both RSP and PSP under reduced conditions. These germ cell NGF-like proteins were apparently different from similarly prepared murine and human NGFs (13 kDa) in their molecular weight. Furthermore, neurite outgrowth from pheochromocytoma cells (PC-12), a functional bioassay for NGF-like activity, was stimulated by addition of RSP and PSP to the culture media of the PC-12 cells. These results demonstrate mitogenic activity in germ cell proteins (RSP and PSP) and identify a NGF-like protein(s) which is associated with most of this activity.

3T3 Cells↗
PubMed1991

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

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 micrograms/ml pachytene spermatocyte protein, whereas maximal stimulation of sulfated glycoprotein-2 and transferrin secretion from Sertoli cells was observed at a dose of 100 micrograms/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.

Animals↗
PubMed1991

Pachytene spermatocytes regulate the secretion of Sertoli cell protein(s) which stimulate Leydig cell steroidogenesis.

The influence of germ cells (pachytene spermatocytes and round spermatids) on the secretion by Sertoli cells of the proteinaceous factor(s) which stimulates Leydig cell steroid biosynthesis was investigated. Sertoli cells from immature rats were cultured on plastic dishes or on Millipore filters impregnated with reconstituted basement membrane in bicameral chambers. Immature rat Sertoli cell secreted proteins (rSCSP; MW greater than 10,000), from conventional cultures, stimulated 4- to 5-fold steroid biosynthesis in normal rat and MA-10 mouse tumor Leydig cells, respectively. MA-10 cells were then used as a bioassay system for most studies, although purified rat Leydig cells were used in some cases to further confirm results obtained with MA-10 cells. rSCSP collected from both the apical and basal compartment of the chambers were examined for their ability to stimulate Leydig cell steroidogenesis. The Leydig cell stimulatory activity from Sertoli cells was found to be secreted in a polarized manner, with 80% of the total bioactivity found in the basal rSCSP. Addition of pachytene spermatocyte proteins (PSP) in the apical compartment of the chambers inhibited, in a time- and concentration-dependent manner, the basally directed Sertoli cell secretion of the Leydig cell stimulatory protein(s) by 85%. Similar results were obtained when freshly isolated pachytene spermatocytes were directly added on top of Sertoli cell epithelial sheets in the apical compartment of the chambers. In contrast, round spermatid proteins (RSP) did not exhibit a comparable effect to that of PSP in regulating the Sertoli cell secretion of the Leydig cell stimulatory activity. These results demonstrate that the Sertoli cell secreted protein(s) which stimulates Leydig cell steroid biosynthesis is secreted in a basally polarized direction, and its secretion is specifically modulated by pachytene spermatocytes.

Androgens↗
PubMed1990

Modulation of Sertoli cell secretory function by rat round spermatid protein(s).

The influence of rat round spermatid protein(s) (RSP) on protein synthesis and secretory function of Sertoli cells was used in the bicameral chamber system. Round spermatids (RS) were purified from 90-day-old rats by centrifugal elutriation. RS were incubated in a supplement-enriched culture medium that lacked exogenous proteins. The RS-conditioned media were dialysed and lyophilized to obtain RSP. Most de novo protein synthesized under basal conditions by Sertoli cells (18-day-old) was secreted into the apical chamber (apical/basal ratio: 3.42). Follicle-stimulating hormone (FSH, 100 ng/ml) stimulated total protein secretion from Sertoli cells by a factor of 1.54. The RSP (100 micrograms/ml) stimulated total protein secretion from Sertoli cells by a factor of 2.33. The enhancement of total Sertoli cell protein secretion by FSH and RSP additively increased by a factor of 2.82. The combined effect of FSH and RSP on total protein secretion from Sertoli cells was dose dependent and saturated at approximately 200 micrograms/ml of RSP. Polarity of total protein secretion from Sertoli cells (apical/basal ratio: 3.42) was stimulated by RSP predominantly in the apical direction (apical/basal ratio: 8.48). The modulation of radiolabeled Sertoli cell secretory proteins (ceruloplasmin, CP; sulfated glycoprotein-2, SGP-2; testins and transferrin, Tf) by cold (non-labeled) RSP was investigated by immunoprecipitation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The secretion of CP, SGP-2 and Tf was stimulated in a dose-dependent manner by the addition of RSP up to a saturating concentration of between 200 and 300 micrograms/ml, whereas the secretion of Sertoli cell testins did not reach saturation at 300 micrograms/ml RSP. These results indicate that FSH and RSP independently modulate Sertoli cell protein secretion, and that Sertoli cell secretory proteins may differentially respond to RSP stimulation.

Animals↗
PubMed1990

Paracrine stimulation of polarized secretion from monolayers of a neoplastic prostatic epithelial cell line by prostatic stromal cell proteins.

The paracrine influence of prostatic stromal cell proteins on a neoplastic prostate cell line (PA-III) was investigated. We have utilized an in vitro experimental model whereby confluent epithelial sheets of PA-III cells are grown on Matrigel-coated filters in bicameral chambers (Millicell-HA). Confluence of the epithelial sheet was confirmed morphologically by electrical resistance measurements and by impedence of [3H]inulin permeability across paracellular channels. Stromal cells were isolated from the ventral prostate of 50-day-old rats by isopyknic Percoll centrifugation. Purity (92%) of the isolated stromal cells was confirmed by indirect immunofluorescence of vimentin intermediate filaments. Prostatic epithelial cells were negative for vimentin immunofluorescence. Prostatic stromal cell secretory proteins with molecular weights greater than 10,000 were placed in the basal reservoir of the bicameral chambers underneath the confluent epithelial sheets of PA-III in a manner that mimics the relationship between stroma and epithelia in vivo. After 24 h incubation the stromal cell proteins increased the [35S]methionine-labeled protein secretion from the epithelial sheet of cells. Trypsinization of the stromal cell secretory proteins eliminated the stimulatory effect on epithelial protein secretion. In addition, conditioned media from Swiss 3T3 fibroblasts, A431 cells, or bovine serum albumin did not stimulate epithelial protein secretion. Two-dimensional gel electrophoresis of the [35S]methionine-labeled epithelial protein secretion showed that the stromal cell proteins induced the secretion of a novel peptide (SE-1) from the basal domain of the epithelial sheet of cells within the first hour of metabolic labeling. These results indicate that stromal cell secretory proteins contain a stimulatory protein that can induce overall protein secretion as well as the vectorial secretion of a novel peptide from the basal domain of PA-III epithelial cells. These results are consistent with a paracrine interaction between epithelial and stromal cells in the regulation of prostatic secretion.

Adenocarcinoma↗
PubMed1990

Characterization of Sertoli cells cultured in the bicameral chamber system: relationship between formation of permeability barriers and polarized secretion of transferrin.

Sertoli cells from immature rats (18 days old) were cultured on Millipore filters impregnated with reconstituted basement membrane in bicameral chambers. Three types of cultures were obtained: 1) confluent monolayer cultures that formed a permeability barrier (impermeable), 2) confluent monolayer cultures that did not form a permeability barrier (permeable), and 3) subconfluent cultures (permeable). The relationships among fluid equilibrium, electrical resistance, and [3H]inulin transport between the apical and basal reservoirs of the chambers were examined. An impermeable confluent monolayer is defined when the cells of the Sertoli cell epithelial sheet are able to prevent hydrodynamic equilibration of fluid levels between the apical and basal reservoirs of a bicameral chamber. That is, a permeability barrier is present between the two sides of the chamber when fluid levels (volumes) do not change. In the impermeable confluent Sertoli cell monolayers, 7.5 +/- 0.6% of added [3H]inulin diffused across the monolayer during a 6-h collection period versus 13.7 +/- 0.5% in permeable cultures. Conversely, the electrical resistance was higher in the impermeable monolayers (41-71 ohm.cm2) than in the permeable layers (less than 33 ohm.cm2). A reciprocal linear relationship (Y = -4.68(X) + 91.50, r = 0.808) exists between inulin flux and electrical resistance, and this relationship is a function of cell density. Transferrin (Tf) was one of a few proteins detected in the basal medium of bicameral chambers, whereas most de novo synthesized proteins were secreted into the apical reservoir of the chamber. No significant differences in the total amount of Tf secreted by impermeable or permeable monolayers of Sertoli cells were observed. However, the Sertoli cell secretion ratios (apical/basal) of Tf during a 15-20-h collection period were 2.03 and 1.57 for impermeable monolayers plated at 2.4 x 10(6) and 3.6 x 10(6) cells/well, respectively, but less than 1.0 in permeable layers of cells. When fewer than 2 x 10(6) Sertoli cells were plated, the apical/basal polarity of Tf secretion declined to below 1 in a 24-h culture period, even though those chambers contained impermeable monolayers (recognized by the lack of hydrodynamic equilibrium). These results indicate that polarized secretion by Sertoli cells is dependent on (1) plating density and (2) formation of an impermeable epithelial sheet.

Animals↗
PubMed1988

In vitro models of differentiated Sertoli cell structure and function.

Primary cultures of Sertoli cells maintained in conventional cultures on plastic culture vessels do not retain many of the structural and functional properties of their in vivo counterparts. Sertoli cell phenotype is better maintained by incorporating certain environmental parameters, intrinsic to the testis, into the Sertoli cell culture system. These environmental parameters include a) high cell density, b) a unique extracellular matrix, c) a semipermeable support between the basal plasma membrane of the cells and blood-derived nutrients in the interstitium, d) chemically distinct microenvironments at the apical and basal surfaces of the cells, and e) cell-to-cell interactions among Sertoli cells and other testicular cell types. Using three variations of Sertoli cell culture we have demonstrated the importance of each of these environmental parameters in obtaining a better Sertoli cell culture model.

Animals↗
PubMed1988

Pachytene spermatocyte proteins influence Sertoli cell function.

Isolated Sertoli cells were cultured on MatrigelTM-coated Millipore filters in bicameral chambers. The Sertoli cells form confluent epithelial sheets that, by virtue of the Sertoli cell tight junctions, form transepithelial permeability barriers between the apical and basal domains of the cells. These Sertoli cells secrete metabolically labeled proteins in a polarized manner. Three peptides, P1 (pI = 4.5-5.0, MW = 70,000), P2 (pI = 4.5-5.0, MW = 50,000), and P3 (pI = 4.0-4.7, MW = 34,000) are secreted apically from the epithelial sheets of Sertoli cells and are not found in basal secretions from the same Sertoli cells. Pachytene spermatocyte-conditioned medium contains proteins released from the germ cells that are uniquely different from the Sertoli cell-secreted proteins. Addition of the pachytene spermatocyte-conditioned medium to the apical reservoir of the bicameral chambers over an epithelial sheet of Sertoli cells stimulated the synthesis and secretion of total protein, transferrin, and specifically induced peptides S1 and S2 from Sertoli cells. As controls, conditioned medium from 3T3 fibroblasts and round spermatids did not stimulate the Sertoli cells. Hence, the ability of pachytene spermatocyte proteins to induce specific Sertoli cell secretion indicates that the pachytene spermatocytes are able to influence their surrounding milieu, and provides further support to the concept of a paracrine interaction between germ cells and Sertoli cells during spermatogenesis.

Animals↗
PubMed1987

Endocytic activity of Sertoli cells grown in bicameral culture chambers.

Immature rat Sertoli cells were cultured for 7 to 14 days on Millipore filters impregnated with a reconstituted basement membrane extract in dual-environment (bicameral) culture chambers. Electron microscopy of the cultured cells revealed the presence of rod-shaped mitochondria, Golgi apparatus, rough endoplasmic reticulum, and Sertoli-Sertoli tight junctions, typical of these cells in vivo. The endocytic activity of both the apical and basal surfaces of the Sertoli cells was examined by either adding alpha 2-macroglobulin (alpha 2-M) conjugated to 20 nm gold particles to the apical chamber or by adding 125I labeled alpha 2-M to the basal chamber. During endocytosis from the apical surface of Sertoli cells, the alpha 2-M-gold particles were bound initially to coated pits and then internalized into coated vesicles within 5 minutes. After 10 minutes, the alpha 2-M-gold was found in multi-vesicular bodies (MVBs) and by 30 minutes it was present in the lysosomes. The proportion of alpha 2-M-gold found within endocytic cell organelles after 1 hour of uptake was used to estimate the approximate time that this ligand spent in each type of organelle. The alpha 2-M-gold was present in coated pits, coated vesicles, multivesicular bodies, and lysosomes for approximately 3, 11, 22, and 24 minutes, respectively. This indicates that the initial stages of endocytosis are rapid, whereas MVBs and lysosomes are relatively long-lived.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗