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ENDOCRINE AND REPRODUCTIVE

Functional Melatonin Receptors in Rat Ovaries at Various Stages of the Estrous Cycle

Jose M. Soares, Jr.¹, Monica I. Masana, Çaatay Erahin², and Margarita L. Dubocovich

Departments of Molecular Pharmacology and Biological Chemistry (J.M.S., M.I.M., C.E., M.L.D.) and Psychiatry and Behavioral Science (M.L.D.), Northwestern Drug Discovery Program (C.E., M.L.D.), Northwestern University Feinberg School of Medicine, Chicago, Illinois

Received February 19, 2003; accepted April 18, 2003.

	Abstract

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This study investigated the receptor mechanism(s) by which the hormone melatonin directly affects ovarian function. Expression of MT₁ and MT₂ melatonin receptor mRNA was detected in the rat ovaries both by reverse transcriptase-polymerase chain reaction and in situ hybridization with digoxigenin-labeled oligoprobes. Specific 2-[¹²⁵I]iodomelatonin binding was significantly higher in ovarian tissue from animals sacrificed during proestrus than in metestrus, suggesting regulation of melatonin receptors by estrogens. Additionally, basal and melatonin-mediated stimulation of guanosine 5'-O-(3-[³⁵S]thio)triphosphate ([³⁵S]GTPS) binding to ovarian sections was higher in proestrus compared with metestrus. During proestrus, both luzindole (0.1 µM) and 4-phenyl-2-propionamidotetraline (4P-PDOT) (0.1 µM), acting as inverse agonists, inhibited basal [³⁵S]GTPS binding to ovarian sections, suggesting the presence of MT₁ constitutively active melatonin receptors. In primary cultures of ovarian granulosa cells, melatonin inhibited forskolin-stimulated cAMP accumulation through activation of G_i-coupled melatonin receptors. This inhibition was blocked by both, luzindole, and 4P-PDOT, acting as competitive receptor antagonists. Exposure of granulosa cells in culture to 17-estradiol seems to alter the state of melatonin receptor coupling. Indeed, the efficacy of 4P-PDOT on forskolin-stimulated cAMP formation was reversed from an MT₂ partial agonist in vehicle-treated cells to that of an MT₁ inverse agonist in 17-estradiol (0.1 µM)-treated granulosa cells. We conclude that MT₁ and MT₂ melatonin receptors expressed in antral follicles and corpus luteum may affect steroidogenesis through cAMP-mediated signaling. These results underscore the implications of the levels of ovarian estrogen when melatonin receptor ligands are used as therapeutic agents.

In mammalian species, melatonin affects reproductive function, in part, through activation of receptor sites within the hypothalamic-pituitary-gonadal axis (Reiter, 1980; Malpaux et al., 2001). Most studies investigating the mechanism(s) by which melatonin regulates reproduction have focused in the hypothalamus and pituitary as target tissues (Malpaux et al., 2001), with little attention directed to the role this hormone may play in the ovary itself. Melatonin is found in ovarian follicular fluid (Brzezinski et al., 1987; Ronnberg et al., 1990), suggesting a direct effect of this hormone in ovarian function. The effects of melatonin on ovarian function vary with tissue structure, cell type and whether the species is a seasonal or a nonseasonal breeder. For example, in hamster and rabbit ovaries melatonin inhibits steroidogenesis via changes in cAMP levels, through a direct action on theca, but not granulosa cells (YoungLai, 1978; Tamura et al., 1998). In contrast, melatonin increases progesterone and estrogen production in rat granulosa cells (Fiske et al., 1984) and luteinizing hormone receptor mRNA levels via the mitogen-activated protein kinase pathway and activation of Elk-1 in human granulosa cells (Woo et al., 2001). Together, these findings suggest that melatonin may regulate ovarian function through activation of multiple receptors and signaling pathways on different target cell types.

In mammals, melatonin modulates physiological functions through activation of at least two pharmacological and molecularly distinct melatonin receptors, the MT₁ and MT₂ (Masana and Dubocovich, 2001). Cohen et al. (1978) first reported [³H]melatonin binding sites in cytoplasmic fractions of hamster, rat, and human ovaries. Melatonin binding sites in ovarian tissue were later detected using 2-[¹²⁵I]iodomelatonin (Yie et al., 1995; Clemens et al., 2001). MT₁ and MT₂ melatonin receptor mRNAs were identified in human granulosa cells (Niles et al., 1999); however, the distribution of mRNA expression in ovarian structures has not been reported. Estradiol treatment of immature rats down-regulates MT₁ melatonin receptors in ovaries as detected using an anti-human MT₁ melatonin receptor antibody (Clemens et al., 2001). These results suggested that endogenous steroids during the estrous cycle may regulate melatonin receptors in selected target areas. The goal of this study was to demonstrate the expression and function of MT₁ and MT₂ melatonin receptors in adult rat ovaries at two stages of the estrous cycle, i.e., when steroids levels are high (proestrus) and low (metestrus), and to examine the effect of estrogen on the pharmacology of melatonin receptors in ovarian granulosa cells in culture.

	Materials and Methods

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Chemicals and Drugs
Melatonin, GDP, CAMP, and 17-estradiol were purchased from Sigma-Aldrich (St. Louis, MO). Pertussis toxin was from Calbiochem-Novabiochem (La Jolla, CA). Luzindole (2-benzyl-N-acetyl-tryptamine) and 4-phenyl-2-propionamidotetraline (4P-PDOT) were purchased from Tocris Cookson, Inc. (Ellisville, MO). Tissue culture reagents were from Invitrogen (Carlsbad, CA). [³H]cAMP and 2-[¹²⁵I]iodomelatonin were from Amersham Biosciences, Inc. (Piscataway, NJ), and [³⁵S]GTPS was from PerkinElmer Life Sciences (Boston, MA). Stock solutions (10 mM) of melatonin, luzindole, and 4P-PDOT were made in 100% ethanol. Luzindole and 4P-PDOT were diluted to 1 mM in 50% ethanol. Further dilutions to achieve the desired concentrations were made in water.

Animals
Female Sprague-Dawley rats (170–220 g) were purchased from Harlan (Madison, WI) and were kept on a 14:10 light/dark cycle, with controlled temperature (23 ± 2°C). Animals were housed in groups of three to five, in clear plastic cages with food and water ad libitum. Rats were always sacrificed during the light phase of the light/dark cycle, between ZT6 and ZT8) (ZT, Zeitgeber time; ZT0 lights on), when the blood melatonin levels are the lowest (Lewy et al., 1980). All studies were carried out in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health.

Estrous Cycle Evaluation
Vaginal smears were collected between ZT7 and ZT8 to evaluate the stages of the estrous cycle. Vaginal samples were obtained after placing a drop of distilled water in the vagina of the rat. The sample was removed using a pipetter fitted with a plastic tip and placed on a glass slide. The smear was left to dry at room temperature, stained using the Schorr modified method, coverslipped, and viewed under the microscope. Smear evaluations were performed daily until three consecutive 4-to 5-day estrous cycles were obtained. The estrous cycle is characterized by four phases: proestrus with numerous nucleated epithelial cells, some squamous epithelial cells and few leukocytes; estrus with many clusters of squamous epithelial cells; metestrus with some nucleated and squamous epithelial cells and abundant leukocytes; and diestrus with few cells and presence of thick mucous.

Quantitative Receptor Autoradiography Using 2-[¹²⁵I]Iodomelatonin
Rats were sacrificed during proestrus (n = 6) and metestrus (n = 6), and ovaries were dissected and processed for receptor autoradiography as described previously (Hunt et al., 2001), with modifications. Briefly, ovarian sections (20 µm) mounted on gelatin-coated slides were air-dried and then incubated with 50 mM Tris-HCl buffer (pH 7.4), containing 4 mM CaCl₂ in the absence or presence of 3 µM melatonin at 25°C for 30 min. Slides were then incubated with 2-[¹²⁵I]iodomelatonin (10–500 pM) (2000 Ci/mmol; Amersham Biosciences, Inc.) in the same buffer with or without melatonin 3 µM for 90 min. Sections were washed twice (5 min) in ice-cold 50 mM Tris-HCl buffer and once in ice-cold distilled water, air-dried, and placed in X-ray cassettes along with ¹⁴C standards and apposed to Biomax-MS (Eastman Kodak, Rochester, NY) film at room temperature for 2 weeks. Using a computer-based image analysis system (Bioquant98; R&M Biometrics, Inc., Nashville, TN), the optical densities of the autoradiograms were measured. For each 2-[¹²⁵I]iodomelatonin concentration (10–500 pM), the mean density readings from three sections of the same animal were converted to the amount of radioligand bound using the standard reference curve. The protein content in tissue sections was determined from tissue equivalents supplied by the manufacturer of ¹⁴C standards. Nonspecific binding was defined with 3 µM melatonin. Saturation curves were generated by nonlinear regression analysis using GraphPad Prism, version 3.03 for Windows (GraphPad Software, Inc., San Diego, CA).

RNA Isolation and Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
Rats were killed by decapitation. Ovaries and mammary glands were dissected and removed immediately, frozen quickly in isomethylbutane, and stored at –80°C until use. Total RNA from rat ovaries and mammary glands (n = 6) was isolated using TRIzol reagent (Invitrogen).

Synthesis of first-strand cDNA and subsequent PCR amplification were carried out using the Titan One Tube RT-PCR system (Roche Diagnostics, Indianapolis, IN) in a PCR DNA thermal cycler (PerkinElmer Instruments, Shelton, CT) according to manufacturer's instructions. DNase-treated RNA samples were reverse transcribed to cDNA followed by amplification in the same tube using an Expand high-fidelity enzyme blend in the presence of 1.5 mM MgCl₂ and 1 µM of each sense and antisense specific primers. The samples were incubated at 50°C for 30 min followed by thermocycling: 1 cycle of 94°C for 2 min; 10 cycles: 94°C for 30 s, 50°C for 30 s, 68°C for 1 min; 30 cycles: 94°C for 30 s, 50°C for 30 s, 68°C for 1 min, plus elongation of 5 s for each cycle and a final elongation time of 10 min at 68°C. A reverse transcriptase negative reaction was run where the enzyme was replaced by RNase free water for each sample to determine whether the amplification product came exclusively from the RNA. RNA extraction, DNA reverse transcription, and PCR assays were conducted in a laboratory where melatonin receptor clones were never handled.

Forward and reverse primer oligonucleotides were designed from the partial cDNA sequences derived from GenBank (MT₁ receptor, accession no. U14409 [GenBank] ; MT₂ receptor, accession no. U28218 [GenBank] ) and by computer analysis using the PrimerSelect program in the LASER-GENE Navigator software by DNAStar (Madison, WI) as described in Masana et al. (2002). PCR products were run on 1.7% agarose gel and excised from the gel, sequenced and confirmed as MT₁ and MT₂ rat melatonin receptors.

In Situ Hybridization
Ovarian sections (20 µm) from eight rats (four during proestrus and four during metestrus) were processed for in situ hybridization using digoxigenin (DIG)-labeled probes as described previously (Hunt et al., 2001). The specific antisense and sense oligonucleotide probes selected corresponded to regions of the MT₁ and MT₂ melatonin receptor mRNA that had little homology to the heterologous (i.e., MT₁ and MT₂, respectively) sequences or with other published nucleotide sequences (Hunt et al., 2001). Oligonucleotide probes were prepared at the Biotechnology Center of Northwestern University (Chicago, IL) and 3' tailed with DIG-11-dUTP using the DIG-oligonucleotide tailing kit (Roche Diagnostics), according to manufacturer's instructions. Ovarian sections were hybridized with DIG-labeled oligoprobes after methods reported previously (Hunt et al., 2001). The hybridization signal generated by DIG-labeled probes was detected using alkaline phosphatase-conjugated anti-DIG IgG (Roche Diagnostics), followed by chromagen 5-bromo-4-chloro-3-indolylphosphate-p-toluidine salt/nitro blue tetrazolium (Roche Diagnostics) in the presence of 1 mM levamisole.

[³⁵S]GTPS Binding to Rat Ovarian Tissue
Rat ovarian sections prepared as described for quantitative receptor autoradiography were processed for [³⁵S]GTPS binding as described by Sim et al. (1997) with modifications. Tissue sections were first incubated at room temperature for 10 min in assay buffer (50 mM Tris-HCl, containing 3 mM MgCl₂, 0.2 mM EGTA, 100 mM NaCl, and 0.5% bovine serum albumin, pH 7.4, at 25°C) and then for 30 min in the same buffer containing 2 mM GDP. Sections were then incubated with assay buffer containing 2 mM GDP and [³⁵S]GTPS (50 pM, 6000 cpm) in the absence or presence of drugs (0.1–10 µM melatonin, 0.1 µM luzindole, 0.1 µM 4P-PDOT). After 30 min, sections were washed twice for 2 min in ice-cold 50 mM Tris-HCl, pH 7.4, and once for 30 s in deionized water. Slides were air-dried overnight and placed in X-ray cassettes along with ¹⁴C standards and apposed to Biomax-MR (Eastman Kodak) film at room temperature for 48 h. Optical density is expressed as arbitrary units.

Primary Culture of Rat Granulosa Cells
Mature female rats (3–4 months old) were injected with 20 to 25 IU of pregnant mare's serum gonadotropin and sacrificed 48 h later by decapitation (Mukumoto et al., 1995). The ovaries were removed under the microscope and placed in 0.5 M sucrose for 30 min, followed by 6 mM EGTA for 15 min. Ovarian follicles were punctured using a needle under microscope, and cells were collected and spun down (600g) on a bench centrifuge. The pellet was resuspended in F-12 medium and viable cells (250,000/well) were incubated in a 12-well plate in Ham's F-12/Dulbecco's modified Eagle's medium with 5% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin in a 5% CO₂, 95% air mixture at 37°C for 5 to 6 days (until 70–80% confluence). Media were changed every 2 days. Viable granulosa cells were identified in cultures by assessment of cell morphology after hematoxylin-eosin and Masson trichromic staining and by determining cellular lipid droplet deposition after Sudan black staining. Both methods revealed that 85% of the cell population were granulosa cells.

2-[¹²⁵I]Iodomelatonin Binding to Granulosa Cell Membranes. Granulosa cells were washed with phosphate-buffered saline, collected in LIFT buffer (0.25 M sucrose, 10 mM potassium phosphate, and 1 mM EDTA, pH 7.4) and pelleted by centrifugation (25,000g, 10 min). Membrane pellets were resuspended (1 mg/ml) in Tris-HCl buffer (50 mM Tris-HCl, pH 7.4, and 10 mMgCl₂). Membrane suspensions were incubated with 2-[¹²⁵I]iodomelatonin (150 pM) for 60 min at 25°C (nonspecific binding defined with 10 µM melatonin). Reactions were terminated by the addition of ice-cold 50 mM Tris-HCl buffer, pH 7.4, and rapid filtration over glass-fiber filters (Schleicher & Schuell, Keene, NH) soaked in 0.5% polyethylenimine (Fluka/Sigma-Aldrich) solution. Each filter was washed twice with 5 ml of ice-cold Tris-HCl buffer and counted in a gamma counter.

cAMP Accumulation. Granulosa cells were stimulated with 20 µM forskolin for 10 min in the absence or presence of 0.1 nM to 1 µM melatonin, or luzindole (0.1–1 µM) and 4P-PDOT (0.1–1 µM). When indicated, granulosa cells were treated with 1 µg/ml pertussis toxin for 15 h. The number of viable granulosa cells was similar in both pertussis toxin-treated and control plates, as determining by trypan blue exclusion. The effect of estrogen on melatonin receptors function was investigated in granulosa cells cultured in charcoal-stripped serum media for 1 day. Cells were treated with vehicle or 0.1 µM 17-estradiol (concentration above the K_D value for estradiol binding to both the estrogen receptors and ; Razandi et al., 1999) in serum-free media for 2 h and then were stimulated with 3 µM forskolin for 10 min. The amount of cAMP in the cells was determined by radioreceptor binding (50,000 cpm of [³H]cAMP/tube) using cAMP as standard and purified regulatory subunit of protein kinase A as binding protein. After incubation at 0°C for 2 h, the reaction was terminated by vacuum filtration using glass fiber filters (Schleicher & Schuell) soaked in 0.5% polyethylenimine (v/v). Filters were washed with ice-cold 50 mM Tris-HCl, dried for 2 h, and the radioactivity was counted by liquid scintillation.

Data Analysis
EC₅₀ values were calculated from concentration-response curves by nonlinear regression using GraphPad Prism, version 3.02 for Windows (GraphPad Software, Inc.). Data were analyzed using two-way analysis of variance or when appropriate "t" tests. Bonferroni correction was performed on all post hoc tests. The level of significance was set at p < 0.05.

	Results

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Specific 2-[¹²⁵I]Iodomelatonin Binding to Rat Ovarian Sections. Figure 1 shows saturation curves of specific 2-[¹²⁵I]iodomelatonin binding to ovarian sections from female rats sacrificed during proestrus and metestrus determined by quantitative receptor autoradiography. The density of 2-[¹²⁵I]iodomelatonin binding sites was significantly higher during proestrus (B_max = 1.5 ± 0.1 fmol/mg of protein, n = 6) than during metestrus (B_max = 1.1 ± 0.1 fmol/mg of protein, n = 6) (p < 0.05). However, no differences in the affinity of 2-[¹²⁵I]iodomelatonin were detected between proestrus (K_D = 30.4 ± 11 pM, n = 6) and metestrus (K_D = 25.5 ± 12 pM, n = 6).

View larger version (19K): [in this window] [in a new window]   Fig. 1. Saturation analysis of 2-[125I]iodomelatonin binding determined by quantitative binding autoradiography in ovarian sections from rats sacrificed during proestrus and metestrus. Sequential 20-µm sections of ovarian tissue were incubated with 2-[125I]iodomelatonin (10–500 pM). Values shown are the mean ± S.E.M. of six independent experiments. 2-[125I]Iodomelatonin binding was significantly affected by the stage of the estrous cycle (F1,52 = 5.86, p < 0.05).

Expression of MT₁ and MT₂ Melatonin Receptor mRNA in Rat Ovaries. MT₁ and MT₂ melatonin receptor cDNAs were amplified from mRNA isolated from Sprague-Dawley rat ovarian tissue by RT-PCR with two rounds of amplification (Fig. 2A). PCR products obtained after the second round of amplification using nested primers, were of the expected size, i.e., 366 bp and 264 bp for MT₁ and MT₂ melatonin receptors, respectively. Sequencing of the PCR products indicated homology with previously described rat MT₁ and MT₂ melatonin receptors (accession nos. U14409 [GenBank] and U28218 [GenBank] , respectively). In the rat mammary gland, no expression of MT₁ or MT₂ mRNA was observed (Fig. 2B).

View larger version (58K): [in this window] [in a new window]   Fig. 2. RT-PCR analysis of MT1 and MT2 melatonin receptors mRNA expression in rat tissue. DNA gel electrophoresis of second amplification products generated from rat ovaries (A) or mammary gland (B). Lanes 1 and 6, DNA molecular weight marker (DNA-HaeIV). Using MT1 nested primers, a band of 366 bp corresponding to expected size for the amplified product of the MT1 melatonin receptor was obtained in ovaries (lane 2) but not in mammary tissue (lane 7) or in the absence of template (lanes 4 and 9). Using MT2 nested primers, a band of 264 bp corresponding to expected size for the amplified product of the MT2 melatonin receptor was obtained from ovarian mRNA (lane 3) but not in mammary tissue (lane 8) or in the absence of template (lanes 5 and 10).

We next localized melatonin receptor mRNA in rat ovaries by in situ hybridization using specific digoxigenin-labeled oligonucleotide probes corresponding to target mRNA sequences of either the rat MT₁ or MT₂ melatonin receptors. The specificity of these labeled oligonucleotides was previously demonstrated in both, the rat suprachiasmatic nucleus (Hunt et al., 2001) and the rat caudal artery (Masana et al., 2002) using homologous and heterologous unlabeled counter-parts. Figures 3 and 4 show the distribution of MT₁ and MT₂ melatonin receptor mRNAs in rat ovarian sections. Hybridization of digoxigenin-labeled oligonucleotide antisense probes to both MT₁ and MT₂ mRNA was observed in granulosa cells of antral follicles: secondary (Figs. 3A and 4A) and tertiary (Figs. 3C and 4C) follicles and in the corpus luteum (stronger signal in granulosa lutein cells than in the peripheral theca lutein cells) (Figs. 3E and 4E). Hybridization of both MT₁ and MT₂ antisense oligonucleotide probes to thecae cells was higher in secondary than in tertiary follicles. Hybridization of sense digoxigenin-labeled oligonucleotide probes was negligible (Figs. 3, B, D, and F, and 4, B, D, and F). No specific signal was observed in interstitial cells and in the ovarian stroma (Figs. 3C and 4C).

View larger version (143K): [in this window] [in a new window]   Fig. 3. Photomicrography of representative sections of ovarian tissue showing in situ hybridization using digoxigenin-labeled antisense oligonucleotides specific for the MT1 melatonin receptor. Hybridization signal for the antisense MT1-specific oligonucleotide was found in cells of secondary follicles (A), tertiary follicles (C), and corpus luteum (E). No hybridization signal using sense oligonucleotides was found (B, D, and F). TC, theca cell; OC, oocyte; GC, granulosa cell. Scale bar, 50 µm (A and B); 100 µm (C–F).

View larger version (155K): [in this window] [in a new window]   Fig. 4. Photomicrography of representative sections of ovarian tissue showing in situ hybridization using digoxigenin-labeled antisense oligonucleotides specific for the MT2 melatonin receptor. Hybridization signal for the MT2 specific oligonucleotides using antisense was found in cells of secondary follicles (A), tertiary follicles (C), and corpus luteum (E). No MT2 specific signal using sense oligonucleotides (B, D, and F). TC, theca cell; OC, oocyte; GC, granulosa cell. Scale bar, 50 µm (A and B); 100 µm (C–F).

[³⁵S]GTPS Binding to Rat Ovarian Sections Determined by Quantitative Autoradiography. [³⁵S]GTPS binding to ovarian sections in the presence of GDP (2 mM) was significantly higher during proestrus (basal: 0.310 ± 0.052 units, n = 6) than during metestrus (basal: 0.158 ± 0.016 units, n = 6) (Fig. 5A). Melatonin (0.1–10 µM) increased in a concentration-dependent manner [³⁵S]GTPS binding to ovarian sections during both phases of the estrous cycle (p < 0.0001) (Fig. 5A). The percentage of increase in [³⁵S]GTPS binding induced by melatonin was identical in both phases. No significant interaction between the stage of the cycle and the melatonin concentration was observed.

View larger version (46K): [in this window] [in a new window]   Fig. 5. [35S]GTPS binding to rat ovarian tissue during metestrus (A and C) and proestrus (B and D) determined by quantitative autoradiography. Sections were incubated with assay buffer containing 2 mM GDP and 50 pM [35S]GTPS, in the presence or absence of melatonin (A and B) luzindole (LUZ), or 4P-PDOT (C and D). A and B, [35S]GTPS binding was significantly affected by melatonin (F3,36 = 9.44, p < 0.0001) and the stage of the estrous cycle (F1,36 = 40.72, p < 0.0001). *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with corresponding basal (B). C and D, [35S]GTPS binding was significantly affected by luzindole and 4P-PDOT (F2,29 = 4.41, p < 0.05) and the stage of the estrous cycle (F1,29 = 13.12, p < 0.005). Values shown are mean ± S.E.M. of six independent experiments. *, p < 0.05; **, p < 0.01 compared with corresponding basal (B); #, p < 0.01 compared with basal in metestrus.

To evaluate whether the higher basal [³⁵S]GTPS binding during proestrus was due, at least in part, to the presence of constitutively active melatonin receptors we used luzindole and 4P-PDOT. These ligands at a concentration of 0.1 µM or higher are known to act as either partial agonists/antagonists on the MT₂ and as inverse agonists/antagonists on the MT₁ melatonin receptors (Masana and Dubocovich, 2001) (Fig. 5B). Both luzindole and 4P-PDOT significantly decreased [³⁵S]GTPS binding to ovarian tissue by 50% of the basal only during proestrus, when the levels of estrogen are higher (Fig. 5B).

Functional Melatonin Receptors in Rat Granulosa Cells. Primary cultures of rat ovarian granulosa cells were used to assess melatonin receptor density and modulation of forskolin-stimulated cAMP formation. The density of 2-[¹²⁵I]iodomelatonin binding sites in granulosa cells was determined after 6 days in culture. Specific 2-[¹²⁵I]iodomelatonin binding to granulosa cell membranes using a saturating concentration of radioligand (500 pM) and defined with melatonin (3 µM) was 5.1 ± 1.5 fmol/mg of protein (n = 3).

Basal levels of cAMP in granulosa cells in culture (1.3 ± 0.6 pmol/well, n = 3) were determined by radioreceptor binding to the purified regulatory subunit of protein kinase A. Forskolin (20 µM, 10 min) increased cAMP levels to 82.3 ± 8.1 pmol/well (n = 8). Melatonin (0.1 nM–1 µM) inhibited forskolin-stimulated cAMP accumulation with an EC₅₀ value of 2.3 nM and a maximum inhibition of 53.4% at 0.1 µM melatonin. This effect was blocked by pretreatment for 15 h with pertussis toxin (1 µg/ml) (Fig. 6). To assess whether the melatonin-mediated inhibition of forskolin-stimulated cAMP formation was receptor-mediated, we used luzindole (0.1–1 µM) and 4P-PDOT (0.1–1 µM). Neither ligand affected forskolin-stimulated cAMP accumulation when tested alone; however, all concentrations completely blocked the melatonin (10 nM)-mediated inhibition of forskolin-stimulated cAMP accumulation (Fig. 7).

View larger version (17K): [in this window] [in a new window]   Fig. 6. Effect of pertussis toxin on melatonin-mediated inhibition of forskolin-stimulated cAMP accumulation in rat granulosa cells. Intact granulosa cells attached to plates were incubated in the presence of 30 µM rolipram for 50 min and then exposed to medium containing 20 µM forskolin for 10 min in the presence of increasing concentrations of melatonin (0.1 nM–1 µM). Pertussis toxin pretreatment (1 µg/ml, for 15 h) completely abolished the melatonin-mediated inhibition of forskolin-induced cAMP accumulation (F5,42 = 7.77 p < 0.0001). Forskolin stimulation was similar in control (82.3 ± 8.1 pmol//well, n = 8) and in pertussis toxin-treated cells (96.2 ± 22.7 pmol/well, n = 3). Values shown are mean ± S.E.M. of at least three experiments run in duplicate. *, p < 0.001 compared with pertussis toxin-treated.

View larger version (30K): [in this window] [in a new window]   Fig. 7. Effect of luzindole and 4P-PDOT on melatonin-mediated inhibition of forskolin-stimulated cAMP accumulation in rat granulosa cells. Intact granulosa cells attached to plates were incubated with rolipram (30 µM) for 50 min in the absence or presence of luzindole (A) or 4P-PDOT (B). Cells were then exposed to media containing forskolin (20 µM) and melatonin (MLT) (10 nM) for 10 min. Both luzindole (A) and 4P-PDOT (B) at 0.1 and 1 µM blocked melatonin effect. Values shown are mean ± S.E.M. of three independent experiments. *, p < 0.05 compared with control; #, p < 0.05 compared with melatonin alone.

The effect of estrogen on melatonin receptor function was investigated in granulosa cells stimulated with a submaximal concentration of forskolin (3 µM), which increased cAMP levels to 27.6 ± 7.1 pmol/well (n = 3). In these experiments, granulosa cells were cultured in charcoal-stripped serum media for 1 day and then treated with either vehicle or 0.1 µM 17-estradiol in serum-free media for 2 h (Fig. 8A). Overall, there was a significant interaction between estrogen treatment and 4P-PDOT on forskolin-stimulated cAMP accumulation. Indeed, the efficacy of 0.1 µM 4P-PDOT was dependent on the estradiol treatment. 4P-PDOT significantly decreased forskolin (3 µM)-mediated cAMP accumulation in vehicle-treated cells growing in stripped serum but increased cAMP accumulation in 17-estradiol-treated cells. Figure 8B shows the effect of 4P-PDOT on forskolin-stimulated c-AMP accumulation expressed as the difference () from control in either vehicle- or 17-estradiol-treated cells. These results show that 17-estradiol treatment reversed 4P-PDOT effects on forskolin-stimulated cAMP formation from an inhibitory to a stimulatory effect.

View larger version (21K): [in this window] [in a new window]   Fig. 8. Efficacy of 4P-PDOT on forskolin-stimulated cAMP accumulation in rat granulosa cells in the presence and absence of estrogen. Granulosa cells were grown in serum-stripped media for 1 day and then treated with vehicle or 0.1 µM 17-estradiol for 2 h. Intact cells attached to plates were incubated with rolipram (30 µM) for 50 min. Cells were exposed to forskolin (3 µM) in the absence or presence of 4P-PDOT (0.1 µM) for 10 min. Values shown are mean ± S.E.M. of four independent experiments. A, a significant interaction between estradiol treatment and 4P-PDOT (F1,12 = 13.72, p < 0.005) was observed. *, p < 0.05, compared with control (C), #, p < 0.05 compared with 4P-PDOT in vehicle-treated cells. B, = difference between 4P-PDOT and control. #, p < 0.01 compared with vehicle-treated cells.

	Discussion

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Here, we demonstrated expression of MT₁ and MT₂ melatonin receptors in rat ovarian tissue that are functionally modulated by the levels of steroid fluctuations during two stages of the estrous cycle, i.e., proestrus versus metestrus. In granulosa cells, melatonin activation of a G_i-coupled melatonin receptor, possibly the MT₂, inhibited forskolin-stimulated cAMP accumulation. Together, the results suggest that 17-estradiol promotes the formation of constitutively active melatonin receptors, because the MT₁ inverse agonist 4P-PDOT inhibited [³⁵S]GTPS binding to ovarian sections collected during proestrus and increased forskolin-stimulated cAMP formation in granulosa cells cultured with 17-estradiol. We conclude that estrogens may play a critical role in the regulation, coupling, and signaling of melatonin receptors in rat ovaries.

Melatonin receptors are expressed in ovaries from various mammalian species, including human. High picomolar affinity 2-[¹²⁵I]iodomelatonin binding sites were reported in rat ovaries (Clemens et al., 2001; present study) and human granulosa cells (Yie et al., 1995). The 2-[¹²⁵I]iodomelatonin binding affinity (K_D = 25 pM) for rat ovary receptors determined by quantitative autoradiography in this study compares closely with the affinity (K_D = 83 pM) determined in ovarian membranes from immature rats (Clemens et al., 2001). [³H]Melatonin binds to lower affinity sites (K_D = 6.3 nM; K_D = 550 nM) in cytoplasmic fractions from hamster ovaries (Cohen et al., 1978). These results suggest that the melatonin receptors in membrane and cytosolic ovarian fractions could mediate distinct functions (Cohen et al., 1978). With the caveat that receptor density may vary between species, strain, and even experimental conditions, the density of 2-[¹²⁵I]iodomelatonin binding sites in ovaries and granulosa cells from rat and human are remarkable similar (1.5–5.5 fmol/mg of protein) (Yie et al., 1995; Clemens et al., 2001; present study). The results suggest that direct activation of melatonin receptors in ovaries by endogenous follicular melatonin may participate in the regulation of reproduction (Brzezinski et al., 1987).

A number of factors, including hormonal and ovarian morphological changes may contribute to the increases in melatonin receptor density during proestrus. Follicle-stimulating hormone is known to increase specific 2-[¹²⁵I]iodomelatonin binding in rat granulosa cells (Clemens et al., 2001). Additionally, high levels of estrogen during proestrus may affect melatonin receptor transcriptional or posttranscriptional mechanisms and/or protein translation as demonstrated for other G protein-coupled receptors [-adrenergic receptors, Krall et al. (1978); angiotensin AT₁ receptors, Krishnamurthi et al. (1999)]. Long-term treatment (48 h) with estrogen decreases ₂-adrenoceptor binding and _2A/D-adrenoceptor mRNA expression in rat cerebral cortex (Karkanias et al., 1997) and MT₁ melatonin receptors density in rat ovarian tissue (Clemens et al., 2001). Low receptor density during metestrus could result from down-regulation of melatonin receptors by the estrogen surge during proestrus. High melatonin receptor density during proestrus may also be secondary to ovarian morphology changes. In fact, large antral follicles with high number of granulosa cells expressing melatonin receptor mRNA are observed during proestrus compared with metestrus (Freeman, 1994).

In rat ovaries, melatonin receptors couple to inhibitory G_i proteins. First, melatonin stimulated [³⁵S]GTPS binding to rat ovarian slices, suggesting that melatonin binds to G protein-coupled melatonin receptors. Second, pertussis toxin blocked the melatonin-mediated inhibition of forskolin stimulated cAMP accumulation in rat granulosa cells in culture. Basal [³⁵S]GTPS binding to rat ovarian tissue was significantly higher during proestrus, the stage of the cycle when the levels of estrogen are higher (Freeman, 1994). Increases in basal [³⁵S]GTPS binding during proestrus could result from increases in G_i protein levels (Bouvier et al., 1991), estrogen activation of inhibitory G proteins (Wyckoff et al., 2001), increases in G protein-receptor coupling (Buhimschi et al., 2001) by estrogen, and/or possible increase number of constitutively active G protein-coupled receptors (Roka et al., 1999; Masana and Dubocovich, 2001). Although basal [³⁵S]GTPS binding was higher during proestrus, the ability of melatonin to further stimulate binding was not affected. The affinity (K_D) of 2-[¹²⁵I]iodomelatonin binding to melatonin receptors in rat ovary is in the low picomolar range; however, the concentrations of melatonin necessary to significantly stimulate [³⁵S]GTPS binding were in the high nanomolar to micromolar range. This relative low potency of melatonin could be attributed to a reduced efficiency of receptor coupling by the presence of high concentrations of GDP in the assay, which are known to decrease agonist affinity and potency at G protein-coupled receptors (Pauwels et al., 1997).

We next investigated the specific contribution of the MT₁ and MT₂ melatonin receptors in mediating ovarian functional responses. MT₁ and MT₂ melatonin receptor mRNA expression was detected in both rat ovarian tissue (present study) and human granulosa cells (Niles et al., 1999) by RT-PCR amplification and were found to be heterogeneously distributed in follicular, granulose, and thecae cells and in corpus luteum. The expression of melatonin receptor mRNA in ovarian granulosa layer correlates with the presence of specific 2-[¹²⁵I]iodomelatonin binding sites in rat (Clemens et al., 2001; present study) and human (Yie et al., 1995; Niles et al., 1999) granulosa cells. Functional melatonin receptors were demonstrated in hamster ovary theca, but not interstitial cells (Tamura et al., 1998). Together, these results suggest that activation of melatonin receptors in the ovaries may mediate steroidogenesis (Fiske et al., 1984; Brzezinski et al., 1992) and luteolysis (Hearn and Webley, 1987). Specific 2-[¹²⁵I]iodomelatonin binding seems to be regulated by estrogens, because the density of 2-[¹²⁵I]iodomelatonin binding sites was significantly increased during proestrus. Whether this increase reflects up-regulation of the MT₁ or/and MT₂ melatonin receptors is not known. We did not find expression of either MT₁ or MT₂ melatonin receptors mRNA in the mammary gland. These results suggest that the low-affinity 2-[¹²⁵I]iodomelatonin binding sites reported in this tissue (Recio et al., 1994) may represent a different melatonin receptor or acceptor site.

Using selective and specific melatonin receptor ligands, we assessed the functional role of MT₁ and MT₂ receptors in rat ovarian tissue. First, we investigated whether the high basal level of [³⁵S]GTPS in ovaries collected during proestrus was due to the presence of constitutively active melatonin receptors. To test this hypothesis, we used luzindole and 4P-PDOT, which act as inverse agonists at MT₁ receptors in systems were melatonin receptors exist in constitutively active form (Dubocovich et al., 1997; Browning et al., 2000; Masana and Dubocovich, 2001; Ersahin et al., 2002). Indeed, both ligands inhibited basal [³⁵S]GTPS binding to rat ovarian tissue during proestrus, which is compatible with the presence of constitutively active MT₁ melatonin receptors during this stage of the cycle. The effect of these ligands cannot be attributed to the presence of melatonin on the receptor, as at the time the animals were sacrificed, serum melatonin is very low (Lewy et al., 1980) and the effect was observed only during proestrus. The presence of MT₁ constitutively active receptors was previously demonstrated in rat arteries (Ersahin et al., 2002). We conclude that high estrogen levels during proestrus may affect the spontaneous activation of MT₁ melatonin receptors, resulting from increases in G protein-receptor coupling (Buhimschi et al., 2001) and/or increased expression of G_i proteins (Bouvier et al., 1991).

In rat granulosa cells, melatonin inhibited forskolin-stimulated cAMP accumulation through activation of a receptor coupled to pertussis toxin-sensitive G proteins. To determine the melatonin receptor type mediating this response we used luzindole and 4P-PDOT known to act as MT₂ melatonin receptor partial agonists and/or antagonists or MT₁ inverse agonists, depending on ligand concentration, the level of receptor expression and system analyzed (Dubocovich et al., 1997; Browning et al., 2000; Masana and Dubocovich, 2001). These ligands did not potentiate on their own stimulation of cAMP formation, which is compatible with the absence of constitutively active melatonin receptors. However, both competitive melatonin receptor antagonists blocked the melatonin-mediated inhibition of forskolin (20 µM) stimulation of cAMP formation, suggesting activation of a melatonin receptor. The effect on cAMP accumulation was possibly mediated through activation of the MT₂ melatonin receptor because it was blocked by an MT₂-selective concentration of 4P-PDOT (Dubocovich et al., 1997; Masana and Dubocovich, 2001).

During proestrus MT₁ melatonin receptors are in constitutively active form, suggesting a role for sex steroids in its regulation. In rat granulosa cells cultured in estrogen-free serum conditions, activation of melatonin receptors by 4P-PDOT probably acting as an MT₂ melatonin receptor partial agonist (Lotufo et al., 2001), inhibited forskolin-stimulated cAMP formation. In contrast, in the presence of 17-estradiol, 4P-PDOT, acting as an MT₁ inverse agonist, increased forskolin-stimulated cAMP levels. Together, these results suggest that estrogen may favor the formation of a spontaneously active state of the MT₁ receptor by an increase in G protein-receptor coupling (Buhimschi et al., 2001), hence increasing ligand-independent activity of this receptor.

In summary, we clearly demonstrated that estrogen is able to modulate melatonin receptor signaling in rat ovaries, possibly by altering the efficacy of melatonin receptor-specific ligands. Cagnacci et al. (2001) reported that melatonin reduces internal carotid artery pressure index and blood pressure in young women and in women undergoing estrogen replacement therapy, but not in its absence. These findings are compatible with a role of estrogen in modulating melatonin responses in humans underscoring the implications of circulating ovarian estrogen on the therapeutic efficacy of melatonin receptor ligands.

	Acknowledgements

 
We thank Dr. L. Van Eldik for the use of laboratory facilities to perform the RT-PCR experiments and Maritess Tinio for editorial assistance.

	Footnotes

 
This work was partially supported by Institutional Funds, National Institutes of Health Grants MH 52685 (to M.L.D.), T32AG00260 (to C.E.), and Companha de Aperfeiçoamento de Pessoal de Nível Superior Fellowship (Brazil) (to J.M.S.).

DOI: 10.1124/jpet.103.049916.

ABBREVIATIONS: MT, melatonin receptors; 4P-PDOT, 4-phenyl-2-propionamidotetralin; GTPS, guanosine 5'-O-(3-thio)triphosphate; ZT, Zeitgeber time; RT-PCR, reverse transcriptase-polymerase chain reaction; DIG, digoxigenin; bp, base pair.

¹ Current address: Department of Gynecology, Escola Paulista de Medicina, Federal University of Sao Paulo, Sao Paulo, Brazil.

² Current Address: Loyola University Medical Center, Department of Pathology, Bldg. 110, 2160 S. First Ave., Maywood, IL 60153.

Address correspondence to: Dr. Margarita L. Dubocovich, Department of Molecular Pharmacology and Biological Chemistry (S215), Northwestern University Feinberg School of Medicine, 303 East Chicago Ave., Chicago, IL 60611-3008. E-mail: mdubo{at}northwestern.edu

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