Molecular Human Reproduction, Vol. 5, No. 5, 396-401,
May 1999
© 1999 European Society of Human Reproduction and Embryology
Inhibitory effects of nitric oxide on the expression and activity of aromatase in human granulosa cells
1 Department of Obstetrics and Gynecology, Akita University School of Medicine, and 2 Akita University College of Allied Medical Science, 1–1–1 Hondo, Akita 010–8543, Japan
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Abstract |
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The aim of the present study was to explore the mechanisms by which nitric oxide (NO) may inhibit aromatase activity of human granulosa cells. Ovarian granulosa–luteal cells, obtained from patients undergoing in-vitro fertilization (IVF) were cultured in the presence of NO-related substances. After 24 h of culture, aromatase activity of the cells was significantly inhibited by treatment with the NO donors, SNAP or NOC12 at
ISOdia10–4 M in a dose-dependent manner. Treatment with NO catabolites or a peroxynitrite-releasing compound, SIN1, had no significant influence. Treatment with SNAP at 10–3 M decreased relative aromatase mRNA values by 72% (P < 0.05) and intracellular cyclic AMP concentrations by 53% (P < 0.01). However, treatment with H89, an inhibitor of protein kinase A, did not inhibit aromatase activity. Since there were no significant effects of NO catabolites or peroxinitrite, the inhibitory action of NO donors on aromatase must be related to NO release. The action of NO is, in part, attributable to the down-regulation of aromatase gene transcription. Although NO decreased intracellular cAMP values, down-regulation of aromatase gene transcription may not be mediated by protein kinase A-dependent mechanisms. aromatase/granulosa cells/mRNA/nitric oxide/ovary
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Introduction |
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Nitric oxide (NO), which is formed from L-arginine by a constitutive calcium-dependent or by a pro-inflammatory cytokine-inducible NO synthase in vivo, has been recognized as an important regulator in a number of physiological and pathological processes (Moncada et al., 1991
). In the ovary, both constitutive and inducible NO synthases are expressed (Van Voorhis, et al., 1995; Zackrisson et al., 1996), and hormonally or cytokine-regulated (Ben-Shlomo et al., 1994; Jablonka-Shariff and Olsen, 1997; Matsumi et al., 1998). Recently, NO has been implicated in a variety of ovarian activities, including ovulation (Shukovsky and Tsafriri, 1994; Bonello et al., 1996; Yamauchi et al., 1997), follicular atresia (Chun et al., 1995), luteolysis (Olson et al., 1996; Motta et al., 1997), and steroidogenesis (Van Voorhis et al., 1994).
In general, NO is thought to interact with the iron-containing enzymes, such as soluble guanylate cyclase (Ignarro et al., 1986), cyclo-oxygenase (Salvemini et al., 1993), and a number of the cytochrome P-450 enzymes (Wink et al., 1993; Stadler et al., 1994). Recently, several authors have reported an inhibitory action of NO on aromatase cytochrome P-450 (aromatase), the enzyme responsible for oestrogen biosynthesis. Addition of NO donors inhibited oestradiol secretion and aromatase activity, whereas inhibiting endogenous NO synthase enhanced oestradiol secretion from cultured ovarian granulosa cells of human (Van Voorhis et al., 1994), rat (Olson et al., 1996; Ahsan et al., 1997), and porcine (Masuda et al., 1997). In addition, in the in-vitro perfused rabbit ovaries, inhibition of endogenous NO synthase resulted in a significant elevation in the production of oestradiol stimulated by human chorionic gonadotrophin (HCG), and the concomitant administration of NO donors reversed this action (Yamauchi et al., 1997). These studies have suggested that NO may act as an important down-regulator of ovarian oestrogen synthesis during luteolysis (Olson et al., 1996) or ovulatory phase (Yamauchi et al., 1997).
Based on these reports, the aim of this study was to explore some of the mechanisms by which NO inhibits aromatase activity in human granulosa–luteal cells. Specifically, we sought to determine whether by-products of NO metabolism, including peroxinitrite, inhibited aromatase activity, and whether inhibitory actions of NO on aromatase involved down-regulation of aromatase gene transcription. In addition, we investigated whether NO modulates the intracellular cyclic AMP (cAMP) values of human granulosa–luteal cells.
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Materials and methods |
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Media, probes and chemicals
Medium used for granulosa–luteal cell culture, was purchased from Wako Chemicals (GIT medium; Osaka, Japan). A human aromatase cDNA corresponding to nucleotides 105–1934, isolated from human placenta (Harada, 1988
), was kindly provided by Dr Nobuhiro Harada through Tukuba Life Science Center at the Institute of Physical and Chemical Research (Tsukuba, Japan). A glyceraldehyde-3-phosphate dehydrogenase (G3PDH) cDNA probe was purchased from Toyobo (Osaka, Japan). Two types of NO donors, S-nitroso-L-acetyl penicillamine (SNAP), and ethanamine 1,1-diethyl-2-hydroxy-2-nitrosohydrazine (NOC12), and a peroxinitrite-releasing compound, 3-morpholinosyndnonimine (SIN1), were purchased from Dojindo Laboratories (Kumamoto, Japan). An inhibitor of protein kinase A, N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulphonamide (H89), was purchased from Seikagaku Co. (Tokyo, Japan). The chemicals used were mainly purchased from Sigma Chemical Co (St Louis, MO, USA).
Ovarian follicular cell isolation and cell culture methods
Ovarian granulosa–luteal cells were obtained from women who underwent follicle aspiration for in-vitro fertilization (IVF), after informed consent was obtained according to the guidelines of our Institutional Review Board. All ovarian stimulations for IVF were performed with a standard mid-luteal administration of a gonadotrophin-releasing hormone agonist (GnRHa) (buserelin acetate; Hoechst Japan, Tokyo, Japan) and follicle stimulating hormone (FSH, Fertinorm; Serono Japan, Tokyo, Japan) stimulation protocol. When the leading follicle reached a size of 17 mm in diameter, the administration of GnRHa and FSH was stopped, and 10 000 IU of human chorionic gonadotropin (HCG) was administered. After 34 h following HCG injection, oocytes were recovered by transvaginal echo-guided follicle aspiration.
Follicular fluid obtained from several IVF patients was collected together and used for each experiment. In order to isolate granulosa–luteal cells, follicular fluid obtained was subjected to centrifugation at 3000 g for 20 min, and the cell pellets were suspended in phosphate-buffered saline (PBS; Sigma) containing 0.1% hyaluronidase. After incubation at 37°C for 20 min, the cells were placed on a 50% (v/v) Percoll cushion (Pharmacia LKB Biotechnology, Piscataway, NJ, USA), and subjected to centrifugation at 3000 g for 30 min. The cell pellets were washed twice with PBS and resuspended in GIT medium containing 50 IU/ml penicillin G and 50 µg/ml streptomycin. Approximately 2x104 cells in 500 µl of GIT medium were placed in each well of a collagen-coated 24-well multi-well plate (Celltight C1 Plate 24F; Sumitomo Bakelite Co, Tokyo, Japan). The cells were incubated at 37°C in an atmosphere of 5% CO2 in air. After 24 h of culture, adherent cells were washed with GIT medium, and used for experiments.
Measurement of aromatase activity and oestradiol
Aromatase activity of cultured granulosa–luteal cells was measured by a tritiated water production assay (Garzo and Dorrington, 1984), with slight modifications, e.g. a change of isotope from testosterone to androstenedione and increased incubation time. Briefly, the medium for granulosa–luteal cell culture was replaced with 500 µl of GIT medium containing 2 µCi of 1ß-3H(N) androstenedione (New England Nuclear, Boston, MA, USA), and cultured at 37°C for 4 h. After incubation, the GIT medium was transferred into the tubes, and the cells were washed with 200 µl of PBS. The PBS used for washing was combined with the previous GIT medium, and 250 mg/ml of Norit A-activated charcoal (Wako, Osaka, Japan) was added to each tube. After incubated at 4°C for 2 h, the charcoal was removed by centrifugation at 3000 g for 20 min, and the tritiated water (3H2O) in the supernatant was determined by counting 0.5 ml of aliquots in 10 ml of Aquasol (Amersham International plc, Amersham, UK) in a liquid scintillation counter (LSC950, Aloka, Tokyo, Japan). The radioactivity quantified in each tube was corrected by subtracting the radioactivity of the blank tube, which contained medium with isotope after incubation without cells.
Northern Blot analysis
Aromatase mRNA expression in human granulosa–luteal cells was measured by Northern Blotting analysis. At first, total RNA was isolated by a single-step guanidinium thiocyanate–phenol–chloroform extraction process (Chomczynski and Sacchi, 1987). Approximately 105 cells were suspended in 1 ml of ISOGENTM solution (Nippongene, Tokyo, Japan) in each experimental condition, and total RNA precipitated was dissolved in TE buffer (10 mM Tris–HCl and 1 mM EDTA, pH 7.5).
For Northern Blot analysis, 12 µl of total RNA from each sample was denatured at 65°C in the solution containing 25 µl formamide, 2.5 µl 20x MOPS buffer (0.4 M of 3-(N-morpholino) propanesulfonic acid, 100 mM of sodium acetate, 20 mM of EDTA, pH 7.0), and 8 µl formaldehyde, and fractionated by electrophoresis on 1% agarose gels containing MOPS buffer. The size separated RNA was transferred onto the nylon membranes (Hybond-N+; Amersham International) by the capillary blotting in 0.05 M of sodium hydroxide.
The probes were labelled with [32P]-deoxy-cytosine triphosphate (dCTP) using a random primed DNA labelling kit (Amersham International plc), and purified by passage through a ProbeQuantTM G-50 Micro Column (Pharmacia LKB, Uppsala, Sweden). The membranes were prehybridized in a rapid hybridization buffer (Rapid-hyb buffer RPN 1635; Amersham International plc) for 30 min at 65°C, and hybridization was performed at 65°C for 3 h in the buffer containing the labelled probe (2 ng/ml). After hybridization, the membrane was washed at 37°C for 15 min in a solution of 2 x sodium chloride/sodium citrate (SSC) and 0.1% sodium dodecyl sulphate (SDS), and then washed twice at 65°C for 15 min in a 1 x SSC and 0.1% SDS solution. The membrane was exposed to Kodak X-AR film at –80°C. After the determination of expression of aromatase mRNA, the probes were stripped and the membrane was subjected to autoradiography to show that no radioactivity remained. This membrane was then reprobed with a glyceraldehyde-3-phosphate dehydrogenase (G3PDH) cDNA. The relative amounts of aromatase mRNA expression in each sample were determined as a ratio of the intensity of aromatase mRNA expression to G3PDH mRNA expression on the autoradiogram determined by densitometric analysis using an image scanner, GT-6000 (Epson, Tokyo, Japan) and NIH Image software (National Institute of Health, Bethesda, MD, USA).
Measurement of cAMP
After incubation with or without SNAP for 24 h, granulosa–luteal cells were collected, and ~105 cells were suspended in 250 µl of ice-cold 6% (w/v) trichloroacetic acid (TCA). After sonication for 20 s, cAMP in the cells was extracted three times with water-saturated ethyl ether (2:1 v/v; Wako Chemicals). After removal of ether fraction, the specimens were stored frozen at –30°C until assay. Measurement of cAMP was performed by radioimmunoassay using a commercially available kit (Yamasa, Tokyo, Japan), according to the manufacturer's protocol.
Statistical analysis
Data were expressed as mean ± SEM, and compared by one-way analysis of variance followed by Scheffe's F test for multiple comparison. P < 0.05 was considered to be statistically significant.
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Effects of NO donors and NO-related substances on aromatase activity of cultured human granulosa cells
To investigate effects of NO, aromatase activity in granulosa–luteal cells was measured after incubation with various concentrations of SNAP for 24 h (Figure 1
). Treatment with SNAP at concentrations of 10–4M or 10–3 M caused significant reduction in aromatase activity by 20% (P < 0.05) or 48% (P < 0.01) respectively. To ascertain whether or not the inhibitory effect of the NO donor on aromatase was related to NO released, another type of NO donor (NOC12), NO catabolites (sodium nitrite and sodium nitrate) and a peroxinitrite-releasing compound (SIN1) were added to the culture, and aromatase activity was measured after 24 h (Figure 2). There was no significant influence on aromatase activity by treatment with sodium nitrite, sodium nitrate and SIN1. On the other hand, NOC12 had quantitatively similar inhibitory effects on aromatase activity as SNAP.
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Effects of NO donors on aromatase gene expression of cultured human granulosa cells
To test the effect of NO on aromatase gene transcription, aromatase mRNA expression in granulosa–luteal cells was quantified by Northern Blot analysis after 24 h of culture with SNAP at 10–3 or 10–4 M. As shown in Figure 3
, transcripts of aromatase gene appeared as double bands located at 3.4 and 2.9 kb sites; this finding was consistent with previous reports (Means et al., 1989). Two major species of human aromatase transcripts are thought to be derived from the use of two different polyadenylation signals (Means et al., 1989). After 24 h of incubation with 10–3 M of SNAP, the relative amount of aromatase mRNA expression in granulosa–luteal cells was reduced by 72% (P < 0.05), compared with that of the control.
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Effects of NO donors on intracellular cAMP values, and effects of A kinase inhibition or C kinase activation on aromatase activity in the cultured human granulosa cells
To investigate whether NO modulates intracellular cAMP values, these values were measured by radioimmunoassay after 24 h of incubation with various concentrations of SNAP (Figure 4
) in granulosa–luteal cells. Treatment with 10–3 M of SNAP caused significant reduction of cAMP values by 53% (P < 0.01).
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To test whether inhibition of A kinase or activation of C kinase decreases basal aromatase activity, such activity in granulosa–luteal cells was determined after 24 h of incubation with various concentrations of H89 or tetradecanoyl phorbol acetate (TPA). There was no significant effect on aromatase activity, when up to 20 µM of H89 was added to culture (Figure 5a
). In contrast, addition of TPA 1 nM decreased aromatase activity in a dose-dependent manner (Figure 5b).
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Discussion |
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Basal aromatase activity in cultured human granulosa–luteal cells was inhibited by treatment with two structurally dissimilar NO donors, and these results were in agreement with previous reports (Van Voorhis et al., 1994
). In this study, we further explored the effect of molecules, generated as a result of NO metabolism, including major NO catabolites (nitrite and nitrate) and peroxinitrite (ONOO–). Peroxinitrite, which is formed in the presence of superoxide and NO, has been reported to mediate various forms of tissue injury (Lipton et al., 1993), including induction of apoptosis (Lin et al., 1995). SIN1, although it is not a strong NO donor, is capable of releasing both NO and superoxide simultaneously, hence generating peroxinitrite (Hogg et al., 1992). In the present study, either addition of SIN1 or the NO catabolites to culture had no significant effects on aromatase activities, confirming that the action of NO donors are NO-related.
So far, the molecular mechanisms of inhibitory actions of NO on aromatase has been partially elucidated. Similar to the actions found for hepatic cytochrome P-450 enzymes (Stadler et al., 1994), the inhibitory actions of NO on aromatase are thought to involve two different mechanisms, one of which is the direct effect on the enzyme, and another of which is decreasing mRNA values for the enzymes (Snyder et al., 1996). A direct inhibitory effect of NO appears to be mediated by the formation of a nitrosothiol group of the cysteine residue in aromatase, rather than by the direct binding to the heme iron, because carbon monoxide (a gas known to bind the heme iron in aromatase), had no effect on aromatase activity (Snyder et al., 1996). The present study demonstrated that NO donors caused a significant reduction of aromatase mRNA expression, supporting the hypothesis that the inhibitory action of NO on aromatase is, in part, attributable to the down-regulation of gene transcription.
The precise intracellular mechanisms mediating down-regulation of the aromatase gene transcription are not known. Although NO activates guanylate cyclase in many cells including human granulosa cells, the inhibitory effect of NO on aromatase is not mediated through a cyclic GMP signalling pathway, because addition of cyclic GMP analogues did not inhibit steroidogenesis (Van Voorhis et al., 1994; Masuda et al., 1997). Several recent studies have led to speculation about the existence of cyclic GMP-independent signal transduction pathway for NO actions: e.g. reports that treatment of cell membrane with NO decreases cAMP production (Duhe et al., 1994), and that some of the biological activities of NO may be mediated by cAMP-dependent mechanisms (Wang et al., 1997). Expression of aromatase gene in the ovary is thought to utilize a proximal promoter that is regulated primarily by cAMP (Means et al., 1989; Simpson et al., 1991, 1993), and treatment with NO donors was found to reduce intracellular cAMP values, suggesting that inhibition of basal values of aromatase activity by NO is mediated by a cAMP-dependent signal transduction pathway. However, this hypothesis was not supported by experiments using an inhibitor of protein kinase A, although the use of H89 alone is insufficient to elucidate the relationship between NO and the cAMP pathway.
The down-regulation of aromatase gene transcription in granulosa–luteal cells may be mediated through activation of protein kinase C, because C-kinase activators, such as gonadotrophin-releasing hormone and phorbol 12-myristate 13-acetate, have been reported to decrease basal values of aromatase mRNA in cultured granulosa cells from rats (Fitzpatrick et al, 1997) and ewes (Mcguire et al., 1994). In this study, we observed that the pharmacological activation of protein kinase C with phorbol esters induced reduction of basal aromatase activity in human granulosa–luteal cells in a dose-dependent manner. Whether NO modulates the intracellular calcium concentrations of human granulosa–luteal cells is the subject of our ongoing investigation.
Several studies have demonstrated relative importance of NO in the ovulatory process (Shukovsky and Tsafriri, 1994; Bonello et al., 1996; Yamauchi et al., 1997). However, effects of NO on steroidogenesis during ovulatory phase remains controversial. Although most previous reports have suggested that NO may act as a down-regulator of steroidogenesis in the ovary, contradictory data has been presented (Bonello et al., 1996) showing that administration of a NO synthase inhibitor inhibited oestrogen generation from the in-vitro perfused rat ovary, and have suggested that NO may stimulate oestrogen synthesis. Bonello et al. (Bonello et al., 1996) commented that the reduction of oestradiol may be attributable to the reduced flow rate due to vasoconstrictor effects of NOS inhibitors, and these results cannot be compared with those of most previous studies using isolated in-vitro cells, which lacked an intact vascular system. However, inhibition of endogenous NO synthase in the in-vitro perfused rabbit ovaries resulted in a significant elevation in the production of oestradiol stimulated by HCG (Yamauchi et al., 1997). The discrepancy between the results of two studies may be due to species differences.
Effects of NO on progesterone synthesis have also been investigated. Administration of NO donor inhibited progesterone synthesis of cultured human granulosa cells (Van Voorhis et al., 1994), and we also confirmed these results (data not shown). However, because the effects of NO on progesterone synthesis were one-order weaker than that on oestradiol (Van Voorhis et al., 1994), a role of NO in the regulation of ovarian progesterone synthesis appears to be less significant.
In conclusion, there were no significant effects in the NO catabolites or peroxinitrite on aromatase activity of human granulosa–luteal cells, confirming that inhibitory action of NO donors on aromatase is related to NO release. The action of NO is, in part, attributable to the down-regulation of aromatase gene transcription. Although NO caused reduction in the intracellular cAMP values, the down-regulation of aromatase gene transcription appears to be independent of the protein kinase A inhibition.
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3 To whom correspondence should be addressed
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Submitted on October 26, 1998; accepted on February 8, 1999.
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