Prolonged ABP synthesis by Sertoli cells cultured in defined medium

Sertoli cells from 20 day old rats are stimulated to secrete ABP in cultured by a number of hormones and Vitamin A. Maximum ABP secretion in prolonged serum-free culture is seen when FSH, Testosterone, Insulin and Retinol are added to the medium. The plating conditions and type of medium used also affect the total ABP secretion. The decline of ABP secreted into medium by hormone stimulated cultures in prolonged incubations correlates with the detachment of the cells from the culture flask.     

Metabolism of palmitate in cultured rat Sertoli cells

Isolated rat Sertoli cells were incubated in the presence of [1-14C]palmitate at a cell concentration of 1.54 +/- 0.31 mg protein/flask (n = 7). The oxidation of palmitate was concentration dependent and maximal oxidation was obtained at 0.35 mM-palmitate. At a saturating concentration of palmitate the oxidation was linear for at least 6 h. About 65% of the total amount of palmitate oxidized during 5 h at 0.52 mM-palmitate (109 +/- 44 nmol/flask, n = 5) was recovered as CO2 and the rest as acid-soluble compounds. Almost all radioactive acid-soluble compounds which were secreted by the Sertoli cells were shown to be 3-hydroxybutyrate and acetoacetate. The palmitate recovery in cellular lipids and triacylglycerols was 9.4 +/- 5.1 nmol/flask (n = 5) and 3.5 +/- 2.8 nmol/flask (n = 5) respectively. Addition of glucose had no significant effect on palmitate oxidation but caused a 9-fold increase in esterification of palmitate into triacylglycerols. We conclude that cultured rat Sertoli cells can oxidize palmitate to CO2 and ketone bodies and that fatty acids appear to be a major energy substrate for these cells.     

Glutathione metabolism in cultured Sertoli cells and spermatogenic cells from hamsters

Isolated spermatocytes and spermatids from hamsters contained a large amount of glutathione (GSH) (approximately 40 and 30 nmol GSH/mg protein, respectively), but showed a spontaneous decrease of GSH content during prolonged incubation (t1/2 approximately 35 h). Incubation of the germ cells in the presence of the glutathione biosynthesis inhibitor buthionine sulphoximine (BSO) provided evidence that the cells can perform glutathione synthesis. This synthesis, however, was not sufficient to maintain the GSH content of the isolated cells, or to restore the cellular GSH pool after depletion caused by exposure of the cells to the glutathione S-transferase substrate, diethyl maleate (DEM). Cultured Sertoli cells, containing approximately 10 nmol GSH/mg protein, had a more active BSO-sensitive GSH synthesis system. The Sertoli cells, but also tubule fragments containing Sertoli cells and germ cells, were able to restore their GSH pool after DEM-induced depletion. DEM treatment of the tubule fragments resulted in a 90% decrease of the GSH content of the spermatocytes and spermatids present within the fragments. The GSH levels of the tubule fragments and the enclosed germ cells were restored during a subsequent incubation in the absence of DEM. As indicated above, such a recovery was not observed for isolated spermatocytes and spermatids. The results illustrate the importance of Sertoli cell-germ cell interaction, and point to a role of Sertoli cells in glutathione synthesis by the germ cells.     

Uptake and metabolism of retinol in cultured Sertoli cells: evidence for a kinetic model

When cultured Sertoli cells derived from 20-day-old weanling rats were supplied [3H]retinol bound to serum retinol binding protein-transthyretin complex, [3H]retinol was rapidly incorporated and [3H]retinyl esters were synthesized. Within 28 h after administration, 83% of the labeled retinoids were accounted for as retinyl esters (64% as retinyl palmitate). Sertoli cells derived from vitamin A deficient rats and supplied [3H]retinol in culture under identical conditions likewise incorporated [3H]retinol and synthesized retinyl esters. In contrast to normal Sertoli cells, vitamin A deficient Sertoli cells eventually metabolized virtually all of the cellular [3H]retinol to retinyl esters. The primary metabolic fate of retinol administered to Sertoli cell cultures was the synthesis of retinyl esters under all conditions tested. However, administration of [3H]retinol bound to serum retinol binding protein gave metabolic profiles having a higher proportion of retinyl esters and lower proportions of unresolved polar material than administration of [3H]retinol alone. The kinetics of retinol uptake and intracellular retinyl ester synthesis in cultured Sertoli cells was complex. An initial, rapid phase of [3H]retinol incorporation lasting 30 min was followed by a slower rate of incorporation and a concomitant decrease in the intracellular concentration of [3H]retinol. During the time course the specific activity of [3H]retinyl palmitate eventually exceeded that of intracellular [3H]retinol. These observations suggest that two intracellular pools of retinol may exist in Sertoli cells.     

Methods for growth of cultured cells in serum-free medium

Mitochondria prepared from skeletal muscle are typically contaminated with sarcoplasmic reticulum fragments and salt-soluble proteins. These contaminants can be removed by density-gradient centrifugation in Percoll. The resulting mitochondria retain both their original state three respiratory rates and their respiratory control ratios. The method has also been adapted to prepare mitochondria from very small tissue samples.     

Enhancement of melanotic expression in cultured mouse melanoma cells by retinoids

Retinoic acid (RA), which reduces the rate of cell proliferation in S91 mouse melanoma clone C2 cells, was found to stimulate the expression of their melanotic phenotype. RA treatment also induced the extension of long cellular processes. The RA effects on melanogenesis included stimulation of tyrosinase activity and augmentation of cellular melanin content to levels 3- to 4-fold higher than in untreated cultures at similar cell densities. These effects became apparent after 48 hours of exposure to 10(-5) M RA and increased thereafter. Half-maximal stimulation in cells treated for 6 days occurred at 5 X 10(-7) M RA. Although the degrees of melanogenesis enhancement by RA (10(-5) M) and by alpha-melanocyte stimulatory hormone (2 X 10(-7) M) were similar, the former did not alter the intracellular cAMP level, whereas the latter induced a transient 4-fold increase. In high-passage (p28) cells, as well as in low-passage cells (less than p10) treated with tyrosinase inhibitor phenylthiocarbamate, melanin synthesis was suppressed in the absence and presence of RA, yet the ability of RA to inhibit cell proliferation was not compromised. In the presence of the tumor promotor phorbol myristate acetate (greater than 5 X 10(-9) M) melanin synthesis in control as well as in cells exposed to RA was dramatically inhibited. Phorbol which is not active in tumor promotion had no effect on melanogenesis. In addition to RA, other retinoids, such as 13-cis-retinoic acid, retinyl acetate, the TMMP analog of RA and the phenyl analog of RA, but not the pyridyl analog of RA or retinyl palmitate, also inhibited cell growth and enhanced melanin synthesis.     

In vitro metabolism of testosterone by cultured Sertoli cells and the effect of FSH.

Cultures of Sertoli cells isolated from testes of 18-and 36-day-old Long Evans rats were used to investigate their capacity to metabolize testosterone and the effect of FSH on such metabolism. Three different approaches were used: 1) investigation of the metabolism of radiolabeled testosterone under saturating substrate conditions; 2) study of the metabolism of radiolabeled testosterone utilizing trace amounts of high specific activity substrates; 3) the utilization of radioimmunoassay for measurement of estradiol-17 beta. The following steroids were isolated and identified by recrystallization to constant specific acitvity from the control and FSH-treated cultures; testosterone (unconverted substrate), androstenedione, dihydrotestosterone, 3 alpha-hydroxy-5 alpha-androstan-17-one and 5 alpha-androstane-3 alpha, 17 beta-diol. Radioimmunoassay data suggests that the Sertoli cells produce an estradiol-17 beta-like compound from unlabeled testosterone and that this production is stimulated by FSH. However, the radioactive metabolite from all our studies that behaved chromatographically like estradiol--17 beta failed to crystallize to constant specific activity, while in each experiment, authentic radiolabeled estradiol-17 beta added as recovery tracer did. The data demonstrate that : 1) cultures of Sertoli cells from immature rats have 5 alpha-reductase, 3 alpha- and 17 beta-hydroxysteroid oxidoreductase activities; 2) these enzymes may be affected by FSH; 3) based on radiolabeled metabolic techniques, Sertoli cells were unable to biotransform testosterone to estradiol-17 beta even in the presence of FSH.     

Inhibitory effect of Sertoli cell-cultured media on LH binding to mouse Leydig cells in culture

The aim of this study is to examine the influence of Sertoli cells on LH binding to Leydig cells in culture in immature mice. Leydig cells and Sertoli cells were obtained from the testes of immature C57BL/6Ncrj mice and were cultured in serum-free medium for 7 days. The LH binding to Leydig cells and the FSH binding to Sertoli cells were dependent on incubation time, the number of cells, and the amount of labelled hormone added. The dissociation constant for LH binding to Leydig cells was 7.3 x 10(-10) M. Co-culture of Leydig cells with Sertoli cells for 7 days decreased LH binding to Leydig cells. The binding was 34.9% of that to Leydig cells cultured alone. After cultivation of Leydig cells with spent Sertoli cell-cultured medium (SM) for the last 4 days of the 7-day culture period, LH binding to Leydig cells decreased to as low as 17.4% of that of the controls. For the controls, LH binding was measured in Leydig cells cultured in spent Leydig cell-cultured medium (LM). There was no difference between SM- and LM-cultures in the final survival rate or the percentage of cells showing histochemically demonstrated 3 beta-hydroxysteroid dehydrogenase activity. These data suggest that some factor or factors are secreted from the cultured Sertoli cells and inhibit the binding of LH to Leydig cells in culture.     

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.     

Loading efficacy and binding analysis of retinoids with milk proteins: a short review

In this review, the loading efficacies of retinoids with milk proteins are investigated. It has been shown that milk proteins β-lactoglobulin, α-, and β-caseins bind retinol and retinoic acid via hydrophobic, hydrophilic, and H-bonding contacts causing minor alterations of protein secondary structure. Hydrophobic contact is predominant in retinoid–protein conjugation and several amino acids are involved in complex formation, stabilized by H-bonding network. Loading efficacy of retinoid was about 30–50% with retinol forming more stable protein conjugates. Milk proteins can transport retinoid to target molecules.     

Hepatic retinol metabolism. Distribution of retinoids, enzymes, and binding proteins in isolated rat liver cells

The main retinoids and some binding proteins and enzymes involved in retinol metabolism have been quantified in different types of rat liver cells. Hepatic perisinusoidal stellate cells contained 28-34 nmol of retinoids/10(6) cells, and parenchymal liver cells contained 0.5-0.8 nmol of retinoids/10(6) cells, suggesting that as much as 80% of more of total liver retinoids might be stored in stellate cells with the rest stored in parenchymal cells. Isolated endothelial cells and Kupffer cells contained very low levels of retinoids. More than 98% of the retinoids recovered in stellate cells were retinyl esters. Isolated parenchymal and stellate cell preparations both contained considerable retinyl palmitate hydrolase and acyl-CoA:retinol acyltransferase activities. Parenchymal cells accounted for about 75-80% of the total hepatic content of these two enzyme activities, with the rest located in stellate cells. On a cell protein basis, the concentrations of both of these activities were much greater in stellate cells than in parenchymal cells. In contrast, cholesteryl oleate and triolein hydrolase activities were fairly evenly distributed in all types of liver cells. Large amounts of cellular retinol binding proteins were also found in parenchymal and stellate cells. Although parenchymal cells accounted for more than 90% of hepatic cellular retinol binding protein, the concentration of the protein in stellate cells (per unit protein) was 22 X greater than that in parenchymal cells. Stellate cells were also enriched in cellular retinoic acid binding protein. Thus, both parenchymal and stellate cells contain substantial amounts of retinoids and of the enzymes and intracellular binding proteins involved in retinol metabolism. Stellate cells are particularly enriched in these several components.     

A simple freezing medium for serum-free cultured cells

Methylcellulose was found to protect serum-free cultured cells from the deleterious effects of freezing and thawing. We have formulated a simple medium suitable for freezing serum-free cultured cells that consists of 0.1% methylcellulose, 10% dimethylsulfoxide, and MEM or any other serum-free culture medium.     

FSH stimulation of cytosolic protein synthesis in cultured pig Sertoli cells

The effects of FSH on the cytosolic protein synthesis by a primary culture of immature porcine Sertoli cells were studied. Cells were cultured in a chemically-defined medium for 3 days and, on day 3, they were incubated for 40 h in another medium containing labelled amino-acids either in the presence or absence of 50 ng/ml porcine FSH (pFSH). In control cells, about 107 spots (pI in the range of 5 to 8) were identified by a two-dimensional polyacrylamide gel electrophoresis of the radiolabelled cytosolic proteins. pFSH treatment produced a marked increase of seven proteins whose molecular weights and isoelectric points are respectively comprised between 25 to 58 K and 5.5 to 5.8. In addition, pFSH treatment induced a slight but constant increase of two other proteins (mol. wt: 24 and 12 K and isoelectric point: 5.3).     

Metabolic utilization of linoleate and alpha-linolenate in cultured Sertoli cells.

1. The capacity of cultured Sertoli cells to synthesize long-chain polyunsatured fatty acids (PUFA) from the essential fatty acid (EFA) precursors 18:2 n-6 and 18:3 n-3 was tested, and the concentrations of each EFA required to obtain maximal incorporation into membrane lipids were determined. 2. The two EFA were added to the culture medium as free fatty acids complexed to albumin in a molar ratio of 12:1. 3. When the substrates were added individually, the maximal levels of biosynthesis were obtained with 0.7 micrograms/ml of 18:2 n-6 and 2 micrograms/ml of 18:3 n-3. 4. When the two EFA were added together, clear alterations in the behavior of the desaturases with regard to the n-6 and n-3 fatty acids were observed. 5. It was found that a concentration of 0.35 micrograms/ml of each EFA represented the "ideal" required level in order to ensure optimal incorporation of 22-carbon PUFA into the membrane lipids. 6. These results provide the first data on the definition of EFA requirements for Sertoli cells in culture.     

Ouabain-resistant cells cultured in a synthetic medium

Several strains of kidney and liver cells cultured in a synthetic medium were found to be resistant to ouabain. These cell strains were characterized because this resistance may serve as a good marker in genetic studies on somatic cells in chemically defined conditions in the absence of Na+ related growth factors and hormones. The phenotype was stable in the absence of selection for at least two years, and the original strains before adaptation to the synthetic medium were found to have ouabain sensitivity equal to the corresponding cells in the synthetic medium. The resting membrane potential, Na+,K+-ATPase activity, and growth rate of the resistant cells were similar to those of ouabain-sensitive cells. The resistance of the cells was not affected by serum or antibodies against some cytoskeletal proteins and the sensitivity of the Na+,K+-ATPase was not restored by partial purification of the membranes. Western blotting of the Na+,K+-ATPase of the ouabain-resistant cells showed that the molecular weights of its two subunits and its immunoreactivity were similar to those of the enzyme from the ouabain-sensitive strain. Thus the ouabain resistance is caused not by ouabain-like hormone produced by the cells or change in the cytoskeletal system, but by a mutation resulting in expression of an ouabain-resistant ATPase gene.     

Influence of follicle-stimulating hormone on glucose transport by cultured Sertoli cells

Transport of 3-O-methyl-D-[14C]glucose by Sertoli cells cultured in plastic dishes, is competitively inhibited by glucose (Ki 4 microM). The glucose analogue was therefore used to study glucose transport in these cells in which it is not metabolized. Addition of follicle-stimulating hormone (FSH) (10 micrograms/ml) or dibutyryl cyclic AMP (1 mM) to the cells, increases transport of methylglucose by Sertoli cells. The increased transport results from increased influx and involves decrease in Km without change in Vmax. These changes in the kinetics of transport are seen with both FSH and dibutyryl cyclic AMP. FSH does not stimulate transport of methylglucose in peritubular fibroblasts nor in germ cells. In view of the importance of lactate as a substrate for spermatids (Mita and Hall, 1982) it is proposed that stimulation of the transport of glucose by Sertoli cells in response to FSH is important in the increased production of lactate by these cells in response to FSH and hence is one mechanism by which the tropic hormone enables the Sertoli cell to promote spermatogenesis.     

Characterization of insulin-like growth factor binding proteins of cultured rat astroglial and neuronal cells

Insulin-like growth factor binding proteins produced by cultured rat neurons, astrocytes, and rat cell lines BRL-3A and B104 were compared to binding proteins found in rat serum, using affinity labeling, deglycosylation, and Western ligand blotting studies. Each source elaborated an unique pattern of heterogeneous binding proteins. Some of the binding proteins from different sources behaved similarly in each experimental system suggesting that subsets of these binding proteins may be structurally related. In particular, our data suggest that cultured astrocytes and neurons make the major binding protein produced by BRL-3A cells.     

Collagen biosynthesis in cultured rat testicular Sertoli and peritubular myoid cells.

The incorporation of 3H-proline into protein was regarded as a measure of total protein synthesis and the incorporation into hydroxyproline as indicative of collagen synthesis. Relative collagen synthesis (expressed as percent of total protein synthesized) by Sertoli and peritubular myoid cells cultured from 20-22 day old rat testis was estimated. In both secreted and cellular pools, relative collagen synthesis by Sertoli cells was significantly greater than by peritubular myoid cells. Coculture of Sertoli and myoid cells resulted in a significant increase in relative collagen synthesis when compared to monocultures of each cell type. Addition of serum to peritubular myoid cells resulted in a stronger stimulation of relative collagen production. Sertoli cell extracellular matrix inhibited relative collagen synthesis by peritubular myoid cells in the presence or absence of serum. Radioactivity into hydroxyproline as corrected per cellular DNA also showed similar results. Immunolocalization studies confirmed that both cell types synthesize type I and type IV collagens. These results indicate that stimulation of collagen synthesis observed in Sertoli-myoid cell cocultures is due to humoral interactions, rather than extracellular matrix, and Sertoli cell extracellular matrix regulates serum-induced increase in collagen synthesis by peritubular myoid cells.