Downregulation of progesterone biosynthesis in rat granulosa ... - Nature

International Journal of Impotence Research (2006) 18, 264–274 & 2006 Nature Publishing Group All rights reserved 0955-9930/06 $30.00

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ORIGINAL ARTICLE

Downregulation of progesterone biosynthesis in rat granulosa cells by adlay (Coix lachryma-jobi L. var. ma-yuen Stapf.) bran extracts S-M Hsia1, W Chiang1, Y-H Kuo2 and PS Wang3 1

Graduate Institute of Food Science and Technology, National Taiwan University, Taipei, Taiwan, Republic of China; Department of Chemistry, National Taiwan University, Taipei, Taiwan, Republic of China and 3Department of Physiology, School of Medicine, National Yang-Ming University, Taipei, Taiwan, Republic of China 2

Adlay (Coix lachryma-jobi L. var. ma-yuen Stapf.) has long been used as a traditional Chinese medicine for dysfunctions of the endocrine system and inflammation conditions. However, the effect of adlay seed on the endocrine system has not yet been reported. In the present study, the effects and the mechanisms of methanolic extract of adlay bran (ABM) on progesterone synthesis in rat granulosa cell were studied. ABM was further partitioned with different solvents including water, 1-butanol, ethyl acetate and n-hexane. Four subfractions named ABM-Wa (water fraction), ABM-Bu (1-butanol fraction), ABM-EA (ethyl acetate fraction) and ABM-Hex (n-hexane fraction) were obtained. ABM-Bu was further fractionated using Diaion HP-20 resin column chromatography with gradient elution. Granulosa cells were prepared from pregnant mare serum gonadotropinprimed immature female rats and challenged with different reagents including human chorionic gonadotropin (hCG 0.5 IU/ml), forskolin (10 lM), 8-bromo-adenosine-30 ,50 -cyclic monophosphate (8-Br-cAMP, 1 mM), A23187 (10 lM), phorbol 12-myristate 13-acetate (PMA, 0.01 lM), 25-OHcholesterol (0.1–10 lM) and pregnenolone (0.1–10 lM) in the presence or absence of ABM-Bu (100 lg/ ml). The functions of steroidogenic enzyme including protein expression of the steroidogenic acute regulatory protein (StAR) and cytochrome P450 side-chain cleavage enzyme (P450scc) protein were investigated. Expressions of both P450scc and StAR mRNA have also been explored. We found that ABM decreased progesterone production via an inhibition on (1) the cAMP-PKA and PKC signal transduction pathway, (2) P450scc and 3b-hydroxysteroid dehydrogenase (3b-HSD) enzyme activity, (3) P450scc and StAR protein and mRNA expressions and (4) the phosphorylation of ERK1/2 in rat granulosa cells. International Journal of Impotence Research (2006) 18, 264–274. doi:10.1038/sj.ijir.3901405; published online 27 October 2005 Keywords: adlay; progesterone; StAR; P450scc; ERK1/2; 3b-HSD

Introduction Coix lachryma-jobi L. var. ma-yuen Stapf, commonly called adlay (Job’s tears), is an annual crop. In traditional Chinese medicine, adlay has long been consumed as both a herbal medicine and a food supplement.1 Recent studies have shown some pharmacological effects of adlay extracts. For example, adlay extracts increase the activity of cytotoxic T lymphocytes and natural killer cells in experi-

Correspondence: Dr PS Wang, Department and Graduate Institute of Physiology, School of Life Science, National Yang-Ming University, Shih-Pai, Taipei 11211, Taiwan, Republic of China. E-mail: [email protected] Received 18 July 2005; revised 23 August 2005; accepted 30 August 2005; published online 27 October 2005

mental animals.2 Adlay extracts inhibit growth of Ehrlich ascites sarcoma and their active components have been identified as coixenolides.3 The adlay extracts have been studied to be antiproliferative and chemopreventive on lung or colon cancer in vivo and in vitro.4–6 Methanolic extracts from adlay seeds have been reported to have a moderate antioxidant effect.7,8 Adlay also has a modulating ability to shift the balance from Th2 to Th1 dominance in the T-cell-mediated immune response and may be beneficial for the treatment of allergic disorders.9 In addition, adlay has long been used in the folk medicine of Chinese as a nourishing food, to regulate the female endocrine system.1 Although adlay has many biological functions, the relationship between its action and endocrine function is unclear. Only one study indicated that adlay seed had an ovulatory-active substance.10 The intracellular mechanism by which adlay mediates

Downregulation of progesterone biosynthesis by ABM S-M Hsia et al

steroidogenesis has not been established either. Nevertheless, some medical reports have also suggested that adlay seeds should not be used during pregnancy for some unknown reasons.1,11 Therefore, the effects of adlay seeds on hormones and the female reproductive system remain unclear and deserve further examination. It has been shown that the luteinizing hormone (LH)-increased steroidogenesis of progesterone in granulosa cell is correlated with increased generation of cAMP.12 Recently, the cytochrome P450 side-chain cleavage enzyme (P450scc) and the steroidogenic acute regulatory protein (StAR) have been reported to be important in steroidogenesis.12–14 The StAR protein facilitates cholesterol transfer from the outer to the inner mitochondrial membrane. P450scc catalyses the transformation of cholesterol into pregnenolone in the mitochondria of ovarian cells. The interconversion of pregnenolone to progesterone is catalysed by the microsomal enzyme 3bhydroxysteroid dehydrogenase (3b-HSD).15–17 In the present study, the direct effect of adlay bran methanol extract (ABM) on the production of progesterone in rat granulosa cells was examined. In the present study, we examine whether ABM exerts direct action on PKA or PKC pathways, or on the function and expression of P450scc, StAR or 3bHSD in rat granulosa cells. Cells were treated with human chorionic gonadotropin (hCG), forskolin (adenylate cyclase activator), 8-bromo-cAMP, (8-BrcAMP, cAMP analogue), A23187 (Ca2 þ ionophore) or phorbol 12-myristate 13-acetate (PMA, PKC activator) with or without ABM to examine whether ABM inhibited the PKA or PKC signal transduction pathway. Different doses of 25-OH-cholesterol (substrate for P450scc) and pregnenolone (substrate for 3b-HSD) were added to cells with or without ABM to determine whether ABM affected enzyme activity. The effects of ABM were investigated on the protein or mRNA expression of StAR and P450scc. Also, the phosphorylation of extracellular signal-regulated protein kinase (ERK) 1/2 was investigated in rat granulosa cells.

Materials and methods Reagents Pregnant mare serum gonadotropin (PMSG), Dulbecco’s modified Eagle’s medium (DMEM)/F12, fatty acid-free bovine serum albumin (BSA), penicillin-G, sodium bicarbonate, streptomycin sulphate, hCG, insulin, medium-199 (M199), L-glutamine, N-2-hydroxyethylpiperazine-N0 -2-ethanesulphonic acid (HEPES), A23187, PMA, 3-isobutyl-methyl-xanthine (IBMX), forskolin, 8-bromo-cAMP, 25-OH-cholesterol, pregnenolone, phenylmethylsulphonyl fluoride (PMSF) and b-actin antibodies were purchased from Sigma Chemical Co. (St Louis, MO, USA). Lauryl sulphate

(SDS), bromophenol blue and dithiothretiol were purchased from Research Organics Inc. (Cleveland, OH, USA). Proteinase inhibitor cocktail tablets were purchased from Boehringer Mannheim (Mannheim, Gemany). Trilostane (4, 5-epoxy-17-hydroxy-3-oxoandrostane-2-arbonitile, an inhibitor of 3b-HSD) was provided by Sanofi-Synthelabo, Inc. (Malvern, PA, USA). Anti-P450scc antibody was kindly provided by Dr Bon-Chu Chung (Academia Sinica, Taipei, Taiwan, ROC),18 and anti-StAR antibody by Dr DM Stocco (Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX, USA).19 [3H]-progesterone and [3H]-pregnenolone were obtained from Amersham International plc. (Buck, UK). [a-32P]deoxy-ATP was obtained from NEN Life Science Product (Boston, MA, USA). Antibodies for phospho-ERK1/2 and nonphospho-ERK1/2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The peroxidase-conjugated IgG fraction to mouse IgG and peroxidase-conjugated IgG fraction to rabbit IgG were purchased from ICN Pharmaceuticals, Inc. (Aurora, OH, USA). The cAMP enzyme immunoassay (EIA) system was obtained from Assay Designs, Inc. (Ann Arbor, MI, USA).

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Plant material and sample preparation Adlay was purchased from local a farmer who plated Taichung Shuenyu no. 4 (TCS4) of Coix lachryma-jobi L. var. ma-yuen Stapf in Taichung, Taiwan, in March 2002 and harvested it in July of the same year. After harvest, the air-dried adlay seeds were separated into four different parts including adlay hull, adlay testa, adlay bran and polished adlay. The preparation of adlay extracts was from the previously described method, with minor modifications.7 Adlay brans were blended in powder form and screened through a 20-mesh sieve (aperture 0.94 mm). The powder of adlay bran (100 g) was extracted with 1 l of methanol stirred on a stirring plate at room temperature for 24 h. Contents were filtered through #1 filter paper (Whatman Inc., Hillsboro, OR, USA). The filtrate was concentrated to dryness in vacuum condition to obtain the methanolic extract form and stored at 201C until use. The methanolic extracts from adlay bran were named as ABM. ABM was further partitioned with different solvents including water, 1-butanol, ethyl acetate and n-hexane. Four subfractions named ABM-Wa (water fraction), ABM-Bu (1-butanol fraction), ABM-EA (ethyl acetate fraction) and ABM-Hex (n-hexane fraction) were obtained. The solvent (water, butanol, ethyl acetate, n-hexane) was concentrated to dryness in vacuum condition to give dried powder and stored at 201C until use. ABM-Bu was further fractionated using Diaion HP-20 resin column chromatography with gradient International Journal of Impotence Research

Downregulation of progesterone biosynthesis by ABM S-M Hsia et al 266 Adlay Bran Methanol Extract (ABM) Hexane Fr. (ABM-Hex)

H2O Fr. partitioned (Ethyl acetate/H2O) H2O Fr. partitioned (Butanol/H2O)

EA Fr. (ABM-EA) Butanol Fr. (ABM-Bu)

H2O Fr. (ABM-Wa)

column chromatography A B C D E F G H I J K L M N O P Q R S T U ∗ ∗ ∗ ∗ ∗ :Inhibits progesterone secretion in rat granulosa cells

Figure 1 Scheme for extraction, partitioning and fractionation of anti-progesterone fractions from adlay bran. Fraction (Fr.) G to J showed the most potent activity on inhibition of progesterone secretion.

elution as previously described7 and had 21 fractions assigned as A to U (Figure 1). For in vitro study, powders of adlay extracts were dissolved in DMSO to prepare stocks. The final concentration of DMSO was less than 0.1%.

Preparation of granulosa cells for cell culture Immature female Sprague–Dawley rats were housed in a temperature-controlled room at 22–241C under a constant 12-h light/dark cycle and were given food and water ad libitum. The preparation of granulosa cells was modified from the method described elsewhere.20,21 The immature female rats (22–25 days of age) were injected subcutaneously with PMSG (15 IU/rat). After 48 h, rats were killed by cervical dislocation. Ovaries were excised and transferred into the sterile DMEM/F12 (1:1) medium, containing 0.1% BSA, 20 mM HEPES, 100 IU/ ml penicillin-G and 50 mg/ml streptomycin sulphate. After trimming free fat and connective tissues, the large and medium-sized follicles were punctured with a 26-gauge needle to release granulosa cells. The harvested cells were pelleted and resuspended in growth medium (DMEM/F12 containing 10% fetal calf serum, 2 mg/ml insulin, 100 IU/ml penicillin-G and 100 mg/ml streptomycin sulphate). Cell viability was 490% as determined using a haemocytometer and the Trypan blue method. Granulosa cells were placed in 24-well plates at approximately l 105 cells per well and incubated at 371C with 5% CO2 95% air for 48 h.

Cell culture and treatment The granulosa cells (1 105/well) were placed in 24-well plates and grown for 48 h, washed with 1 ml International Journal of Impotence Research

ice-cold saline and subjected to various treatment regimens. Cells were treated with 0.5 ml aliquots of serum-free BSA-M199 medium (M199 without phenol red, 0.3% BSA, 25 mM HEPES, 4 mM L-glutamine) containing adlay extracts with or without different reagents including hCG (0.5 IU/ml), forskolin (10 mM), 8-Br-cAMP (1 mM), A23187 (10 mM), PMA (0.01 mM), 25-OH-cholesterol (0.1–10 mM) or pregnenolone (0.1–10 mM) at 371C for 2 h. The media were collected and stored at 201C until further analysis for progesterone or pregnenolone by radioimmunoassay (RIA). For studying the accumulation of cAMP in response to adlay extracts, the rat granulosa cells were incubated with IBMX (phosphodiesterase inhibitor, 1 mM) and adlay extracts in the presence or absence of hCG (0.5 IU/ml) for 1 h. At the end of incubation, the cells were homogenized in 0.5 ml of 65% ice-cold ethanol by polytron (PT-3000, Kinematica AG, Luzern, Switzerland), and then centrifuged at 2000 g for 10 min. The supernatants were lysophilized in a vacuum concentrator (Speed Vac, Savant, Holbrook, NY, USA), then reconstituted with assay buffer (0.05 M sodium acetate buffer with 0.01% azide, pH 6.2) before measuring the concentration of cAMP by EIA.

Assay of progesterone, pregnenolone and cAMP The concentrations of progesterone and pregnenolone in the medium were determined by RIA as described elsewhere.22,23 With anti-progesterone serum No. W5, the sensitivity of progesterone RIA was 5 pg per assay tube. Intra- and interassay coefficients of variation (CV) were 4.8% (n ¼ 5) and 9.5% (n ¼ 4), respectively. Anti-pregnenolone antiserum was diluted with 0.1% gelatin-PBS. The sensitivity of the pregnenolone RIA was 16 pg per assay tube. The intra- and interassay coefficients of variation were 2.3% (n ¼ 6) and 3.7% (n ¼ 4), respectively. The intracellular levels of cAMP were measured in rat granulosa cells using EIA kit.

Gel electrophoresis and Western blotting The Western blotting method has been reported previously.24–26 The granulosa cells (2 106 cells) were incubated with medium containing ABM-Bu (0, 100 mg/ml), ABM-Bu-G (0, 100 mg/ml) or hCG (0.5 IU/ml) for 2 h. At the end of incubation, cells were washed twice with ice-cold saline and detached by trypsinization (1.25 mg/ml). The cells were collected and extracted in homogenization buffer (pH 8.0) containing 1.5% Na-lauroylsarcosine, 1 103 M EDTA, 2.5 103 M Tris-base, 0.68% PMSF and 2% proteinase inhibitor cocktail, and then disrupted by ultrasonic sonicator in an icebath. Cell extracts were centrifuged at 13 500 g for 10 min.25 The supernatant fluid was collected and


the protein concentration was determined by the colorimetric method of protein assay according to the Bradford method.27 Extracted proteins were denatured by boiling for 5 min in SDS buffer (0.125 M Tris-base, 4% SDS, 0.001% bromophenol blue, 12% sucrose, and 0.15 M dithiothreitol).28 The proteins (20 mg) in the samples were separated on 12% SDS-polyacrylamide gel electrophoresis (SDSPAGE) at 75 V for 15 min and then at 150 V for 40 min using a running buffer. The proteins were electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (NEN Life Science Products, Inc., Boston, MA, USA) using a Trans-Blot SD semidry transfer cell (170–3940, Bio-Rad, Hercules, CA, USA) at 64 mA (for 8 10 mm membrane) for 45 min in a blotting solution. The membranes were washed in TBS-T buffer (0.8% NaCl, 0.02 M Tris-base, and 0.3% Tween-20, pH 7.6) for 5 min and then blocked by incubation for 120 min in blocking buffer (TBS-T buffer containing 5% nonfat dry milk). Then the membranes were incubated with a mixture of anti-P450scc antibodies (1:2000), anti-StAR protein antibodies (1:1000) and b-actin antibodies (1:2000) in 5% nonfat dry milk of TBS-T buffer overnight at 41C. The other membranes were incubated with anti-phospho-ERK1/2 or antinonphospho-ERK1/2 antibodies (1:2000) in 5% nonfat dry milk of TBS-T buffer overnight at 41C, and after one wash for 15 min and three washes for 5 min each with TBS-T buffer, the membranes were incubated for 1 h with horseradish peroxidaseconjugated goat anti-rabbit IgG (1:6000 dilution) and horseradish peroxidase-conjugated goat antimouse IgG (1:8000 dilution) in 5% nonfat dry milk of TBS-T buffer. The membranes were washed four times with TBS-T buffer, and then the bands for P450scc, StAR, b-actin, phospho-ERK1/2 and nonphospho-ERK1/2 were visualized by chemiluminescence (ECL, Western blotting detection reagents, Amersham International plc. Buck, UK).

Reverse transcription-polymerase chain reaction (RT-PCR) analysis The RT-PCR method has been reported previously.29 The granulosa cells (2 106 cells) were incubated with ABM-Bu (0, 100 mg/ml), ABM-Bu-G (0, 100 mg/ ml) CG (0.51 m/ml) for 30 min. At the end of incubation, the cells were washed twice and total RNA was isolated by RNAlaregt (Blossom, Taipei, Taiwan, ROC) extraction kit. The procedures were conducted according to the manufacturer’s instructions. RNA samples were dissolved in water containing 0.1% diethylpyrocarbonate (DEPC), and quantified by measuring the absorbance at 260 nm. Aliquots containing 100 ng RNA were assayed by the relative-quantitative RT-PCR procedure, which was modified from the method described by RonenFuhrmann et al.30 RT was conducted for 120 min at

371C using 250 ng pd(T) primer and 50 U of M-MuLV reverse transcriptase (BioLabs, Beverly, MA, USA). Minus control of RT was processed to confirm that mRNA samples were not contaminated by cellular DNA. PCR was performed in the presence of 2 mCi of [a-32P]-deoxy-ATP (3000 Ci/mmol), dNTPs (mixture of dATP, dTTP, dGTP and dCTP, 200 mM) and 500 ng appropriate oligonucleotide primers. Oligonucleotide primers for the ribosomal protein L19 served as an internal control. The number of cycles was examined to verify that the amplification was in exponential phase. Following PCR reaction (23 cycles), tracking dye was added to 10–40 ml of PCR reaction mixture (100 ml) for analysis by 5% PAGE.31 The gels were dried and exposed to X-ray film. The PCR oligonucleotide primer pairs were designed based on known cDNA sequences of various target genes. The expected PCR products would be 246 bp for rat StAR cDNA30; 536 bp for rat P450scc and 194 bp for rat RPL19.31 Forward (A, sense) and reverse (B, antisense) primers were:

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Rat P450scc A, 50 -AGAAGCTGGGCAACATGGAGT CAG-30 Rat P450scc B, 50 -TCACATCCCAGGCAGCTGCATG GT-30 Rat StAR A, 50 -GCAGCAGGCAACCTGGTG-30 Rat StAR B, 50 -TGATTGTCTTCGGCAGCC-30 RPL 19 A, 50 -CTGAAGGTCAAAGGGAATGTG-30 RPL 19 B, 50 -GGACAGAGTCTTGATGATCTC-30

Statistical analysis All data were expressed as mean7s.e.m. The treatment means were tested for homogeneity using the analysis of variance (ANOVA), and the differences between specific means were tested for significance by Duncan’s multiple-range test.32 The levels of significance were exhibited as significant (Po0.05) and highly significant (Po0.01), respectively.

Result The effects of ABM subfractions on progesterone production in rat granulosa cells To determine whether ABM subfractions affect progesterone biosynthesis in granulosa cells, different subfractions of ABM (100 mg/ml) were incubated with or without hCG (0.5 IU/ml) for 2 h (Figure 2a). ABM-EA and ABM-Bu treated with or without hCG significantly reduced progesterone production in rat granulosa cells (Po0.05). However, administration of ABM-Bu (0.1–100 mg/ml) with or without hCG at 100 mg/ml significantly inhibited progesterone release in rat granulosa cells (Po0.01) (Figure 2b). The International Journal of Impotence Research

Downregulation of progesterone biosynthesis by ABM S-M Hsia et al 268 5

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Basal (n=6) hCG (0.5 IU/ml, n=6)

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ABM-Bu (µg/ml) Figure 2 Effects of ABM subfractions (ABM-Wa, ABM-Hex, ABM-EA, ABM-Bu) (a) and ABM-Bu (b) on progesterone release in rat granulosa cells incubated for 2 h. Basal (open bar), hCGstimulated (hatched bar). *Po0.05 and **Po0.01 compared with control.

suppressive effect indicates that ABM-Bu may act directly to influence progesterone production in rat granulosa cells.

Characterization of ABM-Bu extracts and their effects on progesterone production in rat granulosa cells Fractionation of ABM-Bu was performed using Diaion HP-20 resin column chromatography with gradient elution and had 21 fractions assigned as A to U. Figure 3 shows the different subfractions of ABM-Bu on progesterone release in rat granulosa cells. ABM-Bu subfractions (G, H, I, J) with or without hCG all inhibited progesterone release in rat granulosa cells (Po0.05). These results indicated the presence of more than one anti-progesterone chemical in the ABM-Bu. Scale-up isolation, purification and structural characterization of these anti-progesterone chemicals are currently underway in our laboratory. International Journal of Impotence Research

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Figure 3 Effects of ABM-Bu subfractions on progesterone release in rat granulosa cells incubated for 2 h. Basal (open bar), hCG-stimulated (hatched bar). *Po0.05 compared with corresponding control.

Effects of ABM on forskolin-, 8-Br-cAMP-, A23187-, PMA- or hCG-evoked progesterone and intracellular cAMP production in rat granulosa cells To determine whether ABM-Bu affects the cAMPPKA or PKC signal transduction pathway in rat granulosa cells, different concentrations of 8-BrcAMP (103 M), forskolin (105 M), A23187 (105 M) or PMA (108 M) were incubated with or without ABM-Bu (10, 100 mg/ml). 8-Br-cAMP, forskolin, A23187 and PMA all stimulated progesterone release to a significant level (Po0.05). ABM-Bu (100 mg/ml) inhibited not only basal but also 8-BrcAMP- and forskolin-stimulated (Po0.05) progesterone release in rat granulosa cells (Figure 4a). ABM-Bu (100 mg/ml) inhibited not only basal but also A23187- and PMA-stimulated (Po0.05 or Po0.01) progesterone release in rat granulosa cells (Figure 4b). When studying the accumulation of cAMP in response to ABM-Bu in rat granulosa cells, incubation of rat granulosa cells with IBMX (1 mM) in the presence or absence of hCG (0.5 IU/ml) for 1 h increased cellular cAMP production. The ABM-Bu (200 mg/ml) inhibited not only basal but also hCGstimulated (Po0.05 or Po0.01) cellular cAMP production by rat granulosa cells (Figure 5).

Downregulation of progesterone biosynthesis by ABM S-M Hsia et al 269 Basal (n=6) Forskolin (10-5, n=6)

16000

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14000 12000 10000 8000 6000

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Figure 5 Effects of ABM-Bu on the basal and hCG-stimulated cellular cAMP production by rat granulosa cells. Cells were incubated with hCG and with or without ABM-Bu for 1 h. Basal (open bar), hCG-stimulated (hatched bar). *Po0.05 and **Po0.01 compared with ABM-Bu at 0 mg/ml.

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Effects of ABM-Bu on steroidogenic enzyme activities (cytochrome P450scc and 3b-HSD) To determine the effect of ABM-Bu on the activities of P450scc and 3b-HSD enzymes in rat granulosa cells, 25-OH-cholesterol (a substrate of P450scc, 107–105 M) and pregnenolone (a substrate of 3bHSD, 107–105 M) were added separately with or without ABM-Bu and then incubated for 2 h. ABMBu decreased not only the basal release of progesterone but also the progesterone response to 25-OHcholesterol or to the pregnenolone (Figure 6). This result indicates that ABM-Bu might have a direct inhibitory effect on P450scc and/or 3b-HSD activity.

Basal (n=6) ABM-Bu (100 µg/ml, n=6)

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Figure 4 Effects of ABM-Bu on the basal, forskolin-, 8-Br-cAMP-, A23187- or PMA-stimulated progesterone release by rat granulosa cells. Cells were incubated with maximal concentrations of forskolin, 8-Br-cAMP (a), A23187 or PMA (b) in the presence or absence of ABM-Bu (100 mg/ml) for 2 h. The levels of progesterone in response to forskolin, 8-Br-cAMP, A23187 and PMA all are significantly (Po0.01) higher than the corresponding basal group. (a) Basal (open bar), 8-Br-cAMP (hatched bar), forskolin (closed bar). (b) Basal (open bar), A23187 (hatched bar), PMA (closed bar). *Po0.05 and **Po0.01 compared with ABM-Bu at 0 mg/ml.

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Enzyme kinetic analysis of cytochrome P450scc and 3b-HSD in rat granulosa cells To further confirm whether ABM-Bu affects P450scc and 3b-HSD activities in granulosa cells, different doses of 25-OH-cholesterol or pregnenolone were

Figure 6 Effects of ABM-Bu on the cytochrome P450scc enzyme and 3b-HSD activities in rat granulosa cells after incubation with different doses of 25-OH-cholesterol (top) or pregnenolone (bottom) for 2 h. Basal (open bar), ABM-Bu-treated (hatched bar). *Po0.05 and **Po0.01 compared with basal group. þþ Po0.01 compared with (precursor ¼ 0 M) group.

International Journal of Impotence Research


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1/[25-OH-Cholesterol] (nM)-1 Figure 7 Kinetic analysis of ABM-Bu inhibition of P450scc function. Double reciprocal plots of data were obtained from cultured granulosa cells challenged with 25-OH-cholesterol (105–108 M).


0.3 0.2 Control (n=6) R=0.87 Vmax=21.28 Km=8.14

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Figure 8 Kinetic analysis of ABM-Bu inhibition on 3b-HSD function. Double reciprocal plots of data were obtained from cultured granulosa cells challenged with pregnenolone (104– 107 M).

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Effects of ABM-Bu and ABM-Bu-G on mRNA expression of P450scc and StAR protein The mRNA expressions of P450scc and StAR protein in rat granulosa cells administrated with ABM-Bu (100 µg/ml, n=6) R=0.90 Vmax=0.84 Km=31.25

ABM-Bu (100 µg/ml, n=6) R=0.95 Vmax=11.12 Km=8.29

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Effect of ABM-Bu on the expression of cytochrome P450scc and StAR protein To determine the effect of ABM-Bu on expressions of P450scc and StAR protein in rat granulosa cells, Western blotting was used to determine which steroidogenic enzymes or proteins were altered at protein level. In Figure 9, b-actin signal (45 kDa) was used as an internal control. Bands at 54 kDa (P450scc) and 30 kDa (StAR) were detected in rat granulosa cells. The results showed that both P450scc and StAR levels were decreased by 2 h treatment with 100 mg/ml of ABM-Bu.

0.6

1/[Progesterone]

incubated with or without ABM-Bu for 2 h. In Figure 7, ABM-Bu (100 mg/ml) decreased 25-OHcholesterol-evoked pregnenolone production by rat granulosa cells. The maximum velocity (Vmax) was 1.23 ng/105 cells/2 h in the control group. In the ABM-Bu group, it was 0.84 ng/105 cells/2 h. The Michaelis constant (Km) was 19.73 nM in the control group and 31.25 in the ABM-Bu group. In Figure 8, ABM-Bu (100 mg/ml) decreased pregnenoloneevoked progesterone production by rat granulosa cells. The Vmax was 21.28 ng/105 cells/2 h in the control group. In the ABM-Bu group, it was 11.12 ng/105 cells/2 h. The Km was 8.14 nM in the control group and 8.29 in the ABM-Bu group. This result indicates that ABM-Bu might have a direct inhibitory effect on P450scc and 3b-HSD enzyme.

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0.6 0.4 0.2 0.0 Control

ABM-Bu (100 mg/ml)

Figure 9 Effect of ABM-Bu on P450scc and StAR protein levels in rat granulosa cells. Cells were treated for 2 h with or without ABM-Bu, and then protein levels of P450scc and StAR were measured by Western blotting. This experiment was repeated three times with similar results. Each point was normalized to the control. *Po0.05 compared with control group.


ABM-Bu and ABM-Bu-G were investigated. L19 was used as an internal control and was not affected by ABM-Bu and ABM-Bu-G. Administration of ABMBu or ABM-Bu-G inhibited basal or forskolinstimulated mRNA expression of P450scc and StAR protein in rat granulosa cells (Figure 10).

Effects of ABM-Bu-G on the phosphorylation of ERK1/2 The phosphorylation of ERK1/2 in rat granulosa cells administered with ABM-Bu-G was investigated. The nonphospho-ERK1/2 was not affected by ABM-Bu-G. Administration of ABM-Bu-G inhibited basal or hCG-stimulated phosphorylation of ERK1/2 in rat granulosa cells (Figure 11).

Discussion The present results demonstrate that ABM and its subfraction (ABM-Bu-G) inhibit the spontaneous and hCG-stimulated secretion of progesterone and decrease the activity of P450scc and 3b-HSD by

acting directly on rat granulosa cells. Administration of ABM-Bu reduced not only the protein expressions of P450scc and StAR but also the mRNA expressions of P450scc and StAR. ABM and its subfraction (ABM-Bu-G) also inhibit ERK1/2 phosphorylated in rat granulosa cells. To our knowledge, this is the first report demonstrating an inhibitory effect of adlay on steroidogenesis and progesterone secretion in vitro, explaining in part the modulatory effect of adlay extracts on female reproductive function. It has been well established that hCG increases cyclic AMP generation and then stimulates progesterone secretion in granulosa cells.19,25,26,33 Some studies have shown that LH or hCG acts via its G protein-coupled receptor to increase intracellular cAMP further to activate the PKA pathway.33,34 In the present study, we showed that ABM-Bu decreased not only 8-Br-cAMP- or forskolin-induced progesterone release but also hCG-induced cellular cAMP production. These results suggested that one of the actions of ABM-Bu went beyond the membrane receptor to

p-ERK1

1

2

3

4

5

6

7

8

44 kDa 42 kDa

p-ERK2 536 bp

P450scc

44 kDa 42 kDa

ERK1

0.0 (n=3)

**

**

0.8

0.2

* ++

0.4

* ++

0.6

**

StAR/L19

1.0

7

8

0.0

1

2

3

4

5

6

+

+

ABM-Bu-G (100 µg/ml)

−

+

−

+

Figure 10 Effect of ABM-Bu and subfraction (ABM-Bu-G) on StAR and P450scc mRNA levels in rat granulosa cells. Cells were treated for 30 min with ABM-Bu (100 mg/ml) or ABM-Bu-G (100 mg/ml) in the presence or absence of forskolin. Lanes 1 and 2, control; lane 3, ABM-Bu (100 mg/ml); lane 4, ABM-Bu-G 100 mg/ ml; lanes 5 and 6, forskolin (105 M); lane 7, ABM-Bu (100 mg/ ml) þ forskolin (105 M); lane 8, ABM-Bu-G (100 mg/ml) þ forskolin (105 M), and then mRNA levels of P450scc and StAR were measured by RT-PCR. This experiment was repeated three times with similar results. *Po0.05 **Po0.01 compared with control group. þ þ Po0.01 compared with forskolin-treated group.

3.0

Phospho-ERK/total ERK

0.2

* ++

* ++

0.6 0.4

−

(n=3)

2.5 2.0 1.5 1.0 0.5

** ++

**

0.8

−

(n=3)

**

1.0

**

P450scc/L19

1.2

hCG (0.5IU/ml)

*

194 bp

L19

ERK2

**

246 bp

StAR

RT-PCR signal ratio

271

0.0

hCG (0.5IU/ml)

−

−

+

+

ABM-Bu-G (100 µg/ml)

−

+

−

+

Figure 11 Effect of ABM-Bu-G on phospho-ERK1/2 and nonphospho-ERK1/2 protein levels in rat granulosa cells. Cells were treated for 2 h with ABM-Bu-G (100 mg/ml) in the presence or absence of forskolin, and then protein levels of phospho-ERK1/2 and nonphospho-ERK1/2 were measured by Western blotting. This experiment was repeated three times with similar results. *Po0.05 and **Po0.01 compared with control group. þþ Po0.01 compared with hCG-treated group.


Downregulation of progesterone biosynthesis by ABM S-M Hsia et al 272

inhibit the formation of cAMP in rat granulosa cells. The decrease of progesterone by adlay bran extracts was not attributed to the cytotoxicity. The administration of 100 mg/ml ABM-Bu or subfractions did not cause release of lactate dehydrogenase (LDH) from granulosa cells (Table 1). In addition, calcium ions and the PKC signal transduction pathway may also play some roles in steroidogenesis in granulosa cells.35–39 In the present study, both A23187 and PMA reversed the inhibition of progesterone production caused by ABM-Bu, indicating that the PKC signal transduction pathway might be involved in progesterone biosynthesis in rat granulosa cells. In rat granulosa cells, progesterone biosynthesis is via the conversion of pregnenolone to progesterone under the catalysation of microsomal enzyme 3bHSD after transformation of cholesterol to pregnenolone by P450scc (the rate-limiting enzyme).40 It has been demonstrated that cholesterol or pregnenolone at 108–106 M stimulates progesterone release by granulosa cells.26 In the present study, we have confirmed that either 25-OH-cholesterol or pregnenolone stimulated progesterone secretion in rat granulosa cells. However, administration of ABM-Bu inhibited progesterone production caused by 25-OH-cholesterol or pregnenolone. These data suggested that the function of P450scc and/or 3b-HSD might be affected by ABM-Bu. To further examine if the function of either P450scc or 3b-HSD is altered by ABM-Bu, we administrated different doses of 25-OH-cholesterol with or without ABMBu. After inhibiting the function of 3b-HSD by trilostane, the pregnenolone accumulation in rat granulosa cells was measured and used as an index of the activity of P450scc. Our kinetic analysis showed that the action of ABM-Bu was consistent with a mixed inhibition mechanism. Apparently, ABM-Bu inhibits the function of P450scc in rat granulosa cells. To investigate if ABM-Bu affected 3b-HSD activity, we examined the progesterone accumulation in rat granulosa cells following challenge with serial doses of pregnenolone. The kinetic analysis showed that ABM-Bu generated a mixed Table 1 Cytotoxic effect of ABM-Bu and ABM-Bu-G on rat granulosa cells Dosage (mg/ml)

Vehicle 1 10 100 200 1000

ABM-Bu (n ¼ 6)

ABM-Bu-G (n ¼ 6)

Cytotoxicity (%)

P-value

Cytotoxicity (%)

P-value

1.00 0.8570.32 1.1470.94 1.0570.53 2.3171.23 10.5272.56

NS NS NS NS NS Po0.01

1.00 0.9570.45 1.0270.27 1.4770.75 2.5171.59 13.2172.23

NS NS NS NS NS Po0.01

NS ¼ nonsignificant difference indicating that P40.05 compared to ABM-Bu or ABM-Bu-G at 0 mg/ml.


inhibition of 3b-HSD. The mixed-type inhibition of the kinetic profile for ABM-Bu suggested the presence of one or more inhibitors in the extract to provide more than one inhibitory mechanism. In steroidogenesis, the rate-limiting step is transformation of cholesterol to pregnenolone in the inner mitochondrial membrane by P450scc. The StAR protein is thought to facilitate cholesterol transfer through the mitochondrial membrane.41,42 The level of StAR protein is important for steroid biosynthesis.43,44 In the present studies, the protein and mRNA expressions of both P450scc and StAR were examined by Western blotting and a semiquantitative RT-PCR assay. Our data about the protein expression of P450scc and StAR revealed that ABM-Bu inhibited the protein expressions of both P450scc and StAR protein. On the basis of our RT-PCR data, both ABM-Bu and ABM-Bu-G inhibited mRNA expression of P450scc and StAR with or without forskolin. As ABM-Bu decreases progesterone production, this result suggests that acute inhibitory effects of ABM-Bu on progesterone secretion might be via an inhibition of both StAR and P450scc protein and mRNA expressions. Recently, ERK1/2 have been recognized as the first intracellular signalling molecules to regulate FSHinduced progesterone synthesis in rat granulosa cells.45 Of the mitogen-activated protein kinase (MAPK) signaling molecules, ERK1/2 have been implicated in the modulation of steroidogenesis in granulosa cells45–47 and other steroidogenic cells.48 Our results indicated that ABM-Bu-G inhibited ERK1/2 phosphorylation in rat granulosa cells. These results imply that ERK1/2 signalling might be involved in the inhibition of progesterone secretion caused by adlay bran extracts in rat granulosa cells. Adlay bran extract is a complex mixture of natural substances. Phytochemistry studies revealed the presence of phytosterols, phenolic acids, phenolic aldehydes, lignan and flavonoids in adlay bran extract.3,7,10,49 We have isolated some isoflavone and flavonoid phytochemicals in adlay bran extracts (unpublished data). Flavonoid phytochemicals have been shown to possess an inhibitory effect on steroidogenic enzymes in human adrenocortical H295R cells.50 Thus, we suggest that the flavonoid phytochemicals in ABM-Bu might play an important role in inhibiting progesterone secretion in granulosa cells. The search for finer chemical compounds of adlay bran to alter steroidogenesis is worthy of further investigation. Progesterone is an ovarian hormone of pregnancy and is responsible for preparing the reproductive tract for zygote implantation and the subsequent maintenance of the pregnant state.40 Nevertheless, some medical reports suggested that adlay seeds could not be used during pregnancy for some unknown scientific reasons.1,11 In the present study, administration of adlay bran extracts significantly inhibited progesterone secretion in rat granulosa cells. Immunoblotting of rat granulosa cells treated

Downregulation of progesterone biosynthesis by ABM S-M Hsia et al S

S

S R

AC

cAMP PLC PKA DAG/IP3 /Ca 2+

Progesterone 3 -HSD 3β-HSD

MEK PKC ERK

SF1 promoter

transcription StAR genes

Pregnenolone

Mitochondrion

P450scc StAR Cholesterol

Cholesterol esters

Figure 12 Hypothetical scheme of ABM-Bu inhibition on progesterone production in rat granulosa cells. S, substances; R, receptor; AC, adenylate cyclase; PKA, protein kinase A; PKC, protein kinase C; IP3, inostiol trisphosphate; MEK, MAPK-ERK kinase; SF-1, steroidogenic factor-1. —, indicates that ABM-Bu could inhibit this step.

with adlay bran extracts has shown an inhibition of P450scc and StAR protein expressions. These results provided a scientific basis to clarify the effect of adlay seeds on progesterone secretion in rat granulosa cells and in part explained the reason why adlay seeds could not be employed during pregnancy. In conclusion, the present results demonstrated that ABM-Bu inhibited progesterone secretion via a cascade of inhibitions, including the reduction of cAMP production, the PKC pathway and the postcAMP pathway. The post-cAMP pathway involved diminishing of P450scc and 3b-HSD activities and also P450scc, StAR protein and mRNA expressions. ABM-Bu also inhibited ERK1/2 protein phosphorylated before the inhibition of steroidogenesis in rat granulosa cells (Figure 12).

Acknowledgments The technical assistance provided by Dr ShiowChwen Tsai is appreciated. This study was supported by three grants (No. DOH92-TD-1002, No. DOH93-TD-1014 and DOH93-TD-F-113-053-(2)) from the Department of Health, Executive Yuan, Taiwan, ROC. The anti-P450scc antibody was kindly provided by Dr BC Chung, Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, ROC. The anti-StAR antibody was kindly provided by Dr DM Stocco, Department of Cell Biology and Biochemistry, Texas Tech University Health Science Center, Lubbock, Texas, USA.

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