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.