Stimulation of progesterone production by phorbol-12-myristate-13-acetate in MA-10 Leydig tumor cells

The tumor-promoting phorbol ester, phorbol-12-myristate-13-acetate (PMA) markedly stimulated progesterone production in MA-10 Leydig tumor cells. A slight but significant increase (35%) in the activity of the cholesterol side-chain cleavage (CSCC) enzyme was observed in mitochondria isolated from the PMA-treated MA-10 Leydig cells when compared to mitochondria isolated from non-treated cells. However, this stimulation of CSCC activity appears to be of limited importance when compared to the 240-fold increase observed in progesterone production following PMA stimulation. In contrast, the inactive phorbol ester 4 alpha-phorbol-12,13-didecanoate (alpha-PD) had no effect on either progesterone production or CSCC activity. PMA had no effect on the conversion of 25-hydroxycholesterol and 22R-hydroxycholesterol into progesterone suggesting that one of the mechanism(s) of PMA action may involve the delivery of cholesterol to the mitochondria and/or the affinity of cholesterol with cytochrome P-450scc. Stimulation of steroidogenesis by PMA was also shown to be inhibited by cycloheximide. When PMA was added together with a submaximal dose of hCG, hCG-stimulated steroidogenesis was inhibited. However, at a maximal dose of human chorionic gonadotropin (hCG), PMA inhibited steroid synthesis at 1 and 2 h but had no significant effect at 3 h. Conversely, PMA had an additive effect on cAMP induced steroidogenesis. It was further demonstrated that PMA resulted in a decrease in the hCG-induced accumulation of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of hCG- and cAMP-stimulated progesterone production in MA-10 mouse Leydig tumor cells by ketoconazole

In this study we attempted to examine the effects of ketoconazole on steroid biosynthesis and to determine which steps in the steroidogenic pathway were blocked using MA-10 Mouse Leydig tumor cells. This cloned cell line produces progesterone as the major steroid following stimulation by hCG or dbcAMP. At a concentration of 1 microM ketoconazole completely inhibited the hCG- and dbcAMP-stimulated progesterone synthesis in MA-10 Leydig cells. The conversion of 25-hydroxycholesterol and 22R-hydroxycholesterol into progesterone was also suppressed by this drug. The presence of ketoconazole inhibited mitochondrial steroid synthesis but required high concentrations of the drug as compared to inhibition in intact cells. No accumulation of pregnenolone was observed in the presence of ketoconazole indicating that the activity of 3 beta-hydroxysteroid dehydrogenase was not affected. We conclude that ketoconazole directly inhibits the activity of cholesterol side-chain cleavage enzyme (CSCC), a rate-determining enzymatic step in steroidogenesis, by interacting with cytochrome P-450scc.     

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.     

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.     

Role of proteoglycans on testosterone synthesis by purified Leydig cells from immature and mature rats.

In order to characterize an involvement of proteoglycans (PG) in the regulation of Leydig cell function, we have examined the effects of para-nitrophenyl-beta-D-xyloside (PNPX), a specific inhibitor of PG synthesis and para-nitrophenyl-beta-D-galactoside (PNPG), an inefficient structural analogue, on testosterone production by purified Leydig cells from immature and mature rats, in the presence or not of various concentrations of hCG during 24 h. Whatever the age, the addition of PNPX induces a decrease of [35S] and [3H] incorporations into cell layer associated-PG; these latter being less numerous (-50 and -25%, respectively in immature and mature rat), and less sulfated (-40%) when compared to control Leydig cells. In immature Leydig cells, the inhibition of PG synthesis decreases both the basal and weakly stimulable-hCG or -(Bu)2cAMP or -LH testosterone synthesis. In mature Leydig cells, the PG inhibition has no effect on testosterone production both in the absence of hCG and in the presence of weak amounts of hCG but increases it in the presence of subsaturating hCG concentrations. Whatever the age, the inhibition of PG synthesis is ineffective in the presence of saturating amounts of either hCG or (Bu)2cAMP. These effects are maintained in the presence of MIX, PMA, but are not observed in the presence of 22R-hydroxycholesterol. Therefore, our results suggest that in rat Leydig cells, the inhibition of PG synthesis affects the signal transduction at a step distal to cyclic AMP and more precisely, the cholesterol supply to the mitochondria by acting on its cellular distribution (free and esterified cholesterol).     

Evidence for the functional coupling of cyclic AMP in MA-10 mouse Leydig tumour cells

A number of studies have indicated that increased production of steroids can be obtained with doses of tropic hormone which do not result in detectable increases in intracellular cAMP. It has been suggested that this may be a result of compartmentalization or functional coupling of cAMP generated by hormone-receptor interactions to specific steroid producing pathways in the cell. In the present study we have stimulated the MA-10 mouse Leydig tumour cell with hCG, dibutyryl cAMP (dbcAMP) and forskolin to determine if functional coupling of cAMP occurs. Treatment with hCG, dbcAMP and forskolin all resulted in significant increases in the production of progesterone, the major steroid produced in these cells. Stimulation with hCG followed by 2D-PAGE analysis of the proteins resulted in the appearance of two proteins in the 30,000 molecular weight range (pI 6.8 and 6.6) and two in the 25,000-27,000 region (pI 5.9-6.0). Stimulation with dbcAMP or forskolin resulted in the appearance of the same proteins seen with hCG, but also in the appearance of two additional proteins, also having molecular weights of approximately 30,000 (pI 6.3 and 6.1). These data indicate that cAMP generated via hCG stimulation, whilst able to generate similar amounts of progesterone, does not stimulate the synthesis of the same proteins as does cAMP added exogenously or generated through indiscriminate activation of adenylate cyclase activity. Thus, it would appear that the gonadotropin activated pathway generates cAMP which remains functionally compartmentalized within the cell.     

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.     

Regulation of progesterone secretion in human syncytiotrophoblast in culture by human chorionic gonadotropin.

In the present investigation we sought to define the specific sites in the pathway of placental progesterone biosynthesis that underlie the action of human chorionic gonadotropin (hCG). When the cells were challenged with dibutryl cAMP (dbcAMP), forskolin or isobutylmethylxanthine, they produced significantly higher amounts of progesterone which in the presence of the hCG antibody was reduced to the level of the control set of cells. Trophoblast cells cultured in serum free medium with 25-hydroxycholesterol (25-OHC) produced increased amounts of progesterone. In the presence of hCG antibody at a concentration which neutralized the secreted hCG, the steroid production was completely blocked, even when the 25-OHC was added to the medium. Also, direct quantitation of the cytochrome P450 SCC enzyme in the absence of hCG indicated a significant decrease. The exogenous addition of low density lipoproteins (LDL) increased the progesterone secretion by the trophoblast cells in culture. Neutralization of hCG by the antibodies, however, drastically reduced the LDL induced progesterone secretion, which was restored by the addition of dbcAMP to the medium. Based on these findings, we suggest a stimulatory effect of hCG on normal trophoblast cells at the level of LDL utilization and cytochrome P450 SCC enzyme. Since dbcAMP could mimic these actions of hCG, the data suggest a possible autocrine/paracrine role of hCG on the trophoblast cells. An additive effect of hCG and cAMP on progesterone secretion observed in our studies, indicate that apart from hCG, adenylate cyclase activity may also be regulated by other factors.     

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.     

The regulation of steroidogenesis is different in the two types of ovine luteal cells

Ovine luteal tissue contains two distinct steroidogenic cell types, small (8-20 microns) and large (greater than 20 microns), which differ based on morphological and biochemical criteria. Unstimulated small cells secrete low levels of progesterone, respond to LH or dibutyryl cAMP (dbcAMP) with enhanced secretion of progesterone, and contain most of the receptors for LH. The unstimulated large cells, conversely, secrete high levels of progesterone, have few, if any, receptors for LH, and do not respond to LH or dbcAMP with increased progesterone secretion. The lack of response to dbcAMP by large cells was investigated. Large cells incubated in the presence of cholesterol, ram serum, or 25-hydroxycholesterol did not demonstrate substrate limitation. Hormone-independent stimulation of adenylate cyclase by cholera toxin or forskolin resulted in increased adenylate cyclase activities (P less than 0.01), cAMP accumulation (P less than 0.05), and the binding of endogenous cAMP (P less than 0.05) by type I cAMP-dependent protein kinase in both small and large cells. These treatments were accompanied by enhanced secretion of progesterone (P less than 0.05) in small cells. In contrast, large cells did not respond with an increase in progesterone secretion under these conditions. These observations suggest that the high rate of secretion of progesterone in unstimulated large cells is not regulated by cAMP.     

Modulation of adrenal cell functions by cadmium salts: 2. Sites affected by CdCl2 during unstimulated steroid synthesis

In previous studies cadmium chloride (CdCl₂) nonlethally inhibited Y-1 adrenal mouse adrenal tumour cell 20-dihydroxyprogesterone (20DHP) secretion, affecting unstimulated and stimulated steroidogenic pathway sites differently. We studied CdCl₂ effects on unstimulated steroidogenesis using Y-1 cells incubated 0.5 h in medium with or without cadmium (using the concentration that inhibited ACTH-stimulated steroid secretion by 50%). Exogenously added 20-hydroxycholesterol (20OHC), 22(R)-hydroxycholesterol (22OHC), 25-hydroxycholesterol (25OHC), pregnenolone (PREG), or progesterone (PROG) were used to bypass any rate-limited steroidogenic pathway sites that CdCl₂ might inhibit. 25OHC is a biologically active nonpathway steroid, while 20OHC, 22OHC, PREG, and PROG are pathway steroids; each increased unstimulated 20DHP secretion nearly 10-fold. Although CdCl₂ could not reduce dibutyryl cyclic AMP- (dbcAMP)-stimulated 20DHP secretion significantly, it did significantly reduce basal and 25OHC-induced 20DHP secretion 25% below untreated levels. When 20OHC, 22OHC, PREG, or PROG were incubated with unstimulated Y-1 cells, their synthesis into 20DHP was unaffected by cadmium. dbcAMP bypasses the plasma membrane enzyme complex that synthesizes intracellular cAMP during exogenous ACTH stimulation; dbcAMP was not inhibited by CdCl₂. The rate-limited step accelerated by cAMP involves plasma membrane and/or cytoplasmic cholesterol transport to and through outer and inner mitochondrial membranes before the cholesterol is synthesized into pregnenolone by side-chain cleavage enzymes on the inner membrane matrix face. Little is known regarding the mechanisms controlling unstimulated steroidogenesis. Under unstimulated conditions the 25-, 20- and 22(R)-monohydroxyls of cholesterol facilitate plasma membrane, cytoplasm and inner and outer mitochondrial solubility, diffusion and/or transport to bypass rate-limited steps and augment unstimulated steroid synthesis. Since conversion of endogenous mitochondrial cholesterol and 25OHC, but not dbcAMP-mobilized cytoplasmic cholesterol, 20OHC or 22OHC conversion, to 20DHP is inhibited by CdCl₂, this suggests that (a) control of mitochondrial cholesterol supplies is independent of the cAMP-regulated mitochondrial steps in the 20DHP steroid synthetic pathway, (b) CdCl₂ specifically inhibited endogenous mitochondrial cholesterol and 25OHC utilization, (c) CdCl₂ toxicity may affect adrenal, testicular, ovarian, and placental basal steroidogenic functions, and (d) 25OHC may be a useful compound to examine unstimulated steroid synthesisAbbreviations ACTH adrenocorticotropin - ANOVA analysis of variance - CdCl₂ cadmium chloride - cAMP cyclic 3,5-adenosine monophosphate - DMSO dimethylsulfoxide - DNA deoxyribonucleic acid - FMEM serum-free Eagle's Minimum Essential Medium - Hepes N-2-hydroxyethyl-piperazine-N-1,2-ethanesulfonic acid - 20OHC 20-hydroxycholesterol - 22OHC 22(R)-hydroxycholesterol - 25OHC 25-hydroxycholesterol - IC_50' concentration inhibiting stimulated steroid secretion by 50% - IU international unit - MEM Eagle's Minimum Essential Medium - P450scc cytochrome P450 side-chain cleavage enzyme - PREG pregnenolone - PROG progesterone - RNA ribonucleic acid - SEM standard error of the mean - SMEM serum-containing Eagle's Minimum Essential Medium - 20DHP 20-hydroxy-4-pregnen-3-one     

Effect of different steroidogenic stimuli on protein phosphorylation and steroidogenesis in MA-10 mouse Leydig tumor cells.

Numerous studies have indicated that treatment of Leydig cells with gonadotropin results in increased levels of intracellular cAMP, binding of cAMP to and activation of protein kinase A, phosphorylation of proteins, synthesis of new proteins and eventually, stimulation of steroidogenesis. In addition, recent studies have indicated that protein phosphorylation is an indispensable event in the production of steroids in response to hormone stimulation in adrenal cells. Because of the important role of phosphorylation in steroidogenic regulation, we investigated the effects of human chorionic gonadotropin (hCG), dibutyryl cyclic AMP (dbcAMP), forskolin and the phorbol ester, phorbol-12-myristate 13-acetate (PMA) on protein phosphorylation in MA-10 mouse Leydig tumor cells. Cells were stimulated with different steroidogenic compounds in the presence of [32P]orthophosphoric acid for 2 h and phosphoproteins analyzed by two-dimensional polyacrylamide gel-electrophoresis (PAGE). Results demonstrated an increase in the phosphorylation of four proteins (22 kDa, pI 5.9; 24 kDa, pI 6.7 and 30 kDa, pI 6.3 and 6.5) in response to 34 ng/ml hCG, 1 mM dbcAMP and 100 microM forskolin. Conversely, treatment of cells with PMA increased the phosphorylation of only one of these proteins (30 kDa, pI 6.3). At least two of these proteins (30 kDa, pI 6.5 and 6.3) appear to be identical to proteins which we and others have shown to be synthesized in response to trophic hormone stimulation in adrenal, luteal and Leydig cells. In addition, they also appear to be identical to adrenal cell mitochondrial proteins demonstrated to be phosphorylated in response to ACTH. These data indicate that proteins similar to those phosphorylated in adrenal cells in response to ACTH are phosphorylated in hormone stimulated testicular Leydig cells and that these proteins may be involved in steroidogenic regulation.     

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.     

Inhibition of gonadotropin-responsive adenylate cyclase in MA-10 Leydig tumor cells by epidermal growth factor

We have previously shown that mouse epidermal growth factor (mEGF) attenuates the increase in intracellular cAMP provoked by human choriogonadotropin (hCG) in MA-10 Leydig tumor cells (Ascoli, M., Euffa, J., and Segaloff, D. L. (1987) J. Biol. Chem. 262, 9196-9203). The studies presented herein were designed to investigate the mechanism(s) responsible for this phenomenon. We show that mEGF attenuates the increase in cAMP accumulation provoked by hCG primarily, if not entirely, by inhibiting adenylate cyclase activity. This phenomenon has some specificity for the agonist used, but it is not cell-specific. Thus, mEGF inhibited hCG-activated adenylate cyclase in MA-10 cells and in rat luteal cells but had no effect on the forskolin-activated enzyme in MA-10 cells or the isoproterenol-activated enzyme in rat luteal cells.     

Production of 25-hydroxycholesterol by testicular macrophages and its effects on Leydig cells

Testicular macrophages secrete 25-hydroxycholesterol, which can be converted to testosterone by neighboring Leydig cells. The purposes of the present studies were to determine the mode of production of this oxysterol and its long-term effects on Leydig cells. Because oxysterols are produced both enzymatically and by auto-oxidation, we first determined if testicular macrophages possess cholesterol 25-hydroxylase mRNA and/or if macrophage-secreted products oxidize cholesterol extracellularly. Rat testicular macrophages had 25-hydroxylase mRNA and converted 14C-cholesterol to 14C-25-hydroxycholesterol; however, radiolabeled cholesterol was not converted to 25-hydroxycholesterol when incubated with medium previously exposed to testicular macrophages. Exposure of Leydig cells to 10 microg/ml of 25-hydroxycholesterol, a dose within the range known to result in high basal production of testosterone when tested from 1 to 6 h, completely abolished LH responsiveness after 2 days of treatment. Because 25-hydroxycholesterol is toxic to many cell types at 1-5 microg/ml, we also studied its influence on Leydig cells during 4 days in culture using a wide range of doses. Leydig cells were highly resistant to the cytotoxic effects of 25-hydroxycholesterol, with no cells dying at 10 microg/ml and only 50% of cells affected at 100 microg/ml after 2 days of treatment. Similar conditions resulted in 100% death of a control lymphocyte cell line. These results demonstrate that 1) testicular macrophages have mRNA for cholesterol 25-hydroxylase and can convert cholesterol into 25-hydroxycholesterol, 2) macrophage-conditioned medium is not capable of auto-oxidation of cholesterol, 3) Leydig cells are highly resistant to the cytotoxic influences of 25-hydroxycholesterol, and 4) long-term treatment with high doses of 25-hydroxycholesterol results in loss of LH responsiveness. These results support the concept that testicular macrophages enzymatically produce 25-hydroxycholesterol that not only is metabolized to testosterone by Leydig cells when present at putative physiological levels but also may exert inhibitory influences on Leydig cells when present for extended periods at very high concentrations that may occur under pathological conditions.     

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.     

Cholesterol side-chain cleavage by mitochondria from the human placenta. Studies using hydroxycholesterols as substrates

The side-chain cleavage of cholesterol by cytochrome P-450_scc in mitochondria from the human placenta was studied using hydroxycholesterol substrates and intermediates of the reaction. 25-Hydroxycholesterol inhibited 3β-hydroxy-5-pregnen-20-one (pregnenolone) production by placental mitochondria. It was converted to pregnenolone at a maximum velocity of only 19% of that for cholesterol. Addition of 20-hydroxycholesterol or 22R-hydroxycholesterol to placental mitochondria caused a lag in pregnenolone synthesis which was concentration dependent. Measurement of the concentration of 20,22R-dihydroxycholesterol during incubation of placental mitochondria with 22R-hydroxycholesterol revealed that the lag in pregnenolone production was caused by accumulation of 20,22R-dihydroxycholesterol. This intermediate of the reaction dissociated from the active site of cytochrome P-450_scc. Only after its concentration had increased, presumably to a level where it could compete with 22R-hydroxycholesterol for binding to cytochrome P-450_scc, was it converted to pregnenolone. These results indicate a lack of kinetic stabilization of the cytochrome P-450_scc-20,22R-dihydroxycholesterol complex with dissociation occurring more rapidly than the final hydroxylation. Similar measurements of side-chain cleavage of 22R-hydroxycholesterol by mitochondria from the bovine adrenal cortex showed that kinetic stabilization of the cytochrome P-450_scc-20,22R-dihydroxycholesterol complex does not occur in that tissue either. The relative hydroxylation rates of 20-hydroxycholesterol, 22R-hydroxycholesterol and 20,22R-dihydroxycholesterol indicate that all three hydroxylations catalysed by human cytochrome P-450_scc occur at approximately the same rate.     

Low concentrations mono-butyl phthalate stimulates steroidogenesis by facilitating steroidogenic acute regulatory protein expression in mouse Leydig tumor cells (MLTC-1)

Di-n-butyl phthalate (DBP) is one of the most dominant phthalate esters and is widely distributed environmental contaminant. Although previous studies have demonstrated that DBP led to a variety of male reproductive abnormalities similar to those caused by androgen receptor antagonists, DBP and its active metabolite, mono-butyl phthalate (MBP), have been demonstrated no affinity for the androgen receptor, but rather exert anti-androgenic effect by altering testosterone biosynthesis. Furthermore, all these results were obtained from very high administrations of DBP or MBP. The purpose of this study was to determine the onset and the site of action of relatively low concentration of MBP on steroidogenesis in vitro. The mouse Leydig tumor cells (MLTC-1) was employed as a cellular model to investigate the effect of MBP on steroidogenesis. Various concentrations of MBP (1, 10, 100 and 1000nmol/l) and its solvent dimethyl sulfoxide (DMSO) were added to the medium for 24h followed by stimulation of some compounds such as human chorionic gonadotrophin (hCG), cholera toxin (CT), forskolin, cAMP analog 8-Br-cAMP, 22(R)-hydroxycholesterol (22R-HC) and pregnenolone. Progesterone in the medium and amounts of intracellular cAMP were measured by RIA. Expression of steroidogenic acute regulatory protein (StAR) was monitored by real-time PCR and Western blotting. The results revealed that the increases of progesterone production in the presence of hCG, CT, forskolin and 8-Br-cAMP were augmented by MBP. In contrast, the levels of intracellular cAMP exhibited no statistical significance when MLTC-1 cells were treated as above. These results implied that the site in the steroid biosynthesis pathway affected by MBP occurs after PKA activation in MLTC-1 cells. Moreover, supplementing the medium with 22R-HC and pregnenolone as progesterone precursors for P450 side chain cleavage enzyme (P450scc) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD), respectively, resulted in no rise in progesterone production, making clear that MBP did not influence the P450scc and 3beta-HSD but on the rate-limiting step, cholesterol transportation into mitochondria. In fact, the above results were confirmed by the upgraded StAR expression in MBP-treated cells. These data support that MBP promotes steroid hormone production by facilitating StAR expression in MLTC-1 cells.