Effects of Cordyceps sinensis on testosterone production in normal mouse Leydig cells.

The stimulatory effect of Cordyceps sinensis (CS) on MA-10 mouse Leydig tumor cell steroidogenesis was previously demonstrated in our laboratory. In the present studies, we further determined the effect of CS on steroidogenesis in purified normal mouse Leydig cells. Different concentrations of CS (0.1-10 mg/ml) were added to Leydig cells without or with human chorionic gonadotropin (hCG) (50 ng/ml), and the steroid production was determined by radioimmunoassay (RIA). The results illustrated that CS stimulated normal mouse Leydig cell steroidogenesis in a dose-dependent relationship. CS at 3 mg/ml significantly stimulated testosterone production (p<0.05). Concerning the temporal relationship, CS at 3 mg/ml stimulated maximal testosterone production between 2 to 3 hr. Interestingly, hCG-stimulated testosterone productions were suppressed by CS in a dose-dependent relationship. CS also reduced dbcAMP-stimulated testosterone productions, which indicated that CS affected signal transduction pathway of steroidogenesis after the formation of cyclic AMP. Moreover, cycloheximide inhibited CS-treated mouse Leydig cell testosterone production, suggesting that new protein synthesis was required for CS-stimulated steroidogenesis.     

Effects of amphetamine on steroidogenesis in MA-10 mouse Leydig tumor cells

Amphetamine influences plasma and testicular testosterone levels. However, there is no evidence that amphetamine can directly influence Leydig cell functions. In the present study, a MA-10 mouse Leydig tumor cell line was used to determine whether and how amphetamine affected Leydig cell steroidogenesis. MA-10 cells were treated with different concentrations of amphetamine without or with human chorionic gonadotropin (hCG) and/or enzyme precursors over different time durations. Steroid production, enzyme activities and StAR protein expression were determined. Amphetamine alone had no any effect on MA-10 cell steroidogenesis. However, amphetamine (10(-11)M and 10(-10)M) significantly enhanced hCG-treated progesterone production at 3 hr in MA-10 cells (p < 0.05). Furthermore, amphetamine significantly induced more progesterone production upon treatment with 22R-hydroxycholesterol (p < 0.05), a precursor of P450 side-chain cleavage enzyme (P450scc). However, amphetamine did not induce more progesterone production when treated with pregnenolone (p > 0.05), a precursor of 3beta-hydroxysteroid dehydrogenase. In addition, the expressions of StAR protein and P450scc enzyme were not significantly different between hCG alone and hCG plus amphetamine treatment in MA-10 cells (p > 0.05). These results suggested that amphetamine enhanced hCG-induced progesterone production in MA-10 cells by increasing P450scc activity without influencing StAR protein and P450scc enzyme expression or 3beta-HSD enzyme activity.     

Transforming growth factor-beta inhibits Leydig cell steroidogenesis in primary culture

The effects of transforming growth factor (TGF) on Leydig cell steroidogenesis in primary culture were investigated. Basal testosterone levels were 3.7 +/- 0.54 ng/ml (mean +/- SE, N = 7). In the presence of hCG (10 ng/ml), testosterone levels increased to 22.77 +/- 3.05 ng/ml. TGF-beta caused a dose dependent inhibition of hCG-stimulated testosterone formation but without effects on basal levels. TGF-beta also inhibited 8-bromo cyclic AMP-induced testosterone formation and hCG-stimulated cyclic AMP formation. In contrast, TGF-alpha had no effect on either basal or hCG-stimulated testosterone formation and did not modify the inhibitory effect of TGF-beta. Present study indicates that TGF-beta can modulate Leydig cell steroidogenesis.     

Effects of hyper- and hypoprolactinemia on gonadotropin secretion, rat testicular luteinizing hormone/human chorionic gonadotropin receptors and testosterone production by isolated Leydig cells

The effect of prolactin (Prl) on gonadotropin secretion, testicular luteinizing hormone (LH)/human chorionic gonadotropin (hCG) receptors, and testosterone (T) production by isolated Leydig cells has been studied in 60-day-old rats treated for 4 days, 4 and 8 weeks with sulpiride (SLP), a dopaminergic antagonist, or for 4 days and 4 weeks with bromocriptine (CB), a dopaminergic agonist. Plasma Prl concentrations were significantly greater in the SLP groups (204 +/- 6 ng/ml) and lower in the CB groups (3.0 +/- 0.2 ng/ml) than those measured in the control groups (54 +/- 6 ng/ml). The plasma concentrations of gonadotropin were not affected by a 4-day treatment with SLP or CB, nor were they after a 4-week treatment with CB. However, the hyperprolactinemia induced by an 8-week treatment with SLP was associated with a reduced secretion of gonadotropin (LH, 16 +/- 4 vs. 35 +/- 6 ng/ml; FSH, 166 +/- 12 vs. 307 +/- 14 ng/ml). In SLP-induced hyperprolactinemia, a 30% increase in the density of the LH/hCG testicular binding sites was observed (178 +/- 12 fmol/mg protein), whereas a 60% decrease was measured in hypoprolactinemia (55 +/- 5 vs. control 133 +/- 5 fmol/mg protein). Plasma T levels were increased in 4-day and 4-week hyperprolactinemic animals (4.3 +/- 0.4 and 3.9 +/- 0.4 ng/ml, respectively), but returned to normal levels in the 8-week group (3.0 +/- 0.5 vs. C: 2.3 +/- 0.2 ng/ml). No T modifications were observed in hypoprolactinemic animals. Two distinct populations of Leydig cells (I and II) were obtained by centrifugation of dispersed testicular cells on a 0-45% continuous Metrizamide gradient. Both possess LH/hCG binding sites. However, the T production from Leydig cells of population II increased in the presence of hCG, whereas that of cell population I which also contain immature germinal cells did not respond. The basal and stimulated T secretions from cell populations I and II obtained from CB-treated animals were similar to controls, whereas from 4 days to 8 weeks of hyperprolactinemia, basal and hCG induced T productions from cell population II decreased progressively. These data show that hyperprolactinemia causes, in a time-dependent manner, a trophic effect on the density of LH/hCG testicular receptors; reduces basal and hCG-stimulated T production from isolated Leydig cells type II; and results in an elevated plasma T concentration which decreases with time. The latter suggests a slower T catabolism and/or an impaired peripheral conversion of T into 5 alpha-dihydrotestosterone (DHT). Although hypoprolactinemia is associated with a marked reduction in testicular LH receptors, it does not affect T production.     

Attenuation of cAMP-mediated responses in MA-10 Leydig tumor cells by genetic manipulation of a cAMP-phosphodiesterase.

In order to assess the effect of increased cAMP degradation on the responsiveness on an endocrine cell, we have obtained stable transfectants of MA-10 Leydig tumor cells that overexpress a mammalian cAMP-phosphodiesterase. Two novel cell lines, designated MA-10(P+8) and MA-10(P+29), that express high levels of the transfected enzyme were characterized. Although the basal levels of cAMP in the mutant cell lines are comparable to those of the wild-type cells, the increase in cAMP accumulation elicited by human choriogonadotropin (hCG) is severely blunted. Further studies with MA-10(P+29) show that the ability of hCG to stimulate adenylyl cyclase activity is normal. The failure of MA-10(P+29) cells to accumulate cAMP in response to hCG can be correlated with a similar reduction in hCG-stimulated steroidogenesis. On the other hand, the maximal steroidogenic response of MA-10(P+29) cells to dibutyryl cAMP, a cAMP analogue that is fairly resistant to phosphodiesterase degradation, is normal. We also show that the ability of these cells to respond to hCG with increased cAMP accumulation and steroid synthesis can be restored with a specific phosphodiesterase inhibitor. These results demonstrate that overexpression of a cAMP-phosphodiesterase in MA-10 cells limits the levels of cAMP attained under hCG stimulation and supresses the steroidogenic response of these cells to hCG. Since gonadotropins increase the cAMP-phosphodiesterase activity in their target cells, these findings also provide evidence that this regulation plays a major role in the modulation of cell responsiveness. Last, these new cell lines should be valuable in the study of the actions of cAMP because they express a conditional and reversible cAMP-resistant phenotype.     

The inhibitory effects of lead on steroidogenesis in MA-10 mouse Leydig tumor cells

Lead is an environmental and occupational pollutant. It has been reported that lead affects the male reproductive system in humans and animals. However, the cellular mechanism of the adverse effect of lead on Leydig cell steroidogenesis remains unknown. To clarify whether lead has a direct effect on Leydig cells and how lead affects Leydig cells, MA-10 cells, a mouse Leydig tumor cell line, were exploited in this study. Lead acetate significantly inhibited hCG- and dbcAMP-stimulated progesterone production in MA-10 cells at 2 h. Steroid production stimulated by hCG or dbcAMP were reduced by lead. The mechanism of lead in reducing MA-10 cell steroidogenesis was further investigated. The expression of Steroidogenic Acute Regulatory (StAR) protein and the activities of P450 side-chain cleavage (P450scc) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) enzymes were detected. Cells were treated with dbcAMP, 22R-hydroxycholesterol or pregnenolone alone or in combination with lead acetate ranging from 10(-8) to 10(-5) M for 2 h. The expression of StAR protein stimulated by dbcAMP was suppressed by lead at about 50%. Progesterone productions treated with 22R-hydroxycholesterol or pregnenolone were reduced 30-40% in lead-treated MA-10 cells. These data suggest that lead directly inhibited steroidogenesis by decreasing StAR protein expression and the activities of P450scc and 3beta-HSD enzymes with a dose-response trend in MA-10 cells. Moreover, cadmium, a calcium channel blocker, abolished inhibitory effect of lead on MA-10 cell steroid production. This indicates that lead might act on calcium channel to regulate MA-10 cell steroidogenesis.     

The regulatory mechanism of Tremella mesenterica on steroidogenesis in MA-10 mouse Leydig tumor cells

Tremella mesenterica (TM), a yellow jelly mushroom, has been traditionally used as tonic food to improve body condition in Chinese society for a long time. We have previously demonstrated that TM reduced in vitro hCG-treated steroidogenesis in MA-10 mouse Leydig tumor cells without any toxicity effect. In the present study, the mechanism how TM suppressed hCG-treated steroidogenesis in MA-10 cells was investigated. MA-10 cells were treated with vehicle, human chorionic gonadotropin (hCG, 50 ng/ml), or different reagents with or without TM to clarify the effects. TM significantly suppressed progesterone production with the presences of forskolin (10 and 100 microM) or dbcAMP (0.5 and 1mM), respectively, in MA-10 cells (p<0.05), which indicated that TM suppressed steroidogenesis after PKA activation along the signal pathway. Beyond our expectation, TM induced the expression of steroidogenic acute regulatory (StAR) protein with or without hCG treatments. However, TM profoundly decreased P450 side chain cleavage (P450scc) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) enzyme activities without any influences on the expression of both enzymes. These inhibitions on steroidogenic enzyme activities might counteract the stimulation of StAR protein expression. In conclusion, results suggest that TM suppressed hCG-treated steroidogenesis in MA-10 cells by inhibiting PKA signal pathway and steroidogenic enzyme activities.     

Expression and regulation of adrenodoxin and P450scc mRNA in rodent tissues

The rate-limiting step in steroidogenesis is the conversion of cholesterol to pregnenolone. This reaction occurs in steroidogenic tissue in the inner mitochondrial membrane, and is mediated by the cholesterol side-chain cleavage enzyme. This enzyme system transfers electrons from NADPH to cholesterol through its three protein components: adrenodoxin reductase, adrenodoxin, and the terminal oxidase, P450scc. We have previously shown that P450scc mRNA is regulated by tropic hormones and cAMP by a cycloheximide-independent mechanism in mouse Leydig tumor MA-10 cells. We now show that the mRNA for adrenodoxin, another component of the cholesterol side-chain cleavage enzyme system, is regulated by tropic hormones and cAMP in MA-10 cells. We cloned rat adrenodoxin cDNA to analyze adrenodoxin mRNA in various rat tissues and in MA-10 cells by RNase protection assays. Adrenodoxin mRNA is found in virtually all rat tissues examined, although it is most abundant in adrenals, ovaries, and testes. MA-10 cells synthesize two species of adrenodoxin mRNA, one of 1.2 kb and the other of 0.8 kb. Both of these adrenodoxin mRNAs are increased approximately six-fold by 1 mM 8-Br-cAMP, five-fold by 10 microM forskolin, and three-fold by both 25 ng/ml hCG and by 100 ng/ml LH. Maximal adrenodoxin mRNA accumulation occurs by 4 h of hormonal stimulation. The cAMP-mediated increase in adrenodoxin mRNA accumulation is independent of protein synthesis, since treatment with cycloheximide or puromycin in the absence or presence of cAMP does not inhibit, and even increases, adrenodoxin mRNA accumulation.     

Regulation of renin angiotensins by gonadotropic hormones in cultured murine Leydig tumor cells. Release of angiotensin but not renin

Renin and angiotensins coexist in various tissues. The mode of control of the extrarenal renin-angiotensin system is not clear. Whether it is renin or angiotensin that is secreted has not been identified. We have investigated gonadotropin-dependent synthesis and subsequent release of the components of the intracellular renin-angiotensin system in a cloned and cultured mouse Leydig tumor cell line (MA-10). Treatment of cultured Leydig cells with bovine luteinizing hormone (bLH, 100 ng/ml) or human chorionic gonadotropin (hCG, 25 ng/ml) resulted in greater than 150- and 40- fold increased formation of angiotensin I and angiotensin II. In cells incubated with bLH or hCG, the majority of AII (up to 90%) was found in the culture medium while most of angiotensin I (greater than 85%) was in the cell lysate. Treatment with gonadotropic hormones (bLH/ hCG) increased renin 35- to 40-fold. Renin activity was confined mainly in the cell lysate even after the stimulation by gonadotropins, and only 1-2% of the total renin activity was detectable in culture medium. These results were interpreted that, in these transformed cells, hormonally-induced renin functions to generate angiotensin I within the Leydig cell and it is the angiotensins which are secreted.     

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.     

Transiently elevated levels of c-fos and c-myc oncogene messenger ribonucleic acids in cultured murine Leydig tumor cells after addition of human chorionic gonadotropin

The gonadotropic hormones LH and human CG (hCG) normally function to stimulate steroidogenesis in testicular and ovarian cells through receptor-mediated activation of adenylate cyclase. These hormones are also important in regulating the development and growth of responsive cells. Such regulation requires tightly controlled gene expression. Herein we demonstrate that hCG induces increases in mRNAs encoding the competence oncogenes c-fos and c-myc in a murine Leydig cell tumor line (MA-10). When stimulated by hCG (40 ng/ml), the mRNA levels of both genes increase rapidly, peaking at 30 min for c-fos and 1 h for c-myc. Both mRNAs fall to near control levels by 3-6 h. This response to hCG is dose-dependent with half-maximal stimulation of these genes occurring at a concentration of 3 ng/ml, approximating the level required for 50% occupancy of the LH/hCG receptors and the ED50 for steroidogenesis. (Bu)2 cAMP (2 mM) elicits responses similar to those produced by hCG. The observation of oncogene control by the gonadotropin hCG provides further insight regarding the pathways by which such hormones may regulate steroidogenesis, growth, and differentiation of endocrine and neoplastic cells.     

A co-culture system reveals the involvement of intercellular pathways as mediators of the lutropin receptor (LHR)-stimulated ERK1/2 phosphorylation in Leydig cells

Co-cultures of lutropin receptor (LHR) positive and negative Leydig cells were used to test the hypothesis that the LHR provokes phosphorylation of the extracellular regulated kinases (ERK1/2) using intracellular and intercellular pathways. Addition of hCG to MA-10 cells (LHR positive) stimulates phosphorylation of the EGF receptor (EGFR) and ERK1/2 whereas addition of hCG to I-10 cells (LHR negative) does not. Addition of hCG to co-cultures of MA-10 and I-10 cells rapidly stimulates the phosphorylation of the EGFR and ERK1/2 in I-10 cells, however. Transfection of interfering constructs shows that the LHR-mediated activation of Fyn in MA-10 cells is necessary for the phosphorylation of the EGFR and ERK1/2 in I-10 cells. This pathway can also be demonstrated in MA-10 cells but the phosphorylation of ERK1/2 in MA-10 cells also involves a second pathway mediated by protein kinase A (PKA). We propose that the LHR-mediated stimulation of the ERK1/2 cascade in Leydig cells depends on two independent pathways. One is intracellular and is mediated by PKA. The second is mediated by Fyn and it involves the release of soluble factors that act to phosphorylate the EGFR in an autocrine/paracrine fashion.     

Effect of human parathyroid hormone on the cAMP production and the endocrine functions of trophoblast cells from first trimester placenta.

Our previous study on teratocarcinoma cells suggested the role of human parathyroid hormone (hPTH) in early development of the placenta. The purpose of this study was to evaluate the possible role of hPTH on the functions of first trimester trophoblast cells. Adenylate cyclase activity in crude membranes from first trimester human placental villous tissue is stimulated 2-fold by hPTH (1-34) (10(-6) mol.l-1) from 265 +/- 32 to 532 +/- 80 pmol of cAMP/mg protein/15 min. A similar stimulation of adenylate cyclase is observed in human term placental villous tissue but not in 3 different choriocarcinoma cell lines. In order to evaluate the possible role of hPTH on the functions of first trimester human trophoblast cells, these cells were isolated by dispase and cultured (2 x 10(5) cells per plate) in DMEM supplemented with 20% fetal calf serum with or without 100 ng/ml of epidermal growth factor (EGF), for 4 d. On d 2 of culture, hPTH (10(-7) mol.l-1) stimulates cAMP production of these cells from 0.52 +/- 0.2 to 2.58 +/- 0.57 pmol.h-1 per 10(6) cells (mean +/- SEM). As compared to control (30 ng/ml), the output of hCG is increased by 1.5- (NS), 2- (P less than 0.01) and 3- (P less than 0.01) fold by EGF, hPTH, and hPTH added with EGF, respectively. Dibutyryl cAMP (10(-3) mol.l-1) increased hCG secretion by 3-fold (P less than 0.05). EGF and hPTH added separately or together significantly stimulated (P less than 0.01) the secretion of free alpha subunit 2-fold from 35 ng/ml to 70 ng/ml. In contrast, hPTH and EGF added separately did not change the secretion of free beta hCG. However, added together, they significantly increased (P less than 0.01) the secretion of free beta hCG after 48 h of culture, maximal stimulation (2.5 fold) being observed at d 4 of culture. In conclusion, human trophoblast cells are target cells for hPTH. hPTH acts in association with EGF in promoting expression of endocrine activity of these cells, such as hCG secretion. Trophoblast cells provide a model for the study of the cooperative effect between a peptide hormone and a growth factor in the regulation of endocrine function.     

Interstitial cell heterogeneity in rat testes. I. Purification of collagenase-dispersed Leydig cells by unit gravity sedimentation and demonstration of binding sites for gonadotropin in light cells versus enhanced steroidogenesis in heavier cells

Two testicular interstitial cell fractions, light and heavier, biochemically and morphologically distinct were obtained by a unit gravity sedimentation procedure. Binding sites for 125I-labeled human chorionic gonadotropin (hCG) were preferentially localized in the light cell fraction (apparent Kd = 2.02 X 10(-10) M; Bmax = 1.17 X 10(-5) nmol/2 X 10(6) cells). These cells did not synthesize testosterone in response to hCG, but the basal release of testosterone was higher than by cells in the heavier fraction (2.49 +/- 0.02 ng/2 X 10(6) cells in the light versus 0.22 +/- 0.00 ng/2 X 10(6) cells in the heavier fraction). The cells in the heavier fraction bound little or no hCG. The binding data from this fraction did not obey saturation kinetics, but testosterone levels were elevated 700-800% in the presence of hCG (i.e. basal value 0.22 +/- 0.00 ng/2 X 10(6) cells versus 1.81 +/- 0.04 ng/2 X 10(6) cells in hCG-stimulated cells). Electron microscopy revealed that heavier cells had features typical of Leydig cells such as large ovoid nucleus with peripherally located heterochromatin, numerous mitochondria with tubular cristae, some lipid droplets, extensively developed smooth endoplasmic reticulum, and well developed Golgi complex. The cells in the light fraction contained an ovoid nucleus with one or more deep infoldings, and their most notable cytoplasmic feature was the presence of numerous vacuoles of varying sizes and shapes. Based upon this and the investigation which follows (Bhalla, V.K., Flasch, M.V., Browne, E.S., Sohal, G.S., and Sharawy, M.M. (1987) J. Biol. Chem. 262, 5322-5332), we conclude that occupancy of high affinity hCG binding sites, generally assumed to be coupled to steroidogenesis, is not necessarily related to the elicitation of this biological response.     

The Leydig cell MEK/ERK pathway is critical for maintaining a functional population of adult Leydig cells and for fertility

MAPK kinase (MEK)1 and MEK2 were deleted from Leydig cells by crossing Mek1(f/f);Mek2(-/-) and Cyp17iCre mice. Primary cultures of Leydig cell from mice of the appropriate genotype (Mek1(f/f);Mek2(-/-);iCre(+)) show decreased, but still detectable, MEK1 expression and decreased or absent ERK1/2 phosphorylation when stimulated with epidermal growth factor, Kit ligand, cAMP, or human choriogonadotropin (hCG). The body or testicular weights of Mek1(f/f);Mek2(-/-);iCre(+) mice are not significantly affected, but the testis have fewer Leydig cells. The Leydig cell hypoplasia is paralleled by decreased testicular expression of several Leydig cell markers, such as the lutropin receptor, steroidogenic acute regulatory protein, cholesterol side chain cleavage enzyme, 17α-hydroxylase, and estrogen sulfotransferase. The expression of Sertoli or germ cell markers, as well as the shape, size, and cellular composition of the seminiferous tubules, are not affected. cAMP accumulation in response to hCG stimulation in primary cultures of Leydig cells from Mek1(f/f);Mek2(-/-);iCre(+) mice is normal, but basal testosterone and testosterone syntheses provoked by addition of hCG or a cAMP analog, or by addition of substrates such as 22-hydroxycholesterol or pregnenolone, are barely detectable. The Mek1(f/f);Mek2(-/-);iCre(+) males show decreased intratesticular testosterone and display several signs of hypoandrogenemia, such as elevated serum LH, decreased expression of two renal androgen-responsive genes, and decreased seminal vesicle weight. Also, in spite of normal sperm number and motility, the Mek1(f/f);Mek2(-/-);iCre(+) mice show reduced fertility. These studies show that deletion of MEK1/2 in Leydig cells results in Leydig cell hypoplasia, hypoandrogenemia, and reduced fertility.     

Lutropin-dependent protein phosphorylation and steroidogenesis in rat tumour Leydig cells

Tumour Leydig cells have been incubated in the presence or absence of lutropin (luteinizing hormone, ;LH'). Stimulation of cells with lutropin (1000ng/ml) in the presence of 1-methyl-3-isobutylxanthine (0.25mm) resulted in increased steroid production and increased protein phosphorylation. When pregnenolone metabolism was inhibited, basal pregnenolone production was 26.9+/-7.4ng/60min per 10(6) cells; stimulated production was 156.1+/-39.5ng/60min per 10(6) cells (means+/-s.d., n=4). Lutropin-dependent phosphorylated proteins of molecular mass 17000, 22000, 24000, 33000 and 57000Da were detected. A significant increase of [(32)P]P(i) incorporation into these phosphorylated proteins was observed concomitant with the increased pregnenolone production. The occurrence of the phosphoproteins in nuclei, mitochondria and postmitochondrial-supernatant was investigated. The 17000Da phosphoprotein was found in the nuclear fraction, whereas the 22000, 24000, 33000 and 57000Da phosphoproteins were localized in the postmitochondrial-supernatant fraction. Of the cholesterol-side-chain-cleavage activity, 80.3+/-6.1% (mean+/-s.d., n=5) was present in the mitochondrial fraction isolated from tumour Leydig cells, and this activity was 2.5-fold increased when cells had been preincubated with lutropin/1-methyl-3-isobutylxanthine (basal production: 194.6+/-28.6ng/30min per mg of protein; lutropinstimulated production: 498.8+/-91.5ng/30min per mg of protein; means+/-s.d., n=3). The similarities in the kinetics of the phosphorylation of proteins and the pregnenolone production after addition of lutropin/1-methyl-3-isobutylxanthine indicate that the phosphoproteins could be involved in the lutropin-dependent increase in steroidogenesis in tumour Leydig cells. It remains to be demonstrated, however, to what extent the phosphoproteins outside the mitochondria can influence the cholesterol-side-chain-cleavage activity inside the mitochondria.     

Reactive oxygen species production by mitochondria in endothelial cells exposed to reoxygenation after hypoxia and glucose depletion is mediated by ceramide

In endothelium, reoxygenation after hypoxia (H/R) has been shown to induce production of reactive oxygen species (ROS) by complex III of the mitochondrial respiratory chain. The purpose of the present study was to test the involvement of ceramide in this phenomenon. Human umbilical vein endothelial cells underwent 2 h of hypoxia (PO2, approximately 20 mmHg) without glucose and 1 h of reoxygenation (PO2, approximately 120 mmHg) with glucose. ROS production was measured by the fluorescent marker 2',7'-dichlorodihydrofluorescein diacetate, and cell death by propidium iodide. We showed that 1) after 1 h of reoxygenation, fluorescence had risen and that ROS production was inhibited by desipramine, an inhibitor of sphingomyelinase, an enzyme responsible for ceramide production (126 +/- 7% vs. 48 +/- 12%, P < 0.05); 2) administration of ceramide (N-acetylsphingosine) per se (i.e., in the absence of H/R) induced ROS production (65 +/- 3%), which was inhibited by complex III inhibitor: antimycin A (24 +/- 3%, P < 0.0001), or stigmatellin (31 +/- 2%, P < 0.0001); 3) hypoxia/reoxygenation-induced ROS production was not affected by either ceramide-activated protein kinase inhibitor dimethyl aminopurine or mitochondrial permeability transition inhibitor cyclosporin A but was significantly inhibited by the antiapoptotic protein Bcl-2 (82 +/- 8%, P < 0.05); 4) ceramide-induced ROS production was also inhibited by Bcl-2 (41 +/- 4%, P < 0.0001). These results demonstrate that in endothelial cells submitted to hypoxia and glucose depletion followed by reoxygenation with glucose, the pathway implicated in mitochondrial complex III ROS production is ceramide dependent and is decreased by the antiapoptotic protein Bcl-2.     

Site-directed mutagenesis of the human chorionic gonadotropin beta-subunit: bioactivity of a heterologous hormone, bovine alpha-human des-(122-145)beta

Human CG contains an alpha-subunit, common to the pituitary glycoprotein hormones, and a hormone-specific beta-subunit, but unlike the pituitary beta-subunits, hCG beta is characterized by an O-glycosylated carboxy-terminal extension. A mutant beta-subunit, des-(122-145)hCG beta, was prepared using site-directed mutagenesis, and the pRSV expression plasmids were transfected into Chinese hamster ovary cells that produce the bovine alpha-subunit (b alpha). The mutant beta-subunit binds to b alpha, and the heterologous gonadotropin, b alpha-des-(122-145)hCG beta, was capable of stimulating steroidogenesis in cultured Leydig tumor cells (MA-10) to the same extent as standard hCG. When compared with the heterologous gonadotropin, b alpha-hCG beta wild type, the hybrid hormone with the truncated hCG beta exhibited equal potency, within the accuracy of the RIAs used to determine hormone concentrations, and gave a similar time course of steroidogenesis. Interestingly, these transformed Leydig cells do not distinguish between the steroidogenic potencies (as measured by progesterone production) of hCG and human LH (hLH) as do some preparations of normal rodent Leydig cells (as measured by testosterone production). However, the MA-10 cells were able to distinguish hCG from hLH based on their cAMP response; the latter produced a greater response at both maximal and submaximal gonadotropin concentrations. The two expressed heterologous gonadotropins were equipotent in their abilities to stimulate cAMP and gave similar time courses of cAMP accumulation in MA-10 cells. Thus, the carboxy-terminal extension of hCG beta is not required for association with the alpha-subunit nor for functional receptor binding, as judged by cAMP accumulation and progesterone production in MA-10 cells.     

Production of ceramides causes apoptosis during early neural differentiation in vitro

To investigate signal transduction pathways leading to apoptosis during the early phase of neurogenesis, we employed PCC7-Mz1 cells, which cease to proliferate and begin to differentiate into a stable pattern of neurons, astroglial cells, and fibroblasts upon incubation with retinoic acid (RA). As part of lineage determination, a sizable fraction of RA-treated cultures die by apoptosis. Applying natural long-chain C(16)-ceramides as well as membrane-permeable C(2)/C(6)-ceramide analogs caused apoptosis, whereas the biologically nonactive C(2)-dihydroceramide did not. Treating PCC7-Mz1 stem cells with a neutral sphingomyelinase or with the ceramidase inhibitor N-oleoylethanolamine elevated the endogenous ceramide levels and concomitantly induced apoptosis. Addition of RA caused an increase in ceramide levels within 3-5 h, which reached a maximum (up to 3.5-fold of control) between days 1 and 3 of differentiation. Differentiated PCC7-Mz1 cells did not respond with ceramide formation and apoptosis to RA treatment. The acidic sphingomyelinase contributed only weakly and the neutral Mg(2+)-dependent and Mg(2+)-independent sphingomyelinases not at all to the RA-mediated production of ceramides. However, ceramide increase was sensitive to the ceramide synthase inhibitor fumonisin B(1), suggesting a crucial role for the de novo synthesis pathway. Enzymatic assays revealed that ceramide synthase activity remained unaltered, whereas serine palmitoyltransferase (SPT), a key enzyme in ceramide synthesis, was activated approximately 2.5-fold by RA treatment. Activation of SPT seemed to be mediated via a post-translational mechanism because levels of the mRNAs coding for the two SPT subunits were unaffected. Expression of marker proteins shows that ceramide regulates apoptosis, rather than differentiation, during early neural differentiation.     

Hormonal regulation of cytochrome P450 enzymes, cholesterol side-chain cleavage and 17 alpha-hydroxylase/C17-20 lyase in Leydig cells

Testosterone biosynthesis in Leydig cells is dependent on two cytochrome P450 enzymes, cholesterol side-chain cleavage (P450scc) and 17 alpha-hydroxylase/C17-20 lyase (P450(17 alpha]. The expression of these two enzymes is differentially regulated by LH acting via its second messenger, cyclic adenosine 3',5'-monophosphate (cAMP), and by specific steroid hormones. P450scc is constitutively expressed in normal mouse Leydig cells and in MA-10 tumor Leydig cells. Chronic cAMP stimulation increases the steady state levels of P450scc mRNA and de novo P450scc protein synthesis. In contrast, cAMP is obligatory for de novo synthesis of P450(17 alpha) in normal mouse Leydig cells; P450(17 alpha) synthesis ceases in the absence of luteinizing hormone or cAMP. MA-10 tumor Leydig cells do not express P450(17 alpha) even after treatment with cAMP. The amount of P450(17 alpha) in Leydig cells is negatively regulated by testosterone acting by two distinct mechanisms. At low concentrations, testosterone acts via the androgen receptor to repress cAMP-induced synthesis of P450(17 alpha), whereas at high concentrations this steroid increases the rate of degradation of the enzyme by an oxygen-mediated mechanism. Both constitutive and cAMP-induced synthesis of P450scc protein and steady state levels of mRNA are modulated by glucocorticoids. In normal mouse Leydig cells, glucocorticoids repress P450scc synthesis and steady state levels of P450scc mRNA, whereas glucocorticoids stimulate P450scc synthesis and levels of P450scc mRNA in the tumor Leydig cells.(ABSTRACT TRUNCATED AT 250 WORDS)