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Melatonin Stimulates the Release of Growth Hormone and Prolactin by a Possible Induction of the Expression of Frog Growth Hormone-Releasing Peptide and Its Related Peptide-2 in the Amphibian Hypothalamus

Laboratory of Integrative Brain Sciences, Department of Biology, Faculty of Education and Integrated Arts and Sciences, Waseda University, Tokyo 169-8050, Japan

Address all correspondence and requests for reprints to: Kazuyoshi Tsutsui, Ph.D., Professor, Laboratory of Integrative Brain Sciences, Department of Biology, Faculty of Education and Integrated Arts and Sciences, Waseda University, 1-6-1 Nishi-Waseda, Shinjuku-ku, Tokyo 169-8050, Japan. E-mail: k-tsutsui{at}waseda.jp.

	Abstract

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We recently identified a novel hypothalamic neuropeptide stimulating GH release in bullfrogs and termed it frog GH-releasing peptide (fGRP). The fGRP precursor encodes fGRP and its related peptides (fGRP-RP-1, -RP-2, and -RP-3), and fGRP-RP-2 also stimulates GH and prolactin (PRL) release. Cell bodies and terminals containing these neuropeptides are localized in the suprachiasmatic nucleus (SCN) and median eminence, respectively. To understand the physiological role of fGRP and fGRP-RP-2, we investigated the mechanisms that regulate the expression of these neuropeptides. This study shows that melatonin induces the expression of fGRP and fGRP-RPs in bullfrogs. Orbital enucleation combined with pinealectomy (Ex plus Px) decreased the expression of fGRP precursor mRNA and content of mature fGRP and fGRP-RPs in the diencephalon including the SCN and median eminence. Conversely, melatonin administration to Ex plus Px bullfrogs increased dose-dependently their expressions. The expression of fGRP precursor mRNA was photoperiodically controlled and increased under short-day photoperiods, when the nocturnal duration of melatonin secretion increases. To clarify the mode of melatonin action on the induction of fGRP and fGRP-RPs, we further demonstrated the expression of Mel_1b, a melatonin receptor subtype, in SCN neurons expressing fGRP precursor mRNA. Finally, we investigated circulating GH and PRL levels after melatonin manipulation because fGRP and fGRP-RP-2 stimulate the release of GH and GH/PRL, respectively. Ex plus Px decreased plasma GH and PRL concentrations, whereas melatonin administration increased these hormone levels. These results suggest that melatonin induces the expression of fGRP and fGRP-RP-2, thus stimulating the release of GH and PRL in bullfrogs.

	Introduction

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SECRETION OF GH AND PROLACTIN (PRL) by the anterior pituitary is under hypothalamic control in vertebrates. In mammals, GH secretion from the somatotrophs is known to be under dual hypothalamic control, the primary stimulatory neurohormone being the 40- to 44-amino acid peptide GHRH, and the primary inhibitory hormone being the 14-amino acid peptide somatostatin. Although these two hypophysiotropins are thought to be the primary regulators of GH, TRH may play a role in GH regulation in some vertebrate species (1). In amphibians, GHRH (2), pituitary adenylate cyclase-activating polypeptide (2, 3), and ghrelin (4) of amphibian origin have been nominated as stimulating factors for GH release. As for PRL release, TRH is considered to be a major PRL-releasing factor in the amphibian hypothalamus (5).

In addition to these findings, the recent discovery of frog GH-releasing peptide (fGRP) and its related peptide (fGRP-RP)-2 has provided the potential for new insight into the regulation of GH and PRL secretion (6, 7, 8). We previously identified in the bullfrog a novel hypothalamic neuropeptide that stimulates GH release in vivo and in vitro and therefore was designated fGRP (6). Molecular cloning of cDNA encoding the precursor of fGRP further revealed that it encodes fGRP and its related peptides (fGRP-RP-1, -RP-2 and -RP-3) (7). fGRP and fGRP-RPs were invariably equipped with -LPXRF (X = L or Q) at their C termini (6, 7, 8) as follows: fGRP, SLKPAANLPLRFamide; fGRP-RP-1, SIPNLPQRFamide; fGRP-RP-2, YLSGKTKVQSMANLPQRFamide; fGRP-RP-3, AQYTNHFVHSLDTLPLRFamide. Cell bodies and terminals containing fGRP and fGRP-RPs are localized in the suprachiasmatic nucleus (SCN) and median eminence (ME), respectively (6, 7, 8). Among fGRP-RPs, fGRP-RP-2 was also effective in stimulating GH and PRL release in vivo and in vitro (8).

When we understand the functional significance of fGRP and fGRP-RP-2 and their physiological roles as key neuropeptides involved in the regulation of GH and PRL release, it is essential to clarify the regulatory mechanism that regulates the expression of fGRP and fGRP-RP-2. Until now a regulatory mechanism(s) governing the expression of fGRP and fGRP-RPs has remained unclear. In this study, we hypothesized that melatonin may be involved in the induction of fGRP and fGRP-RP-2 expression, thus influencing circulating GH and PRL levels. Because the eyes and pineal glands are the major sources of melatonin in amphibians (9, 10, 11), in this study we first analyzed the effects of orbital enucleation (Ex) combined with pinealectomy (Px) (Ex plus Px) on the expression of fGRP precursor mRNA and content of mature fGRP and fGRP-RPs in bullfrogs. Subsequently melatonin was administered to Ex plus Px frogs. We further analyzed the action of endogenous melatonin on the expression of fGRP precursor mRNA by using frogs exposed to short-day (SD) and long-day (LD) photoperiods, thus varying the length of endogenous melatonin secretion by varying the length of the night (melatonin is secreted at night) (12). To identify the mode of melatonin action on the induction of fGRP and fGRP-RPs, we investigated the expression of Mel_1b, a melatonin receptor subtype, in SCN neurons expressing fGRP and fGRP-RPs. We chose Mel_1b because our preliminary study showed that Mel_1b may be expressed in the SCN of the bullfrog hypothalamus. We cloned a cDNA encoding Mel_1b in the bullfrog diencephalon and conducted dual-label in situ hybridization for Mel_1b mRNA and fGRP precursor mRNA. Because fGRP stimulates GH release and fGRP-RP-2 stimulates GH and PRL release in bullfrog, we finally investigated whether melatonin modulates circulating GH and PRL levels via the action of fGRP and fGRP-RP-2. Here we show that melatonin stimulates the release of GH and PRL by inducing the expression of fGRP and fGRP-RP-2 in bullfrogs.

	Materials and Methods

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Experimental animals
Adult male bullfrogs (Rana catesbeiana) housed in a temperature-controlled chamber at 17 C under daily photoperiods of 12-h light and 12-h dark (lights on at 0600 h) were used in this study. Ethical approval for the surgical experiments of this study was given by the committee for the Care and Use of Laboratory Animals of Waseda University (Tokyo, Japan). All efforts were made to minimize the number of animals used and their suffering.

Experimental schedules
The eyes and pineal gland are the major sources of melatonin in frogs (9, 10, 11). Thus, in the first experiment, Ex and Px were conducted to determine possible action of melatonin on the expression of fGRP and fGRP-RPs. Ex was performed by the method described previously (13). As described for Px (14), the pineal gland, located in the diencephalic roof, was removed with fine forceps (Px). In sham-operated bullfrogs (SH), the pineal gland was exposed but not removed. Bilateral Ex bullfrogs underwent Px (Ex plus Px). All surgery was performed under ethyl 3-amino-benzoate methanesulfonate salt (MS-222; Sigma, St. Louis, MO) anesthesia (1.5 g/liter). One week after surgery, SH and Ex plus Px bullfrogs (n = 5 in each group) were terminated by decapitation between 1100 and 1300 h for the collection of diencephalic samples. The fGRP precursor mRNA was quantified by means of real-time quantitative PCR. The concentration of fGRP and fGRP-RPs in the diencephalon was quantified by means of ELISA. The completeness of Ex plus Px was verified at autopsy and confirmed by measuring melatonin in the diencephalon and plasma by means of an RIA. For the measurement of melatonin, separate groups of identically treated bullfrogs of SH and Ex plus Px groups (n = 5 in each group) were also terminated during the dark phase (between 2300 and 0100 h), when endogenous plasma melatonin is high (12). Thus, we verified that our surgical procedures were effective.

In the second experiment, melatonin was administered to Ex plus Px bullfrogs to investigate the action of melatonin on the expression of fGRP and fGRP-RPs. One week after the beginning of treatment, all bullfrogs (n = 5 in each group) were terminated between 1100 and 1300 h and diencephalic samples were collected for quantification of fGRP precursor mRNA.

In the third experiment, we conducted quantitative analyses for the expression of mature fGRP and fGRP-RPs in SCN neurons in SH, Ex plus Px, and Ex plus Px plus melatonin (25 mg/plate) bullfrogs. One week after the beginning of treatment, all bullfrogs (n = 5 in each group) were terminated between 1100 and 1300 h, and the brains were collected. The expression of fGRP and fGRP-RPs in SCN neurons was analyzed by immunocytochemistry.

In the fourth experiment, we manipulated endogenous melatonin to determine its action on fGRP precursor mRNA expression. Melatonin secretion occurs during the dark phase; thus, SD treatment increases the nocturnal duration of endogenous melatonin secretion (12). After 3 wk of exposure to SD (8 h light and 16 h dark; lights on at 0800 h) or LD (16 h light and 8 h dark; lights on at 0400 h) photoperiods, bullfrogs (n = 5 in each group) were terminated between 1100 and 1300 h, and diencephalic samples were analyzed for fGRP precursor mRNA.

In the fifth experiment, the expression of Mel_1b, a melatonin receptor subtype, in the SCN was analyzed to investigate the mode of melatonin action on the induction of fGRP and fGRP-RPs. We measured Mel_1b expression in the bullfrog diencephalon by cloning a corresponding cDNA. Subsequently we determined the coexpression of Mel_1b mRNA and fGRP precursor mRNA by dual-label in situ hybridization.

In the sixth experiment, to investigate the effect of melatonin on plasma GH and PRL concentrations, blood samples were collected from SH, Ex plus Px, and Ex plus Px plus melatonin (25 mg/plate) bullfrogs 1 wk after melatonin treatment. The plasma samples of these groups were analyzed by RIAs to determine plasma GH and PRL concentrations. Finally, the effect of melatonin on the release of GH and PRL from cultured bullfrog anterior pituitaries was determined to test the possibility of direct action of melatonin on the pituitary.

Real-time quantitative PCR of fGRP precursor mRNA
To quantify the expression of fGRP precursor mRNA in the diencephalon, real-time quantitative PCR was conducted by using the LineGene system (BioFlux, Tokyo, Japan). β-Actin was used as a housekeeping gene for the internal standard. The PCR primers used for the amplification of fGRP precursor cDNA fragments were as follows: sense primer, 5'-ATTCCCAATCTACCGCAAC-3' [identical with nucleotides 394–412 in Sawada et al. (7)] and antisense primer, 5'-GTTCTTCCAAATCGCAGTG-3' [complementary to nucleotides 527–545 in Sawada et al. (7)]. The PCR primers used for the amplification of bullfrog β-actin were as follows: sense primer, 5'-GAAATCGTGCGTGACATC-3' (identical with nucleotides 61–78; GenBank accession no. AB094353) and antisense primer, 5'-CTCTGGACACCTGAACCTC-3' (complementary to nucleotides 200–219; GenBank accession no. AB094353). PCR was performed using a SYBR Green real-time PCR master mix (TOYOBO, Osaka, Japan). An external standard curve was generated by dilutions of the target PCR product, which had been purified and its concentration measured previously. The fGRP precursor expression in each reaction was normalized by the expression of β-actin.

ELISA of fGR and fGRP-RPs
Peptides were extracted from the diencephalon of each bullfrog, as described (15, 16, 17, 18, 19). Frozen diencephalic samples were boiled for 7 min and homogenized in 5% acetic acid using a homogenizer (Ultra-Turrax T8 IKA; Labortechnik, Staufen, Germany). The homogenate was centrifuged at 16,000 x g for 30 min. The supernatant was collected and forced through a disposable C-18 cartridge (Sep-Pak Vac 1cc; Waters, Milford, MA). The retained material was then eluted with 60% methanol. The pooled eluate was concentrated in a vacuum evaporator at 40 C, passed through disposable Ultrafree-MC Centrifugal Filter Units (Millipore, Bedford, MA), and subjected to a competitive ELISA by using the antiserum raised against fGRP in a rabbit (6). This antiserum cross-reacts with fGRP and fGRP-RPs (6, 8). The specificity of the antiserum was checked by a competitive ELISA. The IC₅₀ values in the competitive ELISA were estimated as follows; 0.54 pmol for fGRP (SLKPAANLPLRFamide), 0.42 pmol for fGRP-RP-1 (SIPNLPQRFamide), 1.70 pmol for fGRP-RP-2 (YLSGKTKVQSMANLPQRFamide), 0.33 pmol for fGRP-RP-3 (AQYTNHFVHSLDTLPLRFamide), 21.0 pmol for chicken RFamide (LPXRFamide), and more than 1000 pmol for other RFamide peptides, e.g. Carassius RFamide (SPEIDPFWYVGRGVRPIGRFamide) and molluscan RFamide (FMRFamide). For the ELISA, different concentrations of fGRP (1–1000 pmol/ml) and adjusted tissue extracts were added with the antiserum against fGRP (1:1,000 dilution) to each antigen-coated well of a 96-well microplate (multiwell plate for ELISA, H-type; Sumitomo Bakelite Co., Ltd., Tokyo, Japan) and incubated for 1 h at 37 C. After the reaction with alkaline phosphatase-labeled goat antirabbit IgG, immunoreactive products were obtained in a substrate solution of p-nitrophenylphosphate, and the absorbance was measured at 415 nm on a microtiter plate reader (MTP-120; Corona Electric, Ibaraki, Japan).

Immunocytochemistry of fGRP and fGRP-RPs
Immunocytochemical analysis of fGRP and fGRP-RPs was conducted as previously described (6, 8). In brief, SH, Ex plus Px, and Ex plus Px plus melatonin bullfrogs were killed by decapitation (n = 5). After dissection from the skull, the brains were fixed in 4% paraformaldehyde solution overnight and then soaked in a refrigerated sucrose solution (30% sucrose in 0.1 M phosphate buffer) until they sank. Whole brains were embedded in OCT compound (Miles Inc., Elkhart, IN) and frozen sectioned frontally at a 20-µm thickness with a cryostat at –20 C. After blockage of nonspecific binding components with 1% normal goat serum and 1% BSA in PBS (pH 7.2) containing 0.3% Triton X-100, the sections were immersed overnight at 4 C in a 1:1000 dilution of the antiserum raised against fGRP, which cross-reacts with fGRP and fGRP-RPs (6, 8). The primary immunoreaction was followed by a 60-min incubation with rhodamine-conjugated antirabbit IgG (ICN Pharmaceuticals, Inc., Costa Mesa, CA) used at a dilution of 1:1000. The localization of immunoreactive cell bodies and fibers was examined with a fluorescence microscope (Nikon, Melville, NY). The specificity of the staining was assessed by substituting the antiserum with antiserum (1:1000 dilution) that had been preabsorbed by incubation with the antigen (synthetic fGRP, fGRP-RP-1, fGRP-RP-2, or fGRP-RP-3) in a saturating concentration (20 µg/ml) overnight before use.

Mel_1b cDNA cloning
Partial bullfrog Mel_1b, a melatonin receptor subtype, cDNA (185 bp) was obtained from bullfrog diencephalic cDNA by nested PCR using degenerate primers. The primers used for the first- and second-round PCR were as follows: sense primer 1, 5'-TSCCAAACYTYTTTGTBGGMTC-3' (identical with nucleotides 595–616; GenBank accession no. AB302949), sense primer 2, 5'-TACACSATWRCWGTWGTSRTVDTBCATTT-3' (identical with nucleotides 675–703; GenBank accession no. AB302949) and antisense primer 1, 5'-GCAAARAKDACAAABACYACRAACATGGT-3' (complementary to nucleotides 831–859; GenBank accession no. AB302949). These PCR primers were designed on the basis of alignment of Xenopus laevis Mel_1a (GenBank accession no. U31826), Mel_1b (GenBank accession no. U31827), Mel_1c(a) (GenBank accession no. U67880), and Mel_1c(b) (GenBank accession no. U67882) mRNA sequences. The unknown sequences of 5'- and 3'-untranslated regions were analyzed by rapid amplification of cDNA ends methods as previously described (20) with bullfrog Mel_1b-specific primers as follows: sense primer 3, 5'-GCGAAAGGTCAAGTCAG-3' (identical with nucleotides 769–786; GenBank accession no. AB302949), sense primer 4, 5'-CCAAGAATGAAGCCCAGCGA-3' (identical with nucleotides 795–814; GenBank accession no. AB302949), antisense primer 2, 5'-CCAGATGCGGAGATAGCA-3' (complementary to nucleotides 732–749; GenBank accession no. AB302949), and antisense primer 3, 5'-CCACAGTGATTGGCACG-3' (complementary to nucleotides 706–723; GenBank accession no. AB302949).

Dual-label in situ hybridization of Mel_1b mRNA combined with fGRP precursor mRNA
To examine the expression of Mel_1b mRNA in SCN neurons expressing fGRP and fGRP-RPs, dual-label in situ hybridization of Mel_1b mRNA combined with fGRP precursor mRNA was conducted. Partial bullfrog Mel_1b cDNA (corresponding to nucleotides 915–1480; GenBank accession no. AB302949) was obtained by RT-PCR using the following specific primers: sense primer 5, 5'-AAGGTGGCCCCTAAGATTCC-3' (identical with nucleotides 915–934; GenBank accession no. AB302949) and antisense 4: 5'-CAGCCTGTCCATTTTCTGGT-3' (complementary to nucleotides 1475–1494; GenBank accession no. AB302949). The RT-PCR product was subcloned into pBluescript II plasmid vector (Stratagene, La Jolla, CA). Fluorescein-labeled bullfrog Mel_1b antisense and sense RNA probes were generated with RNA labeling kit (Roche Diagnostics, Basel, Switzerland). fGRP precursor antisense and sense RNA probes were generated as previously described (7). In this study, fGRP precursor probes were labeled with digoxigenin RNA labeling kit according to the manufacturer’s instructions (Roche Diagnostics).

Bullfrogs were killed by decapitation; brains were rapidly dissected, embedded in OCT compound, and frozen. Sections were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, rinsed in PBS, acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0), and then washed in 2x saline sodium citrate [SSC; 1x SSC = 150 mM NaCl and 15 mM sodium citrate (pH 7.0)]. Subsequently the sections were prehybridized for 1 h at 50 C in prehybridization buffer consisting of 50% formamide, 2x SSC, 1x Denhardt’s, 0.5 mg/ml yeast transfer RNA, 0.5 mg/ml heparin sodium, and 0.1% sodium pyrophosphate. Hybridization was performed at 50 C for 16 h with fluorescein-labeled Mel_1b RNA probe and digoxigenin-labeled fGRP precursor RNA probe diluted with hybridization buffer (prehybridization buffer supplemented with 10% dextran sulfate). After hybridization, the sections were sequentially washed in 2x SSC, washed in 2x SSC-50% formamide treated with RNase A (20 µg/ml). The sections were washed sequentially in RNase buffer, 2x SSC, and then washed in 1x SSC-50% formamide. Slides were rinsed in buffer 1 [100 mM Tris-HCl (pH 7.5), 150 mM NaCl] and then incubated with buffer 1 containing 2% blocking reagent (Roche Diagnostics). Then sections were incubated with antifluorescein antibody conjugated with alkaline phosphatase (Roche Diagnostics; diluted 1:500, for detection of Mel_1b mRNA expression) and antidigoxigenin monoclonal antibody (Roche Diagnostics; diluted 1:100, for detection of fGRP mRNA expression) at 4 C overnight. The primary immunoreaction was followed by incubation with biotinylated rabbit-antimouse IgG (Dako Cytomation, Glostrup, Denmark; diluted 1:300). Subsequently they were incubated with Alexa 488-streptavidin (Invitrogen, Eugene, OR; diluted 1:300). Sections were then immersed in buffer 2 [100 mM Tris-HCl (pH 9.5), 100 mM NaCl, 50 mM MgCl₂] and then incubated with the solution of nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate at room temperature for 48 h under dark conditions. Control for specificity of in situ hybridization of Mel_1b mRNA or fGRP precursor mRNA was performed by using each sense RNA probe complementary to the antisense probe sequence.

Pituitary cell culture
Dispersed anterior pituitary cells of bullfrogs were prepared as previously described (6, 8, 21). In brief, the completely dispersed cells were then resuspended in 70% medium 199 (Nissui Pharmaceutical, Tokyo, Japan) containing 0.1% BSA. An aliquot of the cell suspension was used to count the cell number. The volume of the cell suspension was adjusted to that 1 ml contained 3.5 x 10⁵ cells. Seventy thousand cells in 200 µl medium were planted in each well of a 96-multiwell plate (Asahi Techno Glass, Tokyo, Japan), and preincubated for 24 h at 23 C in an atmosphere at 95% air-5% CO₂. Preincubated pituitary cells were transferred to the medium containing 10^–12 to 10^–6 M melatonin (Sigma) and incubated for 24 h at 23 C. To confirm the responsiveness of pituitary cells, TRH (Sigma), a PRL and GH secretagogue at the concentration of 10^–8 M, was also used. Anterior pituitary cells were incubated with medium 199 alone as controls. After incubation, each medium was centrifuged, and the supernatant was subjected to RIAs for bullfrog GH (22) and PRL (23).

RIAs of GH and PRL
Bullfrog GH (24) and PRL (25) used as standards and radioligands were previously purified in our laboratory. They were radioiodinated with ¹²⁵I (Na¹²⁵I; Radiochemical Center, Amersham Pharmacia Biotech, Little Chalfont, UK) by the method described previously (22, 23). Intraassay coefficients of variation in the RIA for GH and PRL were 4.1 and 3.8%, respectively; and interassay coefficients of variation were 4.3 and 3.3%, respectively. The RIA results of bullfrog GH and PRL assayed in duplicate were calculated in terms of nanograms per ml plasma or 10⁴ pituitary cells per 24 h.

Statistical analysis
Results of the PCR, ELISA, RIAs, and quantitative immunocytochemistry were expressed as the mean ± SEM and analyzed for significance by one-way ANOVA followed by Duncan’s multiple range test or Student’s t test.

	Results

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Effects of Ex combined with Px on the expressions of fGRP precursor mRNA and mature fGRP and fGRP-RPs
Changes in fGRP precursor mRNA levels in the diencephalon after Ex plus Px were measured by means of real-time quantitative PCR. The fGRP precursor mRNA level was expressed as a ratio of fGRP precursor mRNA concentration to β-actin mRNA concentration. As shown in Fig. 1A, the fGRP precursor mRNA level decreased significantly in Ex plus Px bullfrogs, compared with SH bullfrogs (P < 0.05, Ex plus Px vs. SH). Changes in the concentration of mature fGRP and fGRP-RPs in the diencephalon, which includes SCN neurons expressing fGRP and fGRP-RPs and their terminals in the ME, were measured by competitive ELISA, using the fGRP-antiserum cross-reacting with fGRP and fGRP-RPs. As shown in Fig. 1B, Ex plus Px also induced a significant decrease in the concentration of fGRP and fGRP-RPs on a unit weight basis (milligrams) of diencephalon (P < 0.01, Ex plus Px vs. SH). These changes in fGRP precursor mRNA and mature fGRP and fGRP-RPs after Ex plus Px were closely related to the changes in melatonin in the diencephalon and plasma (Fig. 1, C and D). Ex plus Px was followed by significant decreases in melatonin concentrations in both the diencephalon and plasma (P < 0.05, Ex plus Px vs. SH for diencephalic melatonin; P < 0.01, Ex plus Px vs. SH for plasma melatonin). As shown in Fig. 2, A, C, and G, the significant decreases in fGRP and fGRP-RPs expressions in SCN neurons after Ex plus Px were confirmed histologically by immunocytochemistry (P < 0.01, Ex plus Px vs. SH; Fig. 2G).

View larger version (24K): [in this window] [in a new window]   FIG. 1. Effects of Ex combined with Px (Ex+Px) on the expressions of fGRP precursor mRNA level (A) and mature fGRP and fGRP-RPs (B) in the diencephalon and melatonin concentrations in the diencephalon (C) and plasma (D). Each column and vertical line represent the mean ± SEM of determinations from five individual animals. **, P < 0.01; *, P < 0.05 vs. SH, by Student’s t test.

View larger version (46K): [in this window] [in a new window]   FIG. 2. Effect of Ex combined with Px and melatonin (25 mg/plate) administration to Ex plus Px (Ex+Px) bullfrogs (Ex+Px+M) on the expression of mature fGRP and fGRP-RPs (A, C, and E) in SCN neurons. Preincubation of the fGRP antiserum with a saturating concentration of synthetic fGRP, fGRP-RP-1, fGRP-RP-2, or fGRP-RP-3 was carried out as the control. Preincubation of the fGRP antiserum with synthetic fGRP is shown in B, D, or F. Preincubation of the fGRP antiserum with synthetic fGRP-RP-1, fGRP-RP-2, or fGRP-RP-3 also resulted in the disappearance of the reaction product (data not shown). Scale bars, 25 µm. Quantitative analyses of the expression of mature fGRP and fGRP-RPs in SCN neurons in the SH, Ex plus Px (Ex+Px), and Ex plus Px plus melatonin (Ex+Px+M) bullfrogs (G). The immunoreactivity of individual neurons was measured as a gray-scale value and expressed as the mean density per cell, which was obtained by subtracting background gray values. Three sections including the SCN of each animal were cut. The mean expression of mature fGRP and fGRP-RPs in SCN neurons was calculated and used as the value of each animal. Each column and vertical line represent the mean ± SEM of determinations from five individual animals. **, P < 0.01, SH vs. Ex plus Px; , P < 0.01, Ex plus Px vs. Ex plus Px plus melatonin, by Duncan’s multiple range test.

View larger version (20K): [in this window] [in a new window]   FIG. 3. Effect of melatonin administration on the expression of fGRP precursor mRNA in the diencephalon. Various doses of melatonin (low dose: 1 mg/plate; medium dose: 5 mg/plate, and high dose: 25 mg/plate) were administered to Ex plus Px bullfrogs by means of a SILASTIC brand plate for 1 wk (Ex plus Px plus melatonin; Ex+Px+M). Ex plus Px bullfrog (controls) were implanted with only SILASTIC brand adhesive. Each column and vertical line represent the mean ± SEM of determinations from five individual animals. **, P < 0.01; *, P < 0.05 vs. Ex plus Px, by Duncan’s multiple range test.

View larger version (15K): [in this window] [in a new window]   FIG. 4. Effect of photoperiodic manipulation on the expression of fGRP precursor mRNA in the diencephalon. Bullfrogs were exposed to either LD or SD photoperiods for 3 wk. Each column and vertical line represent the mean ± SEM of determinations from five different animals. *, P < 0.05 vs. LD, by Student’s t test.

View larger version (63K): [in this window] [in a new window]   FIG. 5. Nucleotide sequence and deduced amino acid sequence of bullfrog Mel1b cDNA containing a single open reading frame (GenBank accession no. AB302949). PCR primers used to amplify a partial cDNA for in vitro transcription are underlined. The asterisk indicates the stop codon.

View larger version (84K): [in this window] [in a new window]   FIG. 6. Expression of Mel1b mRNA in SCN neurons. Dual-label in situ hybridization using antisense RNA probes for Mel1b mRNA (A) and fGRP precursor mRNA (C) on the same section. In situ hybridization using sense RNA probes for Mel1b mRNA (B) and fGRP precursor mRNA (D) served as controls. Arrows in A and C indicate identical cells expressing Mel1b mRNA and fGRP precursor mRNA. Arrowheads in A and C indicate the cells expressing only Mel1b mRNA. Similar results were obtained in repeated experiments by using six different bullfrogs. Scale bars, 25 µm.

View larger version (29K): [in this window] [in a new window]   FIG. 7. Effects of melatonin manipulation by means of Ex plus Px (Ex+Px) and Ex plus Px plus melatonin (Ex+Px+M) on the circulating levels of GH (A) and PRL (B). Each column and vertical line represent the mean ± SEM of determinations from eight individual animals. **, P < 0.01, SH vs. Ex plus Px; , P < 0.01, Ex plus Px vs. Ex plus Px plus melatonin, by Duncan’s multiple range test. Effect of melatonin on the release of GH and PRL from cultured bullfrog pituitary cells after a 24-h incubation at 23 C with different concentrations (10–12 to 10–6 M) of melatonin (C and D). TRH (10–8 M) was used for the positive control. Each column and vertical line represent the mean ± SEM of determinations from six different wells. **, P < 0.01 vs. vehicle, by Duncan’s multiple range test.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
fGRP and fGRP-RPs (fGRP-RP-1, -RP-2, and -RP-3) are novel hypothalamic neuropeptides identified in the bullfrogs (6, 7, 8). Cell bodies and terminals containing fGRP and fGRP-RPs are localized in the SCN and ME, respectively (6, 7, 8). Among these neuropeptides, fGRP stimulates GH release and fGRP-RP-2 stimulates both GH and PRL release in bullfrogs (6, 8). Until now, no mechanism regulating the expression of fGRP and fGRP-RP-2 had been determined. We hypothesized that melatonin may be involved in the induction of fGRP and fGRP-RP-2, thus influencing GH and PRL release because fGRP and fGRP-RP-2 increase GH and PRL release (6, 8).

Our hypothesis was confirmed by a combination of experiments involving melatonin manipulation in bullfrogs. In the present study, Ex combined with Px and melatonin replacement were performed. A combination of Ex plus Px decreased the expressions of fGRP precursor mRNA and mature fGRP and fGRP-RPs in the diencephalon including the SCN and ME, concomitant with a decrease in endogenous melatonin in the diencephalon and plasma. In a further experiment, melatonin administration to Ex plus Px bullfrogs increased, in a dose-dependent fashion, fGRP precursor mRNA expression in the diencephalon. The stimulatory effect of melatonin on the expression of mature fGRP and fGRP-RPs in SCN neurons was further confirmed histologically by immunocytochemistry. Furthermore, fGRP precursor mRNA in the diencephalon increased under SD photoperiods. In bullfrogs, as in all vertebrates, the nocturnal secretion of melatonin is night-length-dependent (12). Thus, the increase in fGRP precursor mRNA expression under SD is likely to be due to the increase in the duration of endogenous melatonin secretion. Melatonin administration by means of implants is pharmacological because they produce elevated levels of melatonin throughout the 24-h period of the light-dark cycle. Endogenous melatonin is elevated only during the dark phase of the light-dark cycle. However, the observed effects of melatonin implants on fGRP precursor mRNA and mature fGRP and fGRP-RPs were in the same direction as those resulting from changes in endogenous melatonin. Taken together, we conclude that melatonin derived from the eyes and pineal gland acts as a potent inducing factor of fGRP and fGRP-RPs in bullfrogs. A similar effect of melatonin on the induction of gonadotropin-inhibitory hormone [GnIH (17)], an avian homologous peptide (SIKPSAYLPLRFamide) of fGRP has been reported in quail (14). Because not only fGRP and fGRP-RPs but also GnIH and GnIH-RPs (17, 26, 27, 28) possess a LPXRFamide (X = L or Q) motif at their C-termini, melatonin may induce generally the expression of these LPXRFamide peptides in vertebrates. Our recent study with the hamster confirms this hypothesis because melatonin also regulated the expression of mammalian homologous peptides having a C-terminal LPXRFamide motif, which were identified from the brain of a Siberian hamster, a photoperiodic mammal (Inoue, K., T. Ubuka, K. Ukena, L. Kriegsfeld, and K. Tsutsui, unpublished observation).

To begin to understand the mode of melatonin action on the induction of fGRP and fGRP-RPs, it is imperative to gather data on the distribution of melatonin receptor relative to SCN neurons expressing fGRP and fGRP-RPs. Recent molecular studies (29, 30) led to the identification of four amphibian melatonin receptor subtypes, Mel_1a, Mel_1b, Mel_1c(a), and Mel_1c(b), in Xenopus laevis, which comprise a distinct subfamily with the superfamily of G protein-coupled receptors. In this study, only Mel_1b cDNA was cloned from bullfrog diencephalon using degenerate primers, which were designed on the basis of the alignment of Xenopus laevis Mel_1a, Mel_1b, Mel_1c(a), and Mel_1c(b) mRNA sequences. Amino acid sequence of bullfrog Mel_1b showed the highest sequence similarity to Xenopus laevis Mel_1b (90.8%); however, bullfrog Mel_1b showed 54.7% identity with Xenopus laevis Mel_1a, 72.1% identity with Mel_1c(a), and 73.4% identity with Mel_1c(b). The cloned bullfrog Mel_1b cDNA was used for in situ hybridization of Mel_1b mRNA, providing evidence that at least Mel_1b is expressed in SCN neurons expressing fGRP and fGRP-RPs in bullfrogs. Although we need to investigate whether SCN neurons express other melatonin receptor subtypes in bullfrogs, it can be predicted that melatonin acts directly on SCN neurons expressing fGRP and fGRP-RPs through Mel_1b-mediated mechanisms to induce the expression of fGRP and fGRP-RPs. Nevertheless, we cannot preclude an indirect effect of melatonin on SCN neurons expressing fGRP and fGRP-RPs via synaptic connections because melatonin receptor was apparently also expressed in cells in the SCN, which do not contain fGRP and fGRP-RPs. There is also another possibility that either melatonin or Mel_1b may not be sufficient to induce the expression of fGRP and fGRP-RPs in some SCN neurons.

Previously we demonstrated that fGRP stimulates GH and fGRP-RP-2 stimulates both GH and PRL release in vitro and in vivo in the bullfrog (6, 8). We therefore hypothesized that melatonin may modulate circulating GH and PRL levels via the action of fGRP and fGRP-RP-2. This hypothesis was supported by the findings of the present in vivo study. A combination of Ex plus Px decreased plasma GH and PRL concentrations in bullfrogs. Conversely, melatonin administration to Ex plus Px bullfrogs increased these hormone levels. It is therefore considered that melatonin may be a key factor to regulate GH and PRL release in bullfrogs. To confirm the physiological relevance of melatonin regulation of the expression of fGRP and fGRP-RP-2, future study is needed to clarify whether plasma GH and PRL concentrations are also higher during SD than LD because fGRP precursor mRNA increased in the diencephalon under SD in the present study.

The findings of the present in vitro study ruled out the possibility that melatonin may act directly on the pituitary to influence GH and PRL release. Consequently, it is possible that melatonin acts directly on SCN neurons through its receptor to induce the expression of fGRP and fGRP-RP-2, thus stimulating GH and PRL release. To the best of our knowledge, this is the first report showing melatonin action on the regulation of GH and PRL release via hypothalamic neuropeptides in any vertebrate. This mechanism is supported by a previous avian finding suggesting that melatonin modulates rather central neural pathways involved in the control of GH secretion at the hypothalamic level than the sensitivity of the pituitary gland (31). On the other hand, there is a report that melatonin stimulates to increase GH release and decrease PRL release from cultured fish pituitary cells (32). Griffiths et al. (33) also reported that melatonin reduced GH release from rat pituitary cells. However, the present in vitro study did not find any direct action of melatonin on the pituitary to influence the release of GH and PRL in bullfrog. Such a discrepancy may be due to species-dependent variations.

In conclusion, melatonin stimulates the release of GH and PRL by a possible induction of the expression of fGRP and fGRP-RP-2 in bullfrogs. Melatonin may act on SCN neurons through its receptor to induce the expression of fGRP and fGRP-RP-2. Nevertheless, we cannot exclude the possibility that melatonin may increase the expression of some other neuropeptide(s), thus stimulating the release of GH and PRL.

	Acknowledgments

 
We thank Dr. Takayoshi Ubuka (University of California, Berkeley) for his valuable discussion and technical assistance.

	Footnotes

 
This work was supported in part by Grants 16086206 and 18107002 from the Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture, Japan (to K.T.) and a Research Fellowship from the Japan Society for the Promotion of Science (P06182 to V.S.C.).

The nucleotide sequence data of bullfrog Mel_1b has been submitted to the DNA Database of Japan (DDBJ), European Molecular Biology Laboratory (EMBL), and GenBank Nucleotide Sequence Databases under accession no. AB302949.

Disclosure Statement: The authors have nothing to disclose.

First Published Online December 6, 2007

Abbreviations: Ex, Orbital enucleation; fGRP, frog GH-releasing peptide; fGRP-RP, fGRP-related peptide; GnIH, gonadotropin-inhibitory hormone; LD, long day; ME, median eminence; Mel_1b, a melatonin receptor subtype; PRL, prolactin; Px, pinealectomy; SCN, suprachiasmatic nucleus; SD, short day; SH, sham-operated bullfrogs; SSC, saline sodium citrate.

Received October 18, 2007.

Accepted for publication November 28, 2007.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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