Simultaneous regulation of lactate production and fatty acid metabolism by Resveratrol in rat Sertoli cells.

Sertoli cells provide structural and nutritional support for germ cell development. They actively metabolize glucose and convert it into lactate, which is an important source of energy for germ cells. They also oxidize fatty acids (FA), stored as triacylglycerides (TAGs) within lipid droplets (LD), to fulfill their own energy requirements. So, the combined regulation of lactate production and FA metabolism may be relevant to the physiology of seminiferous tubules. Resveratrol (RSV) is a nutritional supplement found primarily in red grape skin that exhibits multiple beneficial health effects: it is cardioprotective, anti-inflammatory, anticancer, and antiaging. The aim of this study was to evaluate the effect of RSV in Sertoli cells lactate production and lipid metabolism. Sertoli cell cultures obtained from 20-day-old rats were incubated for different times with 10 or 50 µM RSV. RSV treatment increased lactate production and glucose consumption. These increments were accompanied by a rise in GLUT1 expression, which is the main glucose transporter in Sertoli cells. On the other hand, RSV decreased LD content and TAG levels. In addition, an increase in ATGL and FAT/CD36 mRNA levels was observed, which suggests augmented cytoplasmatic FA availability. RSV treatment also increased P-ACC levels, which might indicate that RSV promotes FA transport into the mitochondria to be oxidized. An enhanced expression of LCAD and MCAD, enzymes that participate in the oxidation of FA, was also observed. Altogether, these results suggest that RSV simultaneously regulates Sertoli cells lactate production and lipid metabolism, ensuring an adequate energetic balance both in germ and Sertoli cells.

PPARγ activation regulates lipid droplet formation and lactate production in rat Sertoli cells.

Sertoli cells provide the structural and nutritional support for germ cell development; they actively metabolize glucose and convert it to lactate, which is an important source of energy for germ cells. Furthermore, Sertoli cells can oxidize fatty acids, a metabolic process that is assumed to fulfill their own energy requirements. Fatty acids are stored as triacylglycerides within lipid droplets. The regulation of fatty acid storage in conjunction with the regulation of lactate production may thus be relevant to seminiferous tubule physiology. Our aim is to evaluate a possible means of regulation by the PPARγ activation of lipid droplet formation and lactate production. Sertoli cell cultures obtained from 20-day-old rats were incubated with Rosiglitazone (10 µM), a PPARγ activator, for various periods of time (6, 12, 24 and 48 h). Increased triacylglycerides levels and lipid droplet content were observed, accompanied by a rise in the expression of genes for proteins involved in fatty acid storage, such as the fatty acid transporter Cd36, glycerol-3-phosphate-acyltransferases 1 and 3, diacylglycerol acyltransferase 1 and perilipins 1, 2 and 3, all proteins that participate in lipid droplet formation and stabilization. However, PPARγ activation increased lactate production, accompanied by an augmentation in glucose uptake and Glut2 expression. These results taken together suggest that PPARγ activation in Sertoli cells participates in the regulation of lipid storage and lactate production thereby ensuring simultaneously the energetic metabolism for the Sertoli and germ cells.

Probing the degradation and homogeneity of embedded perovskite semiconducting layers in photovoltaic devices by Raman spectroscopy.

The key challenges for perovskite solar cells include their poor stability and film homogeneity. Studying the degradation and homogeneity of perovskite layers within device structures can be challenging but critical to the understanding of stability and effect of processing in real life conditions. We show that Raman spectroscopy (RS) is a unique and powerful method (simple and fast) to probe the degradation of the perovskite film within the device structure and image perovskite formation. We demonstrate that RS can be used to directly probe chemical (PbI2) and physical (dihydrated phase) degradation of a perovskite film, and estimate the relative amount of the degradation species formed, mapping its distribution with ∼1 µm spatial resolution. This has been applied to mapping a large area perovskite module to characterise the efficacy of PbI2 to perovskite conversion. We also use RS to study the degradation species and kinetics under diverse accelerated degradation conditions (temperature and humidity) in situ. These capabilities are difficult to achieve with other methods, presenting RS as an important tool to gain understanding of the degradation and effect of processing on perovskite-based photovoltaic devices.

Activation of PPAR α and PPAR ß/δ regulates Sertoli cell metabolism.

The purpose of this study was to evaluate the existence of a possible simultaneous regulation of fatty acid (FA) metabolism and lactate production by PPAR α and PPAR ß/δ activation in Sertoli cells (SC). SC cultures obtained from 20-day-old rats were incubated with WY14643 or GW0742-pharmacological activators of PPAR α and PPAR ß/δ respectively. The fatty acid transporter CD36, carnitine palmitoyltransferase 1, long- and medium-chain 3-hydroxyacyl-CoA dehydrogenases mRNA levels were analyzed. An increase in the above-mentioned genes in response to activation of both nuclear receptors was observed. Additionally, PPAR ß/δ activation increased lactate production as a consequence of increased pyruvate availability by inhibiting the Pyruvate Dehydrogenase Complex. Altogether, these results suggest that in SC, PPAR α activation participates in the regulation of FA metabolism. On the other hand, PPAR ß/δ activation regulates FA metabolism and lactate production ensuring simultaneously the energetic metabolism for SC and germ cells.

Different signal transduction pathways elicited by basic fibroblast growth factor and interleukin 1ß regulate CREB phosphorylation in Sertoli cells.

BACKGROUND AND AIM: Basic fibroblast growth factor (bFGF) and interleukin 1ß (IL1ß) belong to the set of intratesticular regulators that provide for the fine-tuning of processes implicated in the maintenance of spermatogenesis. The aim of this study was to investigate if bFGF and IL1ß activate CREB, what signaling pathways may be participating and the possible relationship between CREB activation and the regulation of Sertoli cell function. METHODS: Twenty-day-old rat Sertoli cell cultures were used. RESULTS: Cultures stimulated with bFGF and IL1ß produced a time-dependent increment in phosphorylated CREB levels that reached maximal values in 5- and 15-minute incubations respectively. MEK inhibitors--PD98059 and U0126--blocked the effect of bFGF on phosphorylated CREB while a p38-MAPK inhibitor--SB203580--blocked the effect of IL1ß on phosphorylated CREB. A possible correlation between CREB regulation and two Sertoli cell-differentiated functions, Ldh A and transferrin expression, was explored. PD98059 blocked the ability of bFGF to stimulate Ldh A expression and SB203580 blocked the ability of IL1ß to stimulate Ldh A expression and LDH activity. Concerning transferrin, PD98059 and U0126 were able to inhibit the ability of bFGF to stimulate its secre tion. On the contrary, SB203580 was unable to block IL1ß induced increase in transferrin secretion suggesting that the p38-MAPK pathway does not participate in the mechanism of action of the cytokine to regulate transferrin. CONCLUSIONS: The results presented herein suggest that CREB is stimulated in response to bFGF and IL1ß through p42/p44-MAPK and p38-MAPK pathways and that this transcription factor may be partially responsible for the regulation of Sertoli cell function.

Solitary fibrous tumours: unusual aspects of a rare disease.

BACKGROUND: In literature there are only a few descriptions of the typical presentation of solitary fibrous tumours (SFT) and only a few case reports showing its unusual clinical and radiological features. METHODS: We retrospectively evaluated the computed tomography scans of 36 patients presenting with a histological diagnosis of SFT between 1998 and 2008. RESULTS: We present five cases of SFT with an atypical clinical presentation and radiological features. CONCLUSIONS: SFT can occasionally present with unusual radiological features making a differential diagnosis difficult. Even thought imaging plays a fundamental role in the initial diagnostic approach, final diagnosis in only confirmed by biopsy and histology.

Assessment of pulmonary fibrosis after radiotherapy (RT) in breast conserving surgery: comparison between conventional external beam RT (EBRT) and intraoperative RT with electrons (ELIOT).

The aim of this study was to assess the frequency and the grade of RT-induced pulmonary fibrosis in patients who underwent EBRT compared to patients who underwent ELIOT. One-hundred-seventy-eight patients enrolled in a prospective randomized phase III trial to compare the efficacy of ELIOT (a single dose of 21 Gy prescribed at the 90% isodose) versus EBRT (50 Gy to the whole breast plus a 10 Gy boost to the tumour bed), underwent a spiral 16-detector row Computed Tomography (CT) examination to assess RT-induced pulmonary fibrosis: 83 patients in the EBRT arm and 95 in the ELIOT arm. All patients (age range 48-75 years) were affected by unicentric infiltrating carcinoma of the breast with diameter < 2.5 cm. This study was approved by our Institutional Ethical Committee and informed consent was obtained from each patient. Two observers, blinded to patient's randomization, independently evaluated each CT examination and assigned a fibrosis score (Grades 0 to 3). Inter-observer agreement for the fibrosis score was evaluated and a consensus between observers was obtained. Differences in fibrosis score between the two arms were evaluated by Chi Square test and Odds Ratio (OR) with 95% Confidence Intervals (CI). Pulmonary fibrosis was diagnosed in 42 patients (23.6%): 38 (90%) were in the EBRT arm and 4 (10%) in the ELIOT arm (p < 0.0001); twenty-six of them were Grade 1 (one ELIOT), fifteen were Grade 2 (three ELIOT) and one was Grade 3. The post-radiotherapy risk in the EBRT arm to develop at least Grade 1 fibrosis was 19 times higher than in the ELIOT one (OR: 19.20; 95%CI: 6.46-57.14) and 6 times higher to develop at least Grade 2 (OR: 5.70; 95%CI: 1.56-20.76). In conclusion, CT detected pulmonary fibrosis in patients treated with ELIOT is significantly less frequent compared to patients treated with EBRT.

Breast ductal carcinoma and metastatic lymphoma to the contralateral breast in patient with cutaneous non-Hodgkin lymphoma.

Breast lymphoma is a rare condition, both as a primary and a metastatic manifestation. The primary form has an incidence ranging from 0.04% to 0.5% of all breast neoplasms, whereas the metastatic form has an incidence of 0.07%. We hereby report a clinical case of a patient who presented with cutaneous non-Hodgkin lymphoma (NHL) in the left scapulohumeral region treated with surgery followed by radiotherapy (40 Gy total). Three years following radiotherapy, the patient was diagnosed with a left breast infiltrating ductal carcinoma, treated with conservative surgery and adjuvant therapy. The following year, i.e. four years after the initial diagnosis of NHL, two lymphoproliferative relapses occurred: in the left cutaneous scapulohumeral region at the original site of disease, and in the right breast. The aim of this paper is to highlight an uncommon oncologic disorder such as breast lymphoma, highlighting its clinical and radiological manifestations. Some studies reported a possible aetiological role of radiotherapy in the development of breast cancer following treatment of NHL, and in the development of breast cancer following treatment of Hodgkin lymphoma, which could potentially explain our findings.

Risk factors for complications of CT-guided lung biopsies.

PURPOSE: This study assessed the risk factors for pneumothorax and intrapulmonary haemorrhage after computed tomography (CT)-guided lung biopsies. MATERIALS AND METHODS: CT-guided lung biopsies performed between January 2007 and July 2008 were retrospectively evaluated to select the study cohort. Whenever possible, emphysema was quantified by using dedicated software. Features related to the patient, the lesion and the needle and its intrapulmonary path were recorded, along with the pathology findings and operators' experience. The occurrence of pneumothorax and parenchymal haemorrhage was recorded. Univariate and multivariate statistical analyses were performed to assess the association between risk factors and complications. P values <0.05 were considered significant. RESULTS: In 157/222 of the procedures considered, complications were associated with small lesion size and length of the intrapulmonary needle path. Transfissural course and type of needle were associated with pneumothorax using univariate analysis, whereas transfissural course was associated with intrapulmonary haemorrhage using both univariate and multivariate analysis. Emphysema, nodule type, patient position, access site and needle diameter were not significant. Fine-needle aspirates and operator experience were significantly correlated with inadequate biopsy samples. CONCLUSIONS: The size of the lesion and the length of the intrapulmonary trajectory are risk factors for pneumothorax and parenchymal haemorrhage. The transfissural course of the needles is frequently related to pneumothorax and intrapulmonary haemorrhage, and the type of the needle is related to pneumothorax.

Adenosine regulates Sertoli cell function by activating AMPK.

This work evaluates adenosine effects on Sertoli cell functions, which are different to those resulting from occupancy of purinergic receptors. The effects of adenosine and N(6)-cyclohexyladenosine (CHA) - an A(1) receptor agonist resistant to cellular uptake - on Sertoli cell physiology were compared. Adenosine but not CHA increased lactate production, glucose uptake, GLUT1, LDHA and MCT4 mRNA levels, and stabilized ZO-1 protein at the cell membrane. These differential effects suggested a mechanism of action of adenosine that cannot be solely explained by occupancy of type A(1) purinergic receptors. Activation by adenosine but not by CHA of AMPK was observed. AMPK participation in lactate production and ZO-1 stabilization was confirmed by utilizing specific inhibitors. Altogether, these results suggest that activation of AMPK by adenosine promotes lactate offer to germ cells and cooperates in the maintenance of junctional complex integrity, thus contributing to the preservation of an optimum microenvironment for a successful spermatogenesis.

FSH activates phosphatidylinositol 3-kinase/protein kinase B signaling pathway in 20-day-old Sertoli cells independently of IGF-I.

The gonadotropin FSH plays a key role in the control of Sertoli cell function. The FSH molecular mechanism of action is best recognized for its stimulation of the adenylyl cyclase/cAMP pathway. However, other signaling events have also been demonstrated in Sertoli cells. We have recently presented evidence that FSH can stimulate the phosphatidylinositol 3-kinase/protein kinase B (PI3K/PKB) pathway in 20-day-old Sertoli cells. At the same time, it was proposed that in 8-day-old Sertoli cells the effects of FSH on phosphorylated PKB (P-PKB) levels can be explained by a combination of increased secretion of endogenous IGF-I, decreased IGF-binding protein-3 (IGFBP-3) production, and a synergistic action of FSH on IGF-I-dependent PI3K activation. The aim of the present study was to determine whether the effect of FSH on 20-day-old Sertoli cells is mediated by IGF-I secretion. Twenty-day-old rat Sertoli cell cultures were used. FSH stimulation produced a time-dependent increment in P-PKB levels reaching maximal values in 60-min incubations. IGF-I stimulation was also time-dependent reaching maximal values in 15-min incubations. On the other hand, stimulation of the cultures with FSH showed time-dependent inhibition in phosphorylated mitogen-activated protein kinase (P-MAPK) levels. In sharp contrast, stimulation of the cultures with IGF-I showed time-dependent increments in P-MAPK levels reaching maximal stimulus in 15-min incubations. In order to rule out an IGF-I action on FSH stimulation of P-PKB levels, the effect of a specific IGF-I antibody on the ability of both hormones to increase P-PKB levels was evaluated. As expected, the antibody inhibited IGF-I stimulation of P-PKB levels. However, simultaneous addition of an IGF-I antibody with FSH did not modify the ability of the hormone to increase P-PKB levels. The next set of experiments intended to analyze the relevance of a PI3K/PKB pathway to two biological responses of Sertoli cells to FSH and IGF-I. The PI3K inhibitor, wortmannin, dose-dependently decreased FSH-stimulated lactate and transferrin production. On the other hand, wortmannin was not able to modify the ability of IGF-I to stimulate these metabolic events. In addition, the analysis of the participation of a MAPK pathway in IGF-I regulation of Sertoli cell biological responses showed that the MAPK kinase inhibitors, PD98059 and U0126, decreased IGF-I-stimulated transferrin secretion while not modifying IGF-I-stimulated lactate levels. In summary, results obtained so far support the hypothesis that FSH action on P-PKB levels and Sertoli cell metabolism in 20-day-old animals is not mediated by autocrine regulation of an IGF-I/ IGFBP-3 axis as previously proposed in 8-day-old Sertoli cells.

Assessment of the roles of mitogen-activated protein kinase and phosphatidyl inositol 3-kinase/protein kinase B pathways in the basic fibroblast growth factor regulation of Sertoli cell function.

Basic fibroblast growth factor (bFGF) belongs to the large set of intratesticular regulators that provide the fine tuning of cellular processes implicated in the maintenance of spermatogenesis. The aim of the present study was to determine the participation of mitogen-activated protein kinase (MAPK) and phosphatidyl inositol 3-kinase/protein kinase B (PI3K/PKB) pathways in bFGF regulation of Sertoli cell function. Twenty-day-old rat Sertoli cell cultures were used. Stimulation of the cultures with bFGF showed a time-dependent increment in phosphorylated MAPK and PKB levels that reached maximal values in 5-min incubations. MAPK kinase inhibitors U0126 (U) and PD98059 (PD) and a PI3K inhibitor wortmannin (W) were able to block the stimulatory effects of bFGF on phosphorylated MAPK and PKB levels respectively. The participation of MAPK- and PI3K/PKB-signaling pathways in the regulation by bFGF of two well-known Sertoli cell-differentiated functions, lactate and transferrin production, was next explored. As for lactate production, PD and W did not modify the ability of bFGF to stimulate lactate production. However, a combination of PD and W partially impaired the increase in lactate production elicited by bFGF. The participation of MAPK- and PI3K/PKB-signaling pathways in the regulation by bFGF of glucose uptake and lactate dehydrogenase (LDH) activity was also analysed. In this respect, it was observed that W markedly decreased basal and bFGF-stimulated glucose uptake and that U and PD did not modify it. On the other hand, U and PD decreased the stimulation of LDH activity by bFGF whereas W did not modify it. As for transferrin production, while both MAPK kinase inhibitors partially decreased the ability of bFGF to stimulate transferrin secretion, the PI3K inhibitor did not modify it. In summary, the results demonstrated that bFGF stimulates MAPK- and PI3K/PKB-dependent pathways in rat Sertoli cells. Moreover, these results showed that while bFGF utilizes the MAPK pathway to regulate transferrin production and LDH activity, it uses the PI3K/PKB pathway to regulate glucose transport into the cell.

Regulation of rat Sertoli cell function by FSH: possible role of phosphatidylinositol 3-kinase/protein kinase B pathway.

The FSH molecular mechanism of action is best recognized for its stimulation of the adenylyl cyclase/cAMP pathway via activation of a G protein. Recently, links between cAMP, phosphatidylinositol 3-kinase (PI3K) and protein kinase B (PKB) signaling pathways in thyroid and granulosa cells have been observed. The aim of this study was to investigate the possible role of the PI3K/PKB pathway in FSH regulation of Sertoli cell function. Twenty-day-old rat Sertoli cell cultures were used. An increase in phosphorylated PKB (P-PKB) levels in response to FSH and dibutyryl-cAMP was observed. These increments in P-PKB levels were not observed in the presence of two PI3K inhibitors, wortmannin and Ly 294002. Inhibition of protein kinase A (PKA) by H89 did not decrease FSH stimulation of P-PKB levels. Taken together, these results indicate that FSH increases P-PKB levels in a PI3K-dependent and PKA-independent manner in rat Sertoli cells. In addition, wortmannin partially inhibited the ability of FSH to stimulate two well-known parameters of Sertoli cell function - transferrin secretion and lactate production - at doses equal to or lower than 0.1 microM. Related to lactate production, a decrease in FSH stimulation of lactate dehydrogenase activity and of basal and FSH-stimulated glucose uptake was observed in the presence of wortmannin. These metabolic changes were in most cases accompanied by changes in the levels of P-PKB. Altogether, these results suggest a meaningful role of the PI3K/PKB pathway in the mechanism of action of FSH in rat Sertoli cells.

Regulation of lactate production and glucose transport as well as of glucose transporter 1 and lactate dehydrogenase A mRNA levels by basic fibroblast growth factor in rat Sertoli cells.

By using cultured rat Sertoli cells as a model, both the action of basic fibroblast growth factor (bFGF) on lactate production and the site of this action were studied. bFGF stimulated Sertoli cell lactate production in a dose-dependent manner (basal: 7.3+/-0.5; 0.1 ng/ml bFGF: 7.5+/-0.5; 1 ng/ml bFGF: 7.5+/-0.6; 10 ng/ml bFGF: 10.3+/-1.0; 30 ng/ml bFGF: 15.2+/-1.5; 50 ng/ml bFGF: 15.4+/-1.6 microg/microg DNA). Two major sites for the action of this growth factor were identified. First, bFGF was shown to exert short- and long-term stimulatory effects on glucose transport (basal: 1170+/-102; 30 ng/ml bFGF for 120 min: 1718+/-152 and basal: 718+/-64; 30 ng/ml bFGF for 48 h: 1069+/-69 d.p.m./microg DNA respectively). Short-term bFGF stimulation of glucose transport was not inhibited by the protein synthesis inhibitor cycloheximide. These results indicate that short-term bFGF stimulation of glucose uptake does not involve an increase in the number of glucose transporters. On the other hand, stimulation with bFGF for periods of time longer than 12 h increased glucose transporter 1 (GLUT1) mRNA levels. These increased mRNA levels were probably ultimately responsible for the increments in glucose uptake that are observed in long-term treated cultures. Secondly, bFGF increased lactate dehydrogenase (LDH) activity (basal: 31.0+/-1.4; 30 ng/ml bFGF: 45.7+/- 2.4 mIU/microg DNA). The principal subunit component of those LDH isozymes that favors the transformation of pyruvate to lactate is subunit A. bFGF increased LDH A mRNA levels in a dose- and time-dependent manner. In summary, the results presented herein show that glucose transport, LDH activity and GLUT1 and LDH A mRNA levels are regulated by bFGF to achieve an increase in lactate production. These observed regulatory actions provide unequivocal evidence of the participation of bFGF in Sertoli cell lactate production which may be related to normal germ cell development.

Regulation of lactate production by FSH, iL1beta, and TNFalpha in rat Sertoli cells.

One of the "nurse cell" functions of Sertoli cells is to provide lactate for the energy production in spermatocytes and spermatids. The present study shows that, as in porcine Sertoli cells, interleukin (IL)1beta and follicle-stimulating hormone (FSH) increase lactate production in rat Sertoli cells (basal, 9.1 +/- 1.0; FSH (100 ng/ml), 16.6 +/- 2.0; IL1beta (50 ng/ml), 13.3 +/- 1.6 microg/microg DNA). Increments in glucose uptake (basal, 1083 +/- 70; FSH, 2686 +/- 128; IL1beta, 1899 +/- 74 dpm/microg DNA), lactic dehydrogenase (LDH) activity (basal, 36.6 +/- 4.1; FSH, 52.2 +/- 4.9; IL1beta, 55.3 +/- 5.1 mUI/microg DNA), LDH A mRNA levels, and redistribution of LDH isozymes are involved in these stimulatory effects. Differences in the period required by IL1beta to increase glucose uptake, as compared with the porcine model, have been observed. In addition, tumor necrosis factor alpha (TNFalpha), one of the major stimulators for lactate production in porcine Sertoli cells, does not control the secretion of this glucose metabolite in rat Sertoli cells. Lactate production may be regulated differently among mammals.

Interleukin-1beta regulates nitric oxide production and gamma-glutamyl transpeptidase activity in sertoli cells.

Several cytokines have been involved in the regulation of Sertoli cell function. Further investigations are required to elucidate the role of interleukin-1beta (IL1beta) in Sertoli cell physiology. Twenty-day-old rat Sertoli cell cultures were used to investigate a possible role of IL1beta in the regulation of gamma-glutamyl transpeptidase (gammaGTP) and to elucidate the signaling pathway utilized by this cytokine. GammaGTP is a membrane-bound enzyme that has been involved in amino acid transport across the plasma membrane and in protection from oxidative stress through its importance in the regulation of glutathione levels. Previous studies suggested that IL1beta stimulates NO biosynthesis in other cell types. Therefore, we investigated whether IL1beta modified the level of nitrite, a stable metabolite of NO, in Sertoli cells. Dose-response curves to IL1beta for gammaGTP activity and nitrite production were observed. The increments observed in gammaGTP activity and nitrite production were partially and completely blocked by simultaneous treatment with the NO synthase inhibitor aminoguanidine. Treatment of Sertoli cell cultures with the NO donors sodium nitroprusside and S-nitroso-N-acetylpenicillamine resulted in an increase in gammaGTP activity. The presence of neural, endothelial, and inducible isoforms of NO synthase (NOS) was investigated by a immunohistochemical technique using specific antibodies. The 2 constitutive isoforms were present under basal conditions, and the inducible protein appeared in IL1beta-treated cultures. Finally, translocation of NF-kappaB p65 subunit to the nucleus in IL1beta-treated cultures was observed. These findings suggest that the action of IL1beta on Sertoli cell gammaGTP activity is partially mediated via activation of NF-kappaB and increments in iNOS and cellular production of NO.

Possible involvement of ceramide in the regulation of rat Leydig cell function.

In the present study, a possible role of a ceramide-dependent pathway in the regulation of Leydig cell function was investigated. Intracellular ceramide levels were increased by: (a) adding ceramide analogs; (b) inhibiting ceramidase activity; and (c) adding sphingomyelinase (SMase). The cell-permeable ceramide analogs N-acetyl-, N-hexanoyl- and N-octanoylsphingosine (C2, C6 and C8) were used. As inhibitor of ceramidase activity 1S,2R-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol (MAPP) was used. Sphingomyelinase from S. aureus origin was utilized. Leydig cells were cultured for 3 or 24 h with or without the different drugs (10 microM) and SMase (0.3 U/ml) in the presence or absence of hCG (10 ng/ml). Basal testosterone production was not modified under any of the experimental conditions. A decrease in hCG-stimulated testosterone production was observed at 3 and 24 h in all cases. The inactive analog (N-hexanoyl dihydrosphingosine) did not produce inhibition in hCG-stimulated testosterone production. TNFalpha and IL1beta, two possible inducers of sphingomyelin hydrolysis, produced similar effects on hCG-stimulated testosterone production. In experiments performed in the presence of C6, inhibition in hCG-stimulated cAMP production was observed. The inhibitory effect of ceramide was also observed in dbcAMP-stimulated cultures indicating that this pathway inhibits post-cAMP formation events. To study possible loci for the action of ceramide on the steroidogenic pathway, cells were incubated with C6 and MAPP in the presence of different testosterone precursors. The drugs inhibited testosterone produced from 22(R)-hydroxycholesterol (22R-OHChol), pregnenolone and 17alpha-hydroxyprogesterone (17OHP4) but not from androstenedione (Delta4). These results suggest that a ceramide-dependent pathway regulates hCG-stimulated Leydig cell steroidogenesis at the level of cAMP production and at post-cAMP events.

Effects of basic fibroblast growth factor and nerve growth factor on lactate production, gamma-glutamyl transpeptidase and aromatase activities in cultured Sertoli cells.

Sertoli cells are under the control of FSH and androgens and also respond to polypeptidic factors locally produced. Basic fibroblast growth factor (bFGF) and nerve growth factor (NGF) have been proposed to belong to the large set of intratesticular regulators. The aim of the present investigation was to analyze the effects of bFGF and NGF on lactate production, gamma-glutamyl transpeptidase (gamma-GTP) and aromatase activities. Cultured Sertoli cells dose-dependently responded to bFGF by increasing lactate production and gamma-GTP activity under basal conditions. In FSH-stimulated cultures, a synergistic effect of FSH with bFGF for lactate production was observed. NGF did not produce changes in lactate production or gamma-GTP activity at any dose tested. Both peptides decreased FSH-stimulated aromatase activity. These results provide additional evidence for the participation of bFGF and NGF in the complex network of intratesticular regulators. bFGF has pleiotropic effects on Sertoli cell function while the actions of NGF seem to be more limited.

Effect of N-acetylsphingosine (C2) and the ceramidase inhibitor (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol on the regulation of Sertoli cell function.

In the present study, a possible role of ceramide in the regulation of Sertoli cell function was investigated. Intracellular ceramide levels were increased by adding N-acetylsphingosine (C2) or by inhibiting ceramidase with (1 S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol (MAPP). Cultured Sertoli cells were stimulated for 3 days with different doses of C2, MAPP, and their corresponding inactive analogs. The effect of these drugs was evaluated along four well-known Sertoli cell parameters: lactate and transferrin secretion, gamma-glutamyl transpeptidase (gamma-GTP) activity, and estradiol production. C2 and MAPP increased lactate production and decreased transferrin secretion. The inactive analogs did not produce any effect. In FSH (follicle-stimulating hormone)-stimulated cultures, C2 and MAPP produced a further increment in lactate production and decreased FSH-stimulated transferrin secretion. No effect was observed under basal or FSH-stimulated gamma-GTP activity, and both treatments decreased estradiol production in response to FSH. Results obtained in dbcAMP (dibutyryladenosine 3':5'-cyclic monophosphate)-stimulated cultures suggest that the observed effects of ceramide on transferrin secretion are secondary to a decrease in cAMP production, whereas the effects of ceramide on lactate and estradiol productions are post-cAMP formation regulatory events. In summary, our results show that ceramide can regulate Sertoli cell function. Similar to what has been observed for other signaling molecules, ceramide can interact with the FSH-dependent pathway, but the precise steps involved in this interaction are still unknown.

Effects of purinergic agonists on aromatase and gamma-glutamyl transpeptidase activities and on transferrin secretion in cultured Sertoli cells.

To study the role of extracellular nucleosides and nucleotides in the regulation of Sertoli cells, the effects of agonists which occupy A1 and P2 purinergic receptors on aromatase and gamma-glutamyl transpeptidase (gamma-GTP) activities and on transferrin secretion were tested. Sertoli cell treatment with purinergic agonists for a prolonged period of time (72 h) resulted in an increase in aromatase activity under basal conditions. In cultures stimulated with FSH, purinergic agonists counteracted the inhibitory effect on aromatase activity that long-term treatment with FSH promoted. The effects of prolonged treatments with purinergic agonists on the other two parameters of Sertoli cell function were less pronounced. Neither gamma-GTP activity nor transferrin secretion was modified under basal conditions. On the other hand, under conditions where cell differentiation was favored by FSH treatment, reductions in gamma-GTP activity and transferrin secretion were usually observed. The results obtained in dbcAMP-stimulated cultures suggested that A1 agonists exert their regulatory function at the level of cAMP formation while P2 agonists act at a more distal point. The fact that morphological changes induced by FSH were reversed by both types of agonists, while those induced by dbcAMP were only abrogated by P2 agonists, supports this hypothesis. In summary, these results demonstrate that purinergic agonists may be important in the regulation of Sertoli cell function.