Progesterone attenuates the inhibitory effects of cardiotonic digitalis on pregnenolone production in rat luteal cells

Previous studies have shown that digoxin decreases testosterone secretion in testicular interstitial cells. However, the effect of digoxin on progesterone secretion in luteal cells is unclear. Progesterone is known as an endogenous digoxin-like hormone (EDLH). This study investigates how digitalis affected progesterone production and whether progesterone antagonized the effects of digitalis. Digoxin or digitoxin, but not ouabain, decreased the basal and human chorionic gonadotropin (hCG)-stimulated progesterone secretion as well as the activity of cytochrome P450 side chain cleavage enzyme (P450scc) in luteal cells. 8-Br-cAMP and forskolin did not affect the reduction. Neither the amount of P450scc, the amount of steroidogenic acute regulatory (StAR) protein, nor the activity of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) was affected by digoxin or digitoxin. Moreover, in testicular interstitial and luteal cells, progesterone partially attenuated the reduction of pregnenolone by digoxin or digitoxin and the progesterone antagonist, RU486, blocked this attenuation. These new findings indicated that (1) digoxin or digitoxin inhibited pregnenolone production by decreasing the activity of P450scc enzyme, but not Na(+)-K(+)-ATPase, resulting in a decrease on progesterone secretion in rat luteal cells, and (2) the inhibitory effect on pregnenolone production by digoxin or digitoxin was reversed partially by progesterone. In conclusion, digoxin or digitoxin decreased progesterone production via the inhibition of pregnenolone by decreasing P450scc activity. Progesterone, an EDLH, could antagonize the effects of digoxin or digitoxin in luteal cells.     

Antisteroidogenic effects of hydrogen peroxide on rat granulosa cells

Rat granulosa cells (GCs) were treated with human chorionic gonadotropin (hCG), 8-bromo-adenosine 3',5'-cyclic monophosphate (8-Br-cAMP), forskolin, phorbol 12-myristate 13-acetate (PMA), A23187 or pregnenolone in the absence or presence of hydrogen peroxide (H(2)O(2)). Different doses of trilostane were applied to GCs treated with steroidogenic precursors, that is, 25-hydroxy-cholesterol (25-OH-C) in the absence or presence of H(2)O(2). Results showed that all of the chemicals stimulated the progesterone (PG) release from rat GCs, but the stimulatory effects were inhibited by H(2)O(2) dose-dependently. 25-OH-C stimulated the PG release, which was inhibited by H(2)O(2) in the presence of trilostane. H(2)O(2) attenuated steroidogenic acute regulatory (StAR) protein expression, but did not alter the expression of cytochrome P450 side chain cleavage (P450scc) in Western blotting. This study indicated that H(2)O(2) inhibited PG production by GCs via cAMP pathway, protein kinase C (PKC) and the activities of intracellular calcium, P450scc and StAR protein.     

Mechanisms of digoxin and digitoxin on the production of corticosterone in zona fasciculata-reticularis cells of ovariectomized rats

Previous studies have indicated that digoxin (DG) inhibits testosterone production by rat testicular interstitial cells through both in vivo and in vitro experiments. DG and digitoxin (DT), but not ouabain, inhibit the progesterone, pregnenolone, and corticosterone secretion by rat granulosa cells, luteal cells, and zona fasciculata-reticularis (ZFR) cells, respectively. However, the effect of DG and DT on the enzyme kinetics of cytochrome P450 side chain cleavage enzyme (P450scc), the protein expression of P450scc and steroidogenic acute regulatory protein (StAR), and mRNA expression of StAR are unclear. ZFR cells were prepared from adrenocortical tissues of ovariectomized rats, and then challenged with adrenocorticotropin (ACTH), 8-Br-cAMP, forskolin, A23187, cyclopiazonic acid (CPA), nicotinic acid adenine dinucleotide phosphate (NAADP), trilostane, 25-OH-Cholesterol, progesterone, or deoxycorticosterone in the presence of DG, DT, or ouabain for 1 h. Enzyme kinetics of P450scc, protein expression of acute regulatory protein (StAR) and P450scc, and mRNA expression of StAR were investigated. DG and DT but not ouabain suppressed basal and other evoked-corticosterone release significantly. DG and DT also inhibited pregnenolone production. The Vmax of the DG and DT group was the same as the control group, but the Km was higher in DG- and DT-treated group than in control group. DT and ouabain significant suppressed mRNA expression of StAR. DG and DT had no effect on the P450scc and StAR protein expression at basal state, but diminished ACTH-induced StAR protein expression to basal level. These results indicated that DG and DT have an inhibitory effect on corticosterone production via a Na+, K+-ATPase-independent mechanism by diminishing actions on cAMP-, Ca2+-pathway, competitive inhibition of P450scc enzyme and reduction of StAR mRNA expression.     

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.     

Inhibition of testosterone production by propylthiouracil in rat Leydig cells

Propylthiouracil (PTU) is a thioamide drug used clinically to inhibit thyroid hormone production. However, PTU is associated with some side effects in different organs. In the present study, the acute and direct effects of PTU on testosterone production in rat Leydig cells were investigated. Leydig cells were isolated from rat testes, and an investigation was performed on the effects of PTU on basal and evoked-testosterone release, the functions of steroidogenic enzymes, including protein expression of cytochrome P450 side-chain cleavage enzyme (P450(scc)) and mRNA expression of the steroidogenic acute regulatory protein (StAR). Rat Leydig cells were challenged with hCG, forskolin, and 8-bromo-cAMP to stimulate testosterone release. PTU inhibited both basal and evoked-testosterone release. To study the effects of PTU on steroidogenesis, steroidogenic precursor-stimulated testosterone release was examined. PTU inhibited pregnenolone production (i.e., it diminished the function of P450(scc) in Leydig cells). In addition to inhibiting hormone secretion, PTU also regulated steroidogenesis by diminishing mRNA expression of StAR. These results suggest that PTU acts directly on rat Leydig cells to diminish testosterone production by inhibiting P450(scc) function and StAR expression.     

Dexamethasone inhibits luteinizing hormone-induced synthesis of steroidogenic acute regulatory protein in cultured rat preovulatory follicles

The effect of dexamethasone on LH-induced synthesis of steroidogenic acute regulatory (StAR) protein was studied in a serum-free culture of preovulatory follicles. StAR protein is a steroidogenic tissue-specific, hormone-induced, rapidly synthesized protein previously shown to be involved in the acute regulation of steroidogenesis, probably by promoting the transfer of cholesterol to the inner mitochondrial membrane and the cytochrome P450 side-chain cleavage (P450(scc)) enzyme. Treatment of preovulatory follicles dissected from ovaries of cyclic adult rats on the morning of proestrus with LH for 24 h resulted in a dose-dependent increase in the level of StAR protein that reached a maximum at 10 ng LH/ml. This increase was associated with an increase in progesterone production. Treatment of the follicles with increasing concentrations (1-1000 ng/ml) of dexamethasone suppressed LH (10 ng/ml)-induced StAR protein levels and progesterone production in a dose-dependent manner. The amount of P450(scc) was not affected by this dexamethasone treatment, indicating that the loss of steroidogenic capacity was not a result of inhibition of P450(scc). Dexamethasone also decreased StAR protein levels and progesterone production induced by the adenylate cyclase activator forskolin (10(-5) M) or a cAMP analogue 8-Br-cAMP (0.5 mM). The effects of dexamethasone on 8-Br-cAMP-induced StAR protein levels and progesterone production were blocked by cotreatment of the follicles with glucocorticoid receptor antagonist RU-486. These results demonstrate that dexamethasone inhibits the LH-induced StAR protein levels and that the effects of dexamethasone are mediated by the glucocorticoid receptor.     

Protein phosphatase inhibitor cantharidin inhibits steroidogenesis and steroidogenic acute regulatory protein expression in cultured rat preovulatory follicles.

The effect of cantharidin, a natural toxicant of blister beetles and a strong inhibitor of protein phosphatases types 1 and 2A, on luteinizing hormone (LH)-induced synthesis of steroidogenic acute regulatory (StAR) protein was studied in a serum-free culture of preovulatory follicles. StAR protein is a steroidogenic tissue-specific, hormone-induced, rapidly synthesized protein previously shown to be involved in the acute regulation of steroidogenesis, probably by promoting the transfer of cholesterol to the inner mitochondrial membrane and the cytochrome P450 side-chain cleavage (P450scc) enzyme. Treatment of preovulatory follicles dissected from ovaries of immature rats primed with pregnant mares' serum gonadotropin (10 IU) with LH for 24 h resulted in a dose-dependent increase in the level of StAR protein that reached a maximum at 100 ng LH/ml. This increase was associated with an increase in progesterone production. Treatment of follicles with increasing concentrations (10 - 1000 ng/ml) of cantharidin suppresssed LH (100 ng/ml)-induced StAR protein levels and progesterone production in a dose-dependent manner. The amount of P450scc protein and the conversion of 22R-hydroxycholesterol to progesterone were not affected by cantharidin. This indicates that cantharidin did not interfere with the activity of P450scc. Cantharidin also decreased StAR protein levels and progesterone production induced by the adenylate cyclase activator forskolin (10(-5) M) or a cAMP analog 8-Br-cAMP (0.5 mM). These results demonstrate that cantharidin inhibits the LH-induced StAR protein levels, and, thus, suggest that phosphoprotein phosphatase activity is required for the cAMP-protein kinase A-stimulated steroidogenic activity of the preovulatory follicle.     

Antisteroidogenic effects of hydrogen peroxide on rat granulosa cells

Abstract

Rat granulosa cells (GCs) were treated with human chorionic gonadotropin (hCG), 8-bromo-adenosine 3′,5′-cyclic monophosphate (8-Br-cAMP), forskolin, phorbol 12-myristate 13-acetate (PMA), A23187 or pregnenolone in the absence or presence of hydrogen peroxide (H₂O₂). Different doses of trilostane were applied to GCs treated with steroidogenic precursors, that is, 25-hydroxy-cholesterol (25-OH-C) in the absence or presence of H₂O₂. Results showed that all of the chemicals stimulated the progesterone (PG) release from rat GCs, but the stimulatory effects were inhibited by H₂O₂ dose-dependently. 25-OH-C stimulated the PG release, which was inhibited by H₂O₂ in the presence of trilostane. H₂O₂ attenuated steroidogenic acute regulatory (StAR) protein expression, but did not alter the expression of cytochrome P450 side chain cleavage (P450scc) in Western blotting. This study indicated that H₂O₂ inhibited PG production by GCs via cAMP pathway, protein kinase C (PKC) and the activities of intracellular calcium, P450scc and StAR protein.     

Effects of dehydroepiandrosterone on aldosterone release in rat zona glomerulosa cells

The present study was to investigate the effects and action mechanisms of dehydroepiandrosterone (DHEA) on steroidogenesis in rat adrenal zona glomerulosa cells (ZG). ZG cells were incubated with DHEA in the presence or absence of angiotensin II (AngII), a high concentration of potassium, 8-Br-cAMP, forskolin, 25-OH-cholesterol, pregnenolone, progesterone, deoxycorticosterone, corticosterone, A23187, or cyclopiazonic acid (CPA) at 37°C for 1 h. The concentration of aldosterone or pregnenolone in the culture medium was then measured by radioimmunoassay (RIA). The cells were used to determine the cellular cAMP content. The data demonstrated that: (1) DHEA inhibited AngII-, high concentration of KCl-, forskolin-, 8-Br-cAMP-, 25-OH-cholesterol-, pregnenolone-, progesterone-, deoxycorticosterone-, corticosterone-, A23187-, or CPA-stimulated aldosterone release; (2) DHEA increased 25-OH-cholesterol-stimulated pregnenolone release but not when 25-OH-cholesterol was combined with trilostane; (3) DHEA noncompetitively inhibited aldosterone synthase but showed uncompetitive inhibition of P450scc. These results suggest that DHEA acts directly on rat ZG cells to diminish aldosterone secretion by inhibition of a post-cAMP pathway or by acting on intracellular Ca²⁺ mobilization. In addition it affects the function of post-P450scc steroidogenic enzymes. Ling-Ling Chang and Paulus S. Wang contributed equally to this work.     

Effects of ovarian theca cells on granulosa cell differentiation during gonadotropin-independent follicular growth in cattle

We investigated the effects of theca cells or FSH on granulosa cell differentiation and steroid production during bovine early follicular growth, using a co-culture system in which granulosa and theca cells were cultured on opposite sides of a collagen membrane. Follicular cells were isolated from early antral follicles (2-4 mm) that were assumed to be in gonadotropin-independent phase and just before recruitment into a follicular wave. Granulosa cells were cultured under serum-free conditions with and without theca cells or recombinant human FSH to test their effects on granulosa cell differentiation. Messenger RNA levels for P450 aromatase (aromatase), P450 cholesterol side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), LH receptor (LHr), and steroidogenic acute regulatory protein (StAR) in granulosa cells were measured by real-time quantitative RT-PCR analysis. FSH enhanced aromatase mRNA expression in granulosa cells, but did not alter estradiol production. FSH also enhanced mRNA expression for P450scc, LHr, and StAR in granulosa cells, resulting in an increase in progesterone production. In contrast, theca cells enhanced aromatase mRNA expression in granulosa cells resulting in an increase in estradiol production. Theca cells did not alter progesterone production and mRNA expression in granulosa cells for P450scc, 3beta-HSD, LHr, and StAR. The results of the present study indicate that theca cells are involved in both rate-limiting steps in estrogen production, i.e., androgen substrate production and aromatase regulation, and that theca cell-derived factors regulate estradiol and progesterone production in a way that reflects steroidogenesis during the follicular phase of the estrous cycle.     

Actions of cyclic adenosine monophosphate on the cytodifferentiation of ovarian cells: studies in cultured swine granulosa cells using a novel exogenous adenylate cyclase from Bordetella pertussis

The regulation by cAMP of cholesterol side-chain cleavage activity and the synthesis of immunoisolated cytochrome P-450scc and adrenodoxin proteins was investigated in primary cultures of swine ovarian (granulosa) cells. Administration of a novel adenylate cyclase toxin isolated from Bordetella pertussis increased granulosa-cell cAMP accumulation up to 200-fold over basal. These effects were additive with those of FSH, forskolin, and cholera toxin. In contrast, bacterial extracts BP 347 and BP 348 from mutant strains of B. pertussis that lack either all virulent factors or the adenylate cyclase toxin and hemolysin were devoid of effect. Granulosa-cell cAMP accumulation supported by active bacterial adenylate cyclase was accompanied by 2- to 11-fold, time-dependent increases in [35S]methionine incorporation into immunospecific cytochrome P-450scc and adrenodoxin. These increases in the synthesis of cholesterol side-chain cleavage proteins were associated with enhanced pregnenolone production in response to exogenous sterol substrate, 25-hydroxycholesterol, and augmented progesterone secretion both in the absence and presence of exogenous lipoprotein. Moreover, the effects of Bordetella adenylate cyclase toxin on granulosa cell steroidogenesis were functionally integrated with other regulatory responses, since the non-cAMP dependent effector, estradiol 17-beta, interacted synergistically with bacterial adenylate cyclase in stimulating progesterone production. We conclude that exogenous adenylate cyclase isolated from B. pertussis can be functionally integrated into the cAMP-dependent effector pathway of granulosa cells with a resulting increase in intracellular cAMP concentrations, augmented biosynthesis of progesterone and pregnenolone, enhanced synthesis of immunospecific cytochrome P-450scc and adrenodoxin, and synergistic interactions with a non-cAMP-dependent ovarian effector hormone (estradiol).(ABSTRACT TRUNCATED AT 250 WORDS)     

Oncogene-transformed granulosa cells as a model system for the study of steroidogenic processes

Highly steroidogenic granulosa cell lines were established by transfection of primary granulosa cells from preovulatory follicles with SV40 DNA and Ha-ras oncogene. Progesterone production in these cells was enhanced to levels comparable to normal steroidogenic cells, by prolonged (> 12 h) stimulation with 8-Br-cAMP, forskolin and cholera toxin, which elevate intracellular cAMP. The steroidogenic capacity of individual lines correlated with the expression of the ras oncogene product (p21) and the morphology of the cells. Formation of the steroid hormones was associated with de novo synthesis of the mitochondrial cytochrome P450scc system proteins. Since cholesterol import into mitochondria is essential for steroidogenesis, the expression of the peripheral benzodiazepine receptor (PBR) and the sterol carrier protein 2 was characterized in these cells. The induction of the expression of the genes coding for both proteins appeared to be mediated, at least in part, by cAMP. Stimulation of the PBR by specific agonists enhanced progesterone production in these cells. The phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA) dramatically suppressed the cAMP-induced steroidogenesis, in spite of enhanced intracellular cAMP levels, suggesting that TPA can modify the effects of cAMP. cAMP stimulation suppressed growth of transformed cells concomitantly with induction of steroidogenesis. The transformed cells lacked receptors for the native stimulants, the gonadotropic hormones. After transfection of the cells with a lutropin (LH) receptor expression plasmid, the LH and hCG response was reconstituted. In these newly established cell lines gonadotropins were able to stimulate the formation of cAMP and progesterone in a dose-dependent manner with an ED₅₀ characteristic of the native receptor. High doses caused desensitization to gonadotropins as observed in normal cells. These newly established oncogene-transformed granulosa cell lines can serve as a useful model to study inducible steroidogenesis and the effect of oncogene expression on this process.     

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.