A novel mechanism of action of corticotropin releasing factor in rat Leydig cells

We have recently demonstrated the presence in the rat Leydig cells of a corticotropin releasing factor (CRF) receptor and an inhibitory action of the peptide on human chorionic gonadotropin (hCG)-induced cAMP generation and steroidogenesis. The inhibitory action of CRF was unaffected by pertussis toxin and was completely reversed by 8-bromo-cAMP (Ulisse, S., Fabbri, A., and Dufau, M. L. (1989) J. Biol. Chem. 264, 2156-2163). In this study, we have evaluated the participation of protein kinase C in CRF action in the Leydig cells and the level of the gonadotropin signal pathway affected by CRF. Binding of 125I-labeled ovine CRF to Leydig cell membranes was reduced by GTP and guanyl-5'-yl imidodiphosphate (Gpp(NH)p), in a dose-dependent manner. Phorbol 12-myristate 13-acetate, like CRF, caused time-dependent inhibition of hCG-induced cAMP generation and steroidogenesis. This inhibitory action was reversed by 8-bromo-cAMP. Both CRF and 12-O-tetradecanoylphorbol-13-acetate did not affect 125I-hCG binding. No additive effects of CRF and the phorbol ester were observed in these studies. CRF caused a rapid translocation of protein kinase C in Leydig cells. Preincubation of cells with protein kinase C inhibitors or TPA-induced depletion of protein kinase C prevented the inhibitory actions of CRF and TPA. CRF and TPA were able to inhibit the stimulation of cAMP and testosterone production by cholera toxin and forskolin. Adenylate cyclase stimulation by Gpp(NH)p, luteinizing hormone + Gpp(NH)p, and NaF in crude membranes or by forskolin and manganese in solubilized membranes, prepared from CRF- and TPA-treated cells, was also markedly inhibited. We conclude that CRF receptors interact with a pertussis toxin-insensitive G protein (possibly Gp) in the Leydig cell and that the inhibitory action of CRF on Leydig cell function is exerted mainly on the catalytic subunit of adenylate cyclase through a direct or indirect action of protein kinase C.     

Direct inhibitory effects of estrogens on rat Leydig cells in vitro.

In dispersed rat interstitial cells in vitro both natural and synthetic estrogens inhibited the action of pituitary luteinizing hormone (LH), as assessed by testosterone production. The estrogens also inhibited dibutyryl cyclic AMP induced steroidogenesis, suggesting that one point of inhibition could be distal to the formation of cyclic AMP in the cells. Diethyl stilbestrol and its clinically used sodium phosphate derivative (Honvol), also affected hormone-receptor interaction when tested with rat testicular homogenates. Among estradiol, estradiol benzoate, Honvol and diethyl stilbestrol only the latter at high concentration had toxic effects on Leydig cells as noted from loss of thier viability.     

Effects of antimicrotubular agents in cAMP production and in steroidogenic response of isolated rat Leydig cells

In dispersed rat Leydig cells, colchicine was found to stimulate basal cAMP production and testosterone secretion in a dose and time-dependent manner, but to a lesser extent than LH. However, these drugs are unable to stimulate adenylate cyclase activity in plasma membranes isolated from these cells. The amount of testosterone secreted at 150 min under the influence of colchicine and LH added simultaneously was not different from the amount produced during stimulation by LH alone. It is only after exposure of the cells for 1 hr to colchicine that the accumulation of cAMP in response to LH was inhibited; furthermore, both intracellular and medium testosterone accumulation in response to the hormone were reduced. Similar effects were observed with two other alkaloids, vinblastine and podophyllotoxin. The three drugs also inhibited the stimulation of testosterone secretion by 8-Br-cAMP or choleratoxin. These studies suggest that the state of microtubule polymerization and/or tubulin can influence the process of steroidogenesis in rat Leydig cells.     

Polyvalent inhibitory action of fusidic acid in isolated rat liver cells

The steroidic antibiotic fusidic acid showed a polyvalent action on isolated rat liver cells. It displayed a strong inhibitory capability on protein synthesis in intact cells even stronger than that previously reported in cell-free extracts. Also, it inhibited basal gluconeogenesis and promoted an increase of the membrane permeability to trypan blue. However, the effects on both protein synthesis and basal gluconeogenesis were observed at doses smaller than those required to reduce the cell viability.     

Angiotensin II receptors and inhibitory actions in Leydig cells

Rat Leydig cells possess functional high-affinity receptors for angiotensin II (AII). AII inhibits adenylate cyclase activity in Leydig cell membranes and reduces basal and human chorionic gonadotropin (hCG)-stimulated cAMP pools and testosterone production in intact cells. Treatment of cells with an inhibitory dose of forskolin (10(-9) M) and a submaximal dose of AII caused additive inhibition of hCG-stimulated events. The inhibitory action of AII was largely prevented by pertussis toxin prior to the addition of AII alone or in the presence of hCG. This study and our recent report on inhibitory action of low doses of forskolin, 10(-12)-10(-9) M (Khanum, A., and Dufau, M.L. (1986) J. Biol. Chem. 261, 11456-11459) are indicative of a pertussis toxin-sensitive subunit of adenylate cyclase available for acute regulation of Leydig cell function. 8-bromo-cAMP bypasses the inhibitory effect of forskolin as well as AII. We have, therefore, demonstrated functional AII high-affinity receptor and an acute inhibitory effect of AII on hCG action in Leydig cells. Our results have provided evidence for a pertussis toxin-sensitive guanine nucleotide inhibitory protein as mediator of the effect of AII. These findings further emphasized the importance of the cAMP pathway in the Leydig cells, and studies also suggest that tubular and locally produced AII could negatively modulate luteinizing hormone stimulation of Leydig cells.     

Is cAMP the obligatory second messenger in the action of lutropin on Leydig cell steroidogenesis

Two adenylate cyclase inhibitors: 9-(tetrahydro-2-furyl)adenine and 2'5'-dideoxyadenosine decreased cAMP levels in LH-stimulated immature rat Leydig cells by 20-40%, independent of the concentration of LH. Steroid production was not correlated with this decrease in cAMP, but was increased (146%). The phorbol ester 4 beta-phorbol-12-myristate-13-acetate stimulated steroidogenesis and the phosphorylation of a 17 kD and a 33 kD protein, which was also stimulated by LH, whereas the inactive phorbol ester 4 beta-phorbol-12,13-diacetate did not have any effects. Moreover, the Ca2+-channel blocker diltiazem inhibited LH effects, but had no direct effects on the cholesterol side chain cleavage enzyme. It is concluded that cAMP may not be the only second messenger in LH action, and that other second messenger systems are probably also involved.     

Tachyphylactic action of high doses of pentagastrin

The study of the human LES was performed with manometrical methods for atropine action on gastrine tachyphylaxis. Our study points out that there is a complex self regulating neuronal circuit in the LES contraction. We discuss some hypothesis for the LES control. In particular ACh could activate an inhibitory adrenergic muscarinic receptor.     

The action of high doses of oestradiol on implantation and decidual reaction in the rat

The action of high doses of oestradiol on ovum implantation and decidual reaction was studied in pseudo-pregnant rats. It is shown that anomalies of ovum implantation take place after the injection of 10 μg of oestradiol on the 4th day of pregnancy, in that the decidual reaction is inhibited as demonstrated by ponderal evolution, lower ³H uridine uptake, ultrastructural features and the ploidy level of stromal cell nuclei.     

Modulation and role of Ca2+ in LH and LHRH agonist action in rat Leydig cells

The results of our recent studies on purified rat Leydig cells indicate that there are no major qualitative differences in the stimulating effects of LH and LHRH agonists on steroidogenesis via mechanisms that are dependent on calcium. This was demonstrated by using inhibitors of calmodulin and the lipoxygenase pathways of arachidonic acid metabolism. Using the fluorescent indicator quin-2, it was shown that LH and LHRH agonist increase intracellular calcium levels; LH was more potent than LHRH agonist (max increase in concentrations obtained were 500 nM and 60 nM respectively). This difference was probably the result of a direct effect of cyclic AMP (whose production is stimulated by LH but not by LHRH) because cyclic AMP analogues were as potent as LH in increasing calcium levels. These studies indicate a major role for calcium in the control of steroidogenesis in testis Leydig cells.     

Microsomal ethanol-oxidizing system in purified rat Leydig cells

These studies provide evidence for the presence of a microsomal ethanol oxidizing system in rat Leydig cells. Activity of the microsomal ethanol oxidizing system in Leydig cells was 47.4 +/- 4.1 nmol acetaldehyde per 20 min per mg protein, while activity in crude interstitial cells was 26.0 +/- 5.4 nmol. This suggests that among cells comprising interstitial cells, activity is concentrated in Leydig cells. Activity was linear with respect to protein concentration and incubation time. The highest specific activity was observed in the microsomal fraction. The most effective cofactor was NADPH. The apparent Km for ethanol was 4 mM, suggesting that this system could effectively metabolize ethanol at concentrations found in the blood of males who drink. The apparent Km for NADPH was 11 microM. The activity in Leydig cells was unaffected by 4-methylpyrazole or potassium cyanide, which inhibit alcohol dehydrogenase and catalase activities, respectively. These data provide strong evidence for an enzyme system in Leydig cell microsomes which is capable of metabolizing ethanol.     

Ca-dependent-chloride and potassium currents in rat Leydig cells

Voltage-clamped membrane currents have been investigated from whole-cell patch-clamp recordings performed on single Leydig cells isolated from the adult rat testis. Two outward membrane currents were evoked by depolarizing voltage steps. A potassium current was recorded in cells dialyzed with low (10(-9)-10(-8) M) calcium media. This current was decreased by TEA (10 mM). A chloride current was recorded in cells dialyzed with high (10(-7)-10(-6) M) calcium media. This current was decreased by an external exposure to glutamate. Comparison of the currents at low and high internal calcium concentrations suggests that an increase of the intracellular calcium activates a chloride current.     

Insulin-like peptide 3 stimulates testosterone secretion in mouse Leydig cells via cAMP pathway

Testicular Leydig cells secrete insulin-like peptide 3 (INSL3) and express its receptor, RXFP2. However, the effects of INSL3 on endocrine function of Leydig cells are unknown. The present study examines the effects of INSL3 on mouse Leydig cells taking testosterone and cAMP secretions as endpoints. Leydig cells were isolated from testicular interstitial cells obtained from 8-week-old male mice. Cells were then plated in the presence or absence of mouse, human, canine or bovine INSL3 (0-100ng/ml) for 18h in multiwell-plates (96 wells) in different cell densities (2500, 5000, 10,000 or 20,000 cells per well). The effects of bovine INSL3 (100ng/ml) on testosterone secretion by Leydig cells were examined in the presence or absence of, an adenylate cyclase inhibitor, SQ 22536 (1μM) or INSL3 antagonist (bovine and human; 100ng/ml). Testosterone and cAMP in spent medium were measured by enzyme immunoassay. All INSL3 species tested significantly stimulated the testosterone secretion in Leydig cells, and the maximum stimulation was observed with 100ng/ml bovine INSL3 at the lowest Leydig cell density (2500 cells per well). Moreover, bovine INSL3 (100ng/ml) significantly stimulated the cAMP production from Leydig cells maximally at 1h, and remained significantly elevated even at 18h. SQ 22536 and INSL3 antagonists (bovine and human) significantly reduced INSL3-stimulated testosterone secretion from Leydig cells. Taken together, stimulatory effects of INSL3 on testosterone secretion in Leydig cells are exerted via the activation of cAMP, suggesting a new autocrine function of INSL3 in males.     

D-Aspartate stimulation of testosterone synthesis in rat Leydig cells

D-Aspartate increases human chorionic gonadotropin-induced testosterone production in purified rat Leydig cells. L-Aspartate, D-,L-glutamate or D-,L-asparagine could not substitute for D-aspartate and this effect was independent of glutamate receptor activation. Testosterone production was enhanced only in cells cultured with D-aspartate for more than 3 h. The increased production of testosterone was well correlated with the amounts of D-aspartate incorporated into the Leydig cells, and L-cysteine sulfinic acid, an inhibitor of D-aspartate uptake, suppressed both testosterone production and intracellular D-aspartate levels. D-Aspartate therefore is presumably taken up into cells to increase steroidogenesis. Intracellular D-aspartate probably acts on cholesterol translocation into the inner mitochondrial membrane, the rate-limiting process in steroidogenesis.     

Phospholipid methylation by intact rat Leydig cells

Phospholipid methylation by intact Leydig cells was investigated by determining the incorporation of radioactivity from [3H-methyl] methionine into phospholipids. Leydig cells incorporated significantly more radioactivity into phospholipids than did unpurified testicular cells, non-Leydig testicular cells, or red blood cells. Approximately 40% of the radioactivity was found in phosphatidylcholine, indicating that the methyltransferase pathway for the synthesis of this phospholipid is highly active in rat Leydig cells. Addition of luteinizing hormone to cells preloaded with [3H-methyl] methionine did not alter the rate of phospholipid methylation. However, phospholipid methylation by Leydig cells desensitized by the injection of human chorionic gonadotropin 1 to 7 days previously was reduced by approximately 60%. Inhibition of phospholipid methylation to 75% of normal with homocysteine thiolactone did not affect luteinizing hormone-stimulated androgen production. Further inhibition of phospholipid (and protein) methylation by treatment with homocysteine thiolactone and 3-deazaadenosine significantly reduced luteinizing hormone-stimulated androgen production. The results of this study demonstrate that the methyltransferase pathway for the synthesis of phosphatidylcholine is highly active in intact Leydig cells but is reduced in desensitized Leydig cells. There does not appear to be a close association between the activity of this pathway and the ability of luteinizing hormone to acutely stimulate androgen production.