Regulation of steroidogenesis in subclones of the MA-10 mouse Leydig tumor cell line.

We have previously reported the isolation of a subclone of the MA-10 mouse Leydig tumor cell line (MA-10 LP) which secretes less than 10% of the steroid synthesized by the parent, accumulates comparable amounts of cAMP and has equivalent cholesterol side-chain cleavage activity as the parent population (Kilgore and Stocco (1989) Endocrinology 124, 1210-1216). In the present study we show that addition of exogenous sterol carrier protein 2 (SCP2) to isolated mitochondria was not able to overcome the deficient steroid response of MA-10 LP. We have also demonstrated that human chorionic gonadotropin (hCG)-stimulated cellular events which activate steroid production by subsequently isolated mitochondria require ongoing protein synthesis, release of intracellular calcium and are mediated through the calcium-calmodulin complex. Additionally, mitochondrial sonicates from hCG-stimulated parent cells were able to stimulate steroid production by intact mitochondria isolated from unstimulated parent cells, whereas sonicates from similarly treated MA-10 LP had no effect on steroid production in these mitochondria. Together these data suggest that hCG induces changes in the mitochondria of the parent stock which are not induced to the same extent in the mitochondria of MA-10 LP.     

Multiple contributions from long-chain fatty acid metabolism in Y-1 and MA-10 cells

Acute steroidogenesis in either Y-1 or MA-10 cells is sensitive to different effects of fatty acids compare to a chronic stimulation. A 3-h stimulation of StAR expression in both cell types was completely blocked by NDGA and AA861, each functioning as lipoxygenase inhibitors. However, the acute 15-min stimulation in Y-1 cells was inhibited by these agents by distinct mechanisms. The inhibition by NDGA was reversed by arachidonic, linoleic, and oleic acids. The inhibition by AA861 was insensitive to reversal by these acids. StAR expression was not affected by these short-term inhibitor treatments. These more rapid fatty acid reversible effects of NDGA are consistent with previously reported inhibition of mitochondrial Acyl CoA ligase. This may function in cooperation with StAR and PBR in providing fatty acid regulation of mitochondrial cholesterol transport. The acute effect of AA861 supports the involvement of an NDGA-insensitive lipoxygenase in the acute stimulation of mitochondrial cholesterol metabolism. The activity of MA-10 cells during prolonged treatments with cAMP appears to utilize each of these processes, which depend on different metabolism of fatty acids.     

Effect of cholesterol transport inhibitors on steroidogenesis and plasma membrane cholesterol transport in cultured MA-10 Leydig tumor cells

These studies were directed toward understanding the cellular actions of inhibitor drugs that affect steroidogenesis and cholesterol transport. We investigated the microfilament inhibitor cytochalasin-D, the microtubule inhibitor colchicine, the calmodulin antagonist trifluoperazine, and the inhibitor of acidic vesicle function nigericin. We found that all of these compounds caused dose-dependent inhibition of progesterone synthesis in the MA-10 cells. Each compound also inhibited (Bu)2cAMP-stimulated pregnenolone synthesis, indicating that each inhibited a fundamental process required for steroidogenesis. Each compound was next evaluated for inhibitory actions on cholesterol transport to and from the plasma membrane. On the basis of inhibitor sensitivity, two different categories of cholesterol transport were defined. Transport of newly synthesized or low density lipoprotein-derived cholesterol from the cell interior to the plasma membrane was inhibitor insensitive. Plasma membrane cholesterol internalization, however, was sensitive to all of the inhibitors and did not result because of any drug effect on the acyl-coenzyme-A-cholesterol acyl transferase. Cycling of cholesteryl ester-derived cholesterol through the plasma membrane appeared to occur before its use for steroidogenesis. Thus, inhibition of plasma membrane internalization would prevent utilization of both plasma membrane cholesterol and cholesteryl ester-derived cholesterol, the two major substrate sources for steroid hormone synthesis. Consistent with this interpretation was the finding that inhibition of plasma membrane cholesterol internalization by each inhibitor paralleled the inhibitor's effect on steroidogenesis.     

Effects of genistein, resveratrol, and quercetin on steroidogenesis and proliferation of MA-10 mouse Leydig tumor cells

This study was performed to compare the effects of three well-known phytoestrogens such as genistein, resveratrol, and quercetin on steroidogenesis in MA-10 mouse tumor Leydig cells. Addition of genistein or resveratrol to MA-10 cells resulted in decreases in the cAMP-stimulated progesterone secretion, but quercetin had an opposite response. Steroidogenic acute regulatory (StAR) mRNA expression and StAR promoter activity in transiently transfected MA-10 cells were significantly reduced by genistein or resveratrol, but increased by quercetin. Genistein was found to inhibit MA-10 cell proliferation, while resveratrol and quercetin had no effect. Quercetin-induced increase in cAMP-stimulated progesterone secretion was reversed by ICI 182,780, an estrogen receptor (ER) antagonist. However, ICI 182,780 had no effect on cAMP plus quercetin-stimulated StAR promoter activity. To examine whether non-ER factors are associated with quercetin-stimulated progesterone production, we treated MA-10 cells with EGTA to deprive them of extracellular Ca(2+). We found that EGTA inhibited quercetin-plus cAMP-stimulated progesterone secretion and StAR promoter activity. Blocking of Ca(2+) influx through L- or T-type voltage-gated Ca(2+) channels with verapamil or mibefradil respectively, attenuated quercetin-stimulated progesterone secretion, while they had no effect on quercetin-plus cAMP-stimulated StAR promoter activity. Blocking of intracellular Ca(2+) efflux by sodium orthovanadate, a Ca(2+)-pump inhibitor, blocked quercetin- plus cAMP-stimulated progesterone secretion and StAR promoter activity in MA-10 cells. Finally, EGTA or vanadate reduced quercetin and cAMP-increased in StAR mRNA expression in MA-10 cells, while ICI 182,780 had no effect. Taken together, these results indicate that phytoestrogens have differential effects on steroidogenesis in MA-10 cells.     

Interaction between arachidonic acid and cAMP signaling pathways enhances steroidogenesis and StAR gene expression in MA-10 Leydig tumor cells

Previous studies have demonstrated that trophic hormone stimulation induced cyclic AMP (cAMP) formation and arachidonic acid (AA) release from phospholipids and that both these compounds were required for steroid biosynthesis and steroidogenic acute regulatory (StAR) gene expression in MA-10 mouse Leydig tumor cells. The present study further investigates the synergistic effects of the AA and cAMP interaction on steroidogenesis. To demonstrate cAMP-induced AA release, MA-10 cells were pre-loaded with 3H-AA and subsequently treated with dibutyryl cyclic AMP (dbcAMP). Stimulation with dbcAMP significantly induced AA release in MA-10 cells to a level 145.7% higher than that of controls. Lowering intracellular cAMP concentration by expressing a cAMP-phosphodiesterase significantly reduced human chorionic gonadotrophin (hCG)-induced AA release. The dbcAMP-induced AA release was inhibited significantly by the phospholipase A(2) (PLA(2)) inhibitor dexamethasone (Dex) and also by the protein kinase A (PKA) inhibitor H89, suggesting the involvement of PKA phosphorylation and/or PLA(2) activation in cAMP-induced AA release. The effect of the interaction between AA and cAMP on StAR gene expression and steroid production was also investigated. While 0.2 mM dbcAMP induced only very low levels of StAR protein, StAR mRNA, StAR promoter activity and steroid production, all of these parameters increased dramatically as AA concentration in the culture medium was increased from 0 to 200 microM. Importantly, AA was not able to induce a significant increase in steroidogenesis at any concentration when used in the absence of dbcAMP. However, when used in concert with submaximal concentrations of dbcAMP (0.05 mm to 0.5 mm), AA was capable of stimulating StAR gene expression and increasing steroid production significantly. The results from this study demonstrate that AA and cAMP act in a highly synergistic manner to increase the sensitivity of steroid production to trophic hormone stimulation and probably do so by increasing StAR gene expression.     

Reactive oxygen disrupts mitochondria in MA-10 tumor Leydig cells and inhibits steroidogenic acute regulatory (StAR) protein and steroidogenesis

Reactive oxygen species (ROS) are involved in a variety of pathophysiological conditions of the testis, and oxidative stress is known to inhibit ovarian and testicular steroidogenesis. The site of ROS-mediated inhibition of steroidogenesis in the corpus luteum and MA-10 tumor Leydig cells was shown to be the hormone-sensitive mitochondrial cholesterol transfer step. The purpose of this study was to examine the effects of ROS on steroidogenic acute regulatory (StAR) protein in MA-10 cells and determine the extent to which MA-10 cell mitochondria are sensitive to oxidative stress. cAMP-stimulated progesterone production was inhibited in a dose-dependent manner in MA-10 cells exposed to H(2)O(2). StAR protein, but not mRNA levels, was decreased in parallel to changes in progesterone production. Even at the highest concentrations of H(2)O(2) tested, there was no effect on P450 side-chain cleavage enzyme protein levels. Oxidative stress from exposure to exogenous xanthine oxidase and xanthine resulted in the inhibition of both progesterone production and StAR protein expression. The mature 30- and 32-kDa intramitochondrial forms of StAR were decreased relative to the 37-kDa extramitochondrial precursor form of StAR, indicating that the ROS-mediated inhibition of StAR protein was due, in part, to the inhibition of mitochondrial import and processing. Vital staining with the fluorescent dye tetramethylrhodamine ethyl ester was used to visualize changes in the mitochondrial electrochemical gradient-dependent membrane potential (Deltapsim). ROS caused a significant dissipation of Deltapsi(m) and time-dependent loss of tetramethylrhodamine ethyl ester fluorescence. The inhibitory effects of H(2)O(2) were transient. There was no evidence for ROS-induced cell death, and following H(2)O(2) removal in the presence of continuous treatment with 8-bromo-cAMP, StAR protein levels and progesterone production were restored. In addition, there was no loss of cell viability following treatment with H(2)O(2) or xanthine/xanthine oxidase as determined by trypan blue exclusion. H(2)O(2) did not cause a significant decrease in total cellular ATP levels. These data indicate that oxidative stress-mediated perturbation of the mitochondria and dissipation of Deltapsi(m) results in the inhibition of StAR protein expression and its import, processing, and cholesterol transfer activity. These findings confirm earlier studies demonstrating the requirement for maintenance of an intact Deltapsi(m) for StAR protein function in cholesterol transport. The significant reduction in the 32- to 30-kDa mature forms of StAR, cessation of cholesterol transport, and loss of Deltapsi(m) are consistent with mitochondrial perturbation because of oxidative stress. This mechanism likely contributes to a host of pathophysiological events evident in testicular disorders such as infection, reperfusion injury, aging, cryptorchidism, and varicocele.     

Effects of steroidogenesis inducing protein (SIP) on steroid production in MA-10 mouse Leydig tumor cells: utilization of a non-cAMP second messenger pathway.

We have investigated the effects of steroidogenesis inducing protein (SIP) (Endocrinology (1990) 126, 3043-3052) on steroid production in MA-10 mouse Leydig tumor cells. Our results indicate that SIP results in the stimulation of progesterone production in MA-10 cells to the same extent obtained when maximal doses of luteinizing hormone (LH), human chorionic gonadotropin (hCG) and dibutyryl cAMP (dbcAMP) are used. It was also observed that the increased progesterone production in response to SIP was not accompanied by an increase in intracellular cAMP levels as was seen following hCG stimulation. In addition, stimulation of progesterone production using maximal doses of LH, hCG and dbcAMP could be further increased by the addition of SIP to the incubation medium also indicating that this steroidogenic activity was acting through a differential signal transducing system than these hormones. That SIP was not acting through the cAMP second messenger pathway was also demonstrated by its lack of sensitivity to the neutralizing effects of a monoclonal antibody to LH as well as by its insensitivity to the protein kinase A inhibitor HA 1004 while both of these treatments significantly decreased LH and hCG stimulated steroid production. Lastly, SIP was unable to elicit the induction of several mitochondrial proteins which have previously been shown to be synthesized in MA-10 cells in response to LH, hCG and dbcAMP. Our results indicate that SIP stimulates the production of high levels of steroids through a signal transduction pathway which is distinct from that employed by trophic hormone stimulation in Leydig cells.     

Effects of overexpression of CXCL10 (cytokine-responsive gene-2) on MA-10 mouse Leydig tumor cell steroidogenesis and proliferation

Chemokines have been implicated in tumor growth, angiogenesis, metastasis and the host immune response to malignant cells. Infection and autoimmune disorders can reduce androgen production by Leydig cells and adversely affect spermatogenesis. Cytokine-responsive gene-2 (crg-2) (systematic name CXCL10, also known as interferon-gamma-inducible protein 10 (IP-10)) is a potent chemokine expressed predominantly by macrophages and Leydig cells in the testis. CXCL10 binds to CXCR3 receptor (a G-protein-coupled receptor) and acts via Gialpha protein. We have shown previously that CXCL10 is differentially expressed in normal Leydig cells, inhibited by human chorionic gonadotropin and induced by interferon-gamma, interleukin-1alpha and tumor necrosis factor-alpha. The purpose of the present study was to determine the effects of overexpression of CXCL10 by transfection experiments in MA-10 cells on cell growth, CXCR3 expression, progesterone synthesis and steroidogenic acute regulatory protein (StAR D1, a key regulatory factor in steroidogenesis) gene expression. We cloned the complete CXCL10 cDNA in a mammalian expression vector with the CMV promoter, pcDNA3.1D/V5-His-TOPO, and confirmed its expression with rat CXCL10 antibody and V5 antibody. Results showed large amounts of CXCL10 protein secreted in the medium in the CXCL10 transfectants by Western blotting. The production of CXCL10 mRNA ranged from 30-50-fold more (n=6) in the transfected cells than the control cells, as determined by semiquantitative and real-time RT-PCR. 8-Br-cAMP downregulated CXCL10 mRNA expression and stimulated CXCR3 mRNA expression. Transfection of MA-10 cells with CXCL10 decreased cAMP-induced progesterone synthesis from 38.5+/-1.7 ng/ml (1.5 x 10(5) cells/ml) in control cells to 23.2+/-1.5 ng in transfected cells (P<0.01). 8-Br-cAMP (0.2 mM)-induced StAR D1 mRNA was decreased 30-40% by transfection with CXCL10. Interestingly, overexpression of CXCL10 induced the expression of its receptor CXCR3 gene, as determined by RT-PCR and fluorescence-activated cell sorter (FACS) analysis. Transfection of CXCL10 also significantly decreased insulin-like growth factor-I (IGF-I, 100 ng/ ml)-induced [3H]thymidine incorporation into DNA. These data suggest that CXCL10 also inhibits MA-10 tumor cell proliferation. In conclusion, CXCL10 inhibits StAR D1 expression, decreases progesterone synthesis and inhibits cell proliferation. CXCL10 has the potential to be used in gene therapy for prostate cancer due to its antiangiogenic effect and its inhibitory effect on steroidogenesis.     

Protein tyrosine phosphatases are involved in LH/chorionic gonadotropin and 8Br-cAMP regulation of steroidogenesis and StAR protein levels in MA-10 Leydig cells

The LH signal transduction pathway features the activation of protein tyrosine phosphatases (PTPs) as one of the components of a cascade that includes other well characterized events such as cAMP-dependent protein kinase A (PKA) activation. Moreover, the action of PTPs is required to increase the rate-limiting step in steroid biosynthesis, namely the cAMP-regulated transfer of cholesterol to the inner mitochondrial membrane. Since both PKA activity and steroidogenic acute regulatory (StAR) protein induction are obligatory steps in this transfer of cholesterol, the present study was performed to investigate the role of PTPs in the regulation of PKA activity and StAR expression in response to LH/chorionic gonadotropin (CG) and 8Br-cAMP in MA-10 cells. While the exposure of MA-10 cells to the PTP inhibitor, phenylarsine oxide (PAO), did not modify PKA activity, it partially inhibited the effect of human CG and cAMP analog on StAR protein levels. Time-course studies demonstrated that PAO inhibited cAMP induction of StAR protein and mRNA. At 30 min, the effect on cAMP-stimulated StAR protein levels was a 35% inhibition, progressing to up to 90% inhibition at 120 min of stimulation. The maximal inhibitory effect on cAMP-induced StAR mRNA level was obtained at 60 min (85%). In summary, these results demonstrate that inhibition of PTP activity affected both StAR protein and mRNA synthesis and suggest that the activity of hormone-regulated PTPs is a requirement in the LH signaling cascade that results in the up-regulation of StAR protein and, subsequently, increased steroid synthesis.     

Metabolic clearance rate, production rate, and mammary uptake and metabolism of progesterone in cows.

Tracer kinetic techniques have been used to measure the production rate, metabolic clearance rate and mammary uptake of progesterone in six experiments on two Jersey cowsmthe cows were surgically prepared so that the carotid artery, jugular vein and mammary vein concentrations of progesterone, and udder blood flow, could be determined in conscious animals without anaesthesia or stress. The mean production rate of progesterone was 173 +/- 23-3 (S.ET) mug/min, with values ranging from 80 to 276 mug/min in pregnancy. The metabolic clearance rate was 22-5 +/- 2-0 1/min, or 0-21 +/- 0-025 1/min/kg metabolic body weight. The mammary uptake of progesterone was low, 3-1 +/- mug/min, and udder uptake accounted for about 3% of progesterone production rate. During [3H]A1progesterone infusion, radioactivity was transferred from blood to milk, probably by diffusion down a concentration gradient. Progesterone accounted for more than 88% of the ether-soluble radioactivity recovered from milk.     

A cholesteryl ester hydrolase inhibitor blocks cholesterol translocation into the mitochondria of MA-10 Leydig tumor cells.

The present studies describe an unexpected action of a cholesteryl ester hydrolase inhibitor on MA-10 Leydig tumor cells. These studies were initially intended to use the inhibitor, diethylumbelliferyl phosphate, to block cholesteryl ester hydrolysis and, thus, determine the contributions of this form of cholesterol to steroidogenesis and reveal any direct hormone effects on cholesterol esterification. Although this compound acted as an effective inhibitor of the cholesteryl ester hydrolase in intact MA-10 cells, it inhibited steroidogenesis at lower concentrations and to a greater extent than could be explained by simple inhibition of the ester hydrolase enzyme. This compound proved not to be generally toxic, but blocked some process occurring between cAMP formation and cholesterol side-chain cleavage. The diethylumbelliferyl phosphate block of steroidogenesis was readily bypassed by 22-hydroxycholesterol. These data indicated that the compound inhibited cholesterol transport. The lesion in cholesterol transport was not general, but very specific; cholesterol translocation to the mitochondrial site of cholesterol side-chain cleavage was blocked by this organophosphate compound.     

Ca+2 is an inhibitor of adenylate cyclase in MA-10 Leydig tumor cells

Using a clonal strain of cultured Leydig tumor cells (designated MA-10), we have examined the effects of Ca+2 on the activation of cAMP accumulation and steroid biosynthesis by hCG. Our results show that addition of Ca+2 ionophores (A23187 or ionomycin) leads to inhibition of the activation of cAMP accumulation by hCG. The magnitude of this effect is dependent on the concentrations of ionophore and hCG used, becoming more pronounced as the concentration of hCG increases. A detailed examination of the effects of A23187 and removal of extracellular Ca+2 on the rates of cAMP synthesis and degradation in intact cells revealed that A23187 inhibits the rate of cAMP accumulation activated by hCG, but does not affect the rate of degradation of cAMP. On the other hand, removal of extracellular Ca+2 had no effect on the rate of cAMP accumulation activated by hCG or on the rate of degradation of cAMP. Removal of extracellular Ca+2, however, completely prevented the inhibitory effect of A23187 on the rate of cAMP accumulation. Additional experiments show that the effects of A23187 or removal of extracellular Ca+2 on hCG-activated steroidogenesis closely parallel those described for cAMP accumulation. We conclude that Ca+2 is an inhibitor of the hCG-activated adenylate cyclase in Leydig tumor cells, and that this inhibition imposes a limitation on the ability of hCG to activate steroid biosynthesis.     

Epidermal growth factor modulates intracellular arachidonic acid levels in MA-10 cultured Leydig tumor cells

Epidermal growth factor (EGF) acts on various cell types, including the mouse Leydig tumor cell line MA-10, where it has been shown to stimulate steroidogenesis, apparently in a cAMP-independent manner. In the process of examining other possible signaling pathways for EGF in these cells, we found rapid changes in the intracellular concentration of arachidonic acid (AA) following addition of EGF. For example, a significant increase in AA was detected 1 min after incubating the cells with EGF, with the maximal effect observed at an EGF concentration of 10 ng/ml. In addition, exogenous AA increased steroidogenesis, and the steroidogenesis enhanced by AA and EGF was reduced by lipoxygenase inhibitors, suggesting a possible role of an AA metabolite(s) in promoting steroidogenesis. Consistent with this hypothesis is our observation that several exogenous lipoxygenase metabolites were capable of enhancing progesterone production. The EGF-stimulated steroidogenesis was also inhibited by two phospholipase A2 inhibitors, again confirming a probable role of AA or a metabolite in this process. Therefore, AA appears to be an important intracellular mediator responsible, at least in part, for some of the acute metabolic effects mediated by EGF in MA-10 cells.     

Mechanism of action of progesterone on amphibian oocytes. A possible biological role for progesterone metabolism.

The ability of various steroids to induce maturation of Pleurodeles waltlii oocytes after incubation has been studied. Progesterone is metabolized during the course of maturation. All metabolites isolated are less efficient than progesterone for inducing germinal vesicle breakdown. Progesterone binding to the 'melanosome' fraction has been studied (KD = 4.5 X 10(-8) M at 4 degrees C). 20BETA-Hydroxy-pregn-4-en-3-one, the main progesterone metabolite isolated from the oocyte, also binds to the melanosome fraction, but with a lower affinity (KD = 1.6 X 10(-7) M at 4 degrees C). At high concentration 20beta-hydroxy-pregn-4-en-3-one induces maturation, but at low concentration it is a competitive inhibitor of progesterone. Progesterone metabolism in Pleurodeles oocytes can be interpreted as an inactivation process, and also as a mechanism for inhibitime progesterone action.