Direct evidence for de-novo synthesis of LH receptors in cultured pig granulosa cells in response to FSH

FSH plus insulin, cortisol, and thyroxine (IFT) stimulated incorporation of dense isotope-containing (2H, 13C, 15N) amino acids into soluble 125I-labelled hCG binding sites. Evidence of new synthesis of binding sites appeared as early as 3 h after the beginning of the pulse-labelling period. By 48 h the majority of detectable soluble 125I-labelled hCG binding sites appeared to be newly synthesized. Studies with FSH + IFT and puromycin indicated that FSH + IFT stimulated synthesis of new LH/hCG binding sites, and that internalization or degradation of LH/hCG binding sites may also require protein synthesis.     

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.     

Sertoli cells from immature rats : in vitro stimulation of steroid metabolism by FSH.

Sertoli cells from 10 day old rats convert androstenedione to testosterone and 5α-androstane-3α,17β-diol, testosterone to 17β-hydroxy-5α-androstan-3-one and 5α-androstane-3α,17β-diol, and 17β-hydroxy-5α-androstan-3-one to 5α-andro-stane-3α,17β-diol after 72 hours $\text{in vitro}$. Conversions of androstenedione to testosterone and 5α-androstane-3α,17β-diol, and testosterone to 5α-androstane-3α,17β-diol were 2 to 3 times greater in FSH treated cultures. Steroid conversion was not stimulated significantly by LH or TSH. The results are interpreted as evidence that in young rats Sertoli steroid metabolism is stimulated by FSH, that Sertoli cells are an androgen target and that FSH may induce or facilitate Sertoli androgen responsiveness.     

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.     

Isolation and culture of FSH responsive Sertoli cells.

Methods for the isolation and culture of enriched populations of Sertoli cells from 20-60 day old rats are described. The identity of the Sertoli cells was verified by bright light and electron microscopy. Freshly isolated Sertoli cells specifically bound follicle stimulating hormone (FSH) but not luteinizing hormone (LH) and responded to FSH stimulation with dramatic increase in cyclic AMP level. Isolated Sertoli cells, maintained in culture for 11 days, showed no evidence of proliferation but retained their characteristic ultrastructural features and FSH binding ability. Incubation of cultured cells with FSH resulted in a significant stimulation of cyclic AMP and androgen binding protein (ABP). Since the freshly isolated or cultured cells were predominantly (greater than 80%) Sertoli cells, these results provide direct evidence that the Sertoli cells represent a primary target site for FSH activity in the testes. The culture method also provides a valuable in vitro model for the study of chronic effects of various agents on the Sertoli cell.     

Characteristics of retinol accumulation from serum retinol-binding protein by cultured Sertoli cells

The uptake of retinol was examined in cultured Sertoli cells when retinol was provided as a complex with the transport protein retinol-binding protein (RBP). Sertoli cells accumulated [3H]retinol in a time- and temperature-dependent manner. At 32 degrees C, the rate of retinol accumulation was biphasic. Accumulation was linear for approximately 1 h, but then accumulation continued at a linear but decreased rate for 23 h. The change in rate of retinol accumulation occurred when the cells had accumulated approximately 0.53 pmol of retinol/micrograms of cellular DNA. This amount of retinol was approximately equal to the cellular content of cellular retinol-binding protein (CRBP). Extraction and HPLC analysis of the cell-associated radioactivity yielded retinol and retinyl esters, indicating that a significant proportion of the accumulated retinol was esterified. Excess unlabeled retinol-RBP competed with [3H]retinol-RBP for [3H]retinol delivery to the cells, indicating that RBP delivery of retinol was a saturable and competable process. However, free [3H]retinol associated with Sertoli cells in a noncompetable manner. The transport constant for specific retinol accumulation from RBP was 3.0 microM, suggesting that any change in the normal circulating retinol-RBP level (approximately 2 microM) would directly affect the rate of retinol accumulation. Neither iodinated nor reductively methylated RBP was accumulated by or tightly bound to Sertoli cells. In addition, energy inhibitors and lysosomal poisons had no effect on [3H]retinol accumulation, indicating that RBP delivery of retinol to Sertoli cells did not occur by endocytosis of the retinol-RBP complex. Competition studies indicated, however, that protein recognition is important in the retinol uptake process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and secretion of retinol-binding protein by cultured rat Sertoli cells.

We report here that retinol-binding protein (RBP) is synthesized and secreted by rat Sertoli cells in culture. This was demonstrated in four ways. First, transthyretin (TTR) bound to Sepharose 4B retained a labeled protein from media collected from Sertoli cells provided with 35S-methionine, under the same conditions as authentic RBP is bound. The protein was co-eluted with authentic RBP by pure water. Second, this same radioactive protein co-eluted with pure RBP upon gel filtration. Third, when subjected to SDS-PAGE, the protein again migrated with pure RBP, as shown by radioautography. Finally, Sertoli cells were incubated with 35S-cysteine and the conditioned medium was put over a TTR-Sepharose column to isolate the radioactive protein, as characterized above. Cysteine residues were oxidized to cysteic acid residues, and the protein was submitted for sequencing through the first ten residues. Radioactivity was located only in the fourth residue, where a cysteine residue is found in rat RBP. This indicates that RBP is secreted by the Sertoli cell and may serve as the carrier of retinol to the developing germ cells, which are known to be dependent upon vitamin A.     

Processing of follitropin and its receptor by cultured pig Sertoli cells. Effect of monensin

Immature pig Sertoli cells, cultured in a chemically defined medium, are able to maintain many of their functional characteristics for at least two weeks. This model was used to investigate the binding, internalization and degradation of 125I-labelled human follitropin (hFSH) and the effects of pig FSH (pFSH) on its own receptors. The binding of 125I-labelled hFSH was dependent on time, temperature and concentration. At 4 degrees C, the apparent steady state was reached in 8-12 h and remained constant for at least 24 h, whereas at 33 degrees C the apparent equilibrium was reached in 4-6 h. Thereafter the total binding declined and by 24 h it was less than 50% of the maximum binding. At 33 degrees C the binding for the hormone to its surface receptor was followed by internalization of the hormone (half-life approximately equal to 1 h) and its degradation (half-life approximately equal to 3 h). The receptor-mediated internalization of hFSH was blocked by phenylarsine oxide. In the presence of the ionophore monensin (20 microM) the rates of binding and internalization were not modified but the degradation rate was much lower (half-life approximately equal to 18 h). Thus, in the presence of monensin, maximum binding increased twofold to threefold, and remained constant for 24 h. This increase was mainly due to an increase of the internalized hormone. When Sertoli cells were exposed to pFSH there was a loss of its own receptor, which was both dose-dependent (ED50 = 250 ng/ml) and time-dependent (t 1/2 = 14 h). Cycloheximide did not modify the FSH-induced down-regulation, whereas monensin enhanced the down-regulation process. These results show that FSH, like other ligands, is internalized and degraded by its target cells and indicate that the hormone-mediated down-regulation is related to the internalization process. However, the discrepancy between the rate of internalization and of hormone-induced down-regulation, suggests that some of the internalized receptors are recycled.     

Age-dependent changes in the in-vitro response of a pig Sertoli cell-enriched population to FSH

The response of pig Sertoli cell-enriched cultures to FSH was investigated by measuring plasminogen activator (PA) secretion in culture, throughout the nonpubertal and prepubertal periods. Sertoli cell-enriched populations could be isolated from birth until a testicular weight of 56 g. FSH elicited a dose-dependent increase in PA secretion by pig Sertoli cell-enriched cultures. The ED50 was minimal for cells coming from testes weighing 10-22 g, and increased more than 2-fold for cells from heavier testes. This suggests that, at the end of the non-pubertal period, an increased FSH sensitivity is important for initiation of spermatogenesis in this species, and that during the prepubertal period Sertoli cells become less sensitive to FSH. The FSH-stimulated PA secretion increased about 10-fold from a testicular weight of 25 g onwards, i.e. when primary spermatocytes appear in seminiferous tubules.     

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.     

Induction of refractoriness to thyrotropin stimulation in cultured thyroid cells. Dependence on new protein synthesis.

Cultured dog thyroid cells were used to investigate the mechanism by which previous exposure to thyrotropin (TSH) induces refractoriness to further TSH stimulation of cellular adenosine 3'-5'-monophosphate (cAMP). Refractoriness of the cAMP response to TSH could not be overcome by exposure of the cells to supramaximal stimulatory concentrations of TSH. Although an unknown factor present in human and fetal calf serum was found to inhibit the thyroid cell cAMP response to TSH, this factor could not account for refractoriness because refractoriness could be induced in the absence of serum. Induction of thyroid refractoriness did not appear to be related to cellular concentrations of cyclic AMP, because equal refractoriness was produced by TSH alone or TSH plus the phosphodiesterase inhibitor, 3-isobutyl-1-methyl xanthine. In addition, preincubation of thyroid cells in 10(-4) M cAMP did not result in subsequent refractoriness. Recovery from the refractory process required almost 24 h. Short term (15 min) stimulation with TSH did not produce thyroid cell refractoriness, and reversal of the stimulation was obtained by thorough washing of the cells. Long term TSH stimulation (16 h), however, resulted in both supramaximal cAMP response to TSH, and inclusion of TSH together with cycloheximide did not produce refractoriness. Cyclic AMP phosphodiesterase activity in thyroid cell homogenate was unaltered by TSH or dibutyryl cyclic AMP pretreatment of the cells for up to 24 h, or cycloheximide for up to 4 h. In contrast, TSH-stimulated, but not F--stimulated, adenylate cyclase activity was reduced in thyroid cell homogenates after preincubation of the cells in TSH. Refractoriness to TSH stimulation was not associated with an alteration in the binding of 125I-TSH to cultured thyroid cells. These studies suggest that the thyroid cAMP response to TSH is modulated by an inhibitory mechanism dependent upon new protein synthesis. TSH stimulation itself increases the degree of this inhibition through a mechanism not involving cAMP.     

Activation of protein kinase C increases proteoglycan synthesis in immature rat Sertoli cells

In order to determine the signal transduction pathways involved in the regulation of proteoglycan (PG) synthesis in immature rat Sertoli cells (SC), we have examined the effect of the tumor promoter phorbol ester PMA (phorbol myristate acetate) on [35S]sulfate and [3H]glucosamine incorporation into PG molecules neosynthesized by cultured rat SC. PMA induced a dose- and time-dependent stimulation of labeled cell-associated PG as determined by quantitative solid phase assay. The overall effect of PMA resulted from enhancement of both glycosylation and catabolism of cell PG, this latter effect leading to a drastic decrease of their residence time in the membrane. Besides these quantitative effects, activation of protein kinase C by PMA induced qualitative changes as reflected by increase in relative proportion of heparan sulfate PG (HSPG) in cell membrane PG. In light of our previous results suggesting an inverse relationship between PG synthesis and FSH responsiveness in immature rat Sertoli cells, the PMA-induced upregulation of cell membrane PG, and particularly HSPG, could constitute one mechanism involved in the repression of FSH-stimulated steroidogenesis induced by PKC activation.     

Nociceptin is upregulated by FSH signaling in Sertoli cells in murine testes

In postnatal testes, follicle-stimulating hormone (FSH) acts on somatic Sertoli cells to activate gene expression directly via an intracellular signaling pathway composed of cAMP, cAMP-dependent protein kinase (PKA), and cAMP-response element-binding protein (CREB), and promotes germ cell development indirectly. Yet, the paracrine factors mediating the FSH effects to germ cells remained elusive. Here we show that nociceptin, known as a neuropeptide, is upregulated by FSH through cAMP/PKA/CREB pathway in Sertoli cells in murine testes. Chromatin immunoprecipitation from Sertoli cells shows that CREB phosphorylated at Ser133 associates with prepronociceptin gene encoding nociceptin. Analyses with Sertoli cells and testes demonstrates that both prepronociceptin mRNA and the nociceptin peptide are induced after FSH signaling is activated. In addition, the nociceptin peptide is induced in testes after 9days post partum following FSH surge. Thus, our findings may identify nociceptin as a novel paracrine mediator of the FSH effects in the regulation of spermatogenesis.     

Brefeldin A inhibits protein synthesis in cultured cells.

The fungal metabolite brefeldin A (BFA) is known to disrupt the Golgi apparatus resulting in redistribution of Golgi proteins to the endoplasmic reticulum and inhibition of protein secretion. BFA was found to inhibit protein synthesis in rat glioma C6 cells by up to 70% between 0.1 and 1 microgram/ml. Inhibition was both time-dependent and reversible. BFA inhibited protein synthesis to varying degrees in a number of other cell lines but not in BFA-resistant marsupial kidney cells. The same concentrations of BFA which inhibited protein synthesis, also blocked the inhibitory effects of Pseudomonas exotoxin and ricin on BFA-sensitive cells. BFA, however, was unable to block the inhibition of protein synthesis by the toxins in the resistant marsupial kidney cells.