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Endocrinology, doi:10.1210/en.2007-1357
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Endocrinology Vol. 149, No. 5 2149-2158
Copyright © 2008 by The Endocrine Society

Interplay between the Endocannabinoid System and GnRH-I in the Forebrain of the Anuran Amphibian Rana esculenta

Rosaria Meccariello, Maria Fosca Franzoni, Rosanna Chianese, Erika Cottone, Donatella Scarpa, Daniela Donna, Gilda Cobellis, Alda Guastalla, Riccardo Pierantoni and Silvia Fasano

Dipartimento di Studi delle Istituzioni e dei Sistemi Territoriali (R.M.), Università di Napoli "Parthenope," 80133 Napoli, Italy; Dipartimento di Biologia Animale e dell’Uomo (M.F.F., E.C., D.D., A.G.), Laboratorio di Anatomia Comparata, Università degli Studi di Torino, 10123 Torino, Italy; and Dipartimento di Medicina Sperimentale (R.C., D.S., G.C., R.P., S.F.), Sez. "F. Bottazzi," II Università di Napoli, 80138 Napoli, Italy

Address all correspondence and requests for reprints to: Professor Riccardo Pierantoni, Seconda Università di Napoli, Dipartimento di Medicina Sperimentale, Sez. "F.Bottazzi," via Costantinopoli 16, 80138 Napoli, Italy. E-mail: riccardo.pierantoni{at}unina2.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The morphofunctional relationship between the endocannabinoid system and GnRH activity in the regulation of reproduction has poorly been investigated in vertebrates. Due to the anatomical features of lower vertebrate brain, in the present paper, we chose the frog Rana esculenta (anuran amphibian) as a suitable model to better investigate such aspects of the reproductive physiology. By using double-labeling immunofluorescence aided with a laser-scanning confocal microscope, we found a subpopulation of the frog hypothalamic GnRH neurons endowed with CB1 cannabinoid receptors. By means of semiquantitative RT-PCR assay, we have shown that, during the annual sexual cycle, GnRH-I mRNA (formerly known as mammalian GnRH) and CB1 mRNA have opposite expression profiles in the brain. In particular, this occurs in telencephalon and diencephalon, the areas mainly involved in GnRH release and control of the reproduction. Furthermore, we found that the endocannabinoid anandamide is able to inhibit GnRH-I mRNA synthesis; buserelin (a GnRH agonist), in turn, inhibits the synthesis of GnRH-I mRNA and induces an increase of CB1 transcription. Our observations point out the occurrence of a morphofunctional anatomical basis to explain a reciprocal relationship between the endocannabinoid system and GnRH neuronal activity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE MULTIPLE EFFECTS produced in humans and, more generally in mammals, by Cannabis sativa bioactive components are mediated by G protein-coupled specific cannabinoid (CB) receptors characterized in the mammalian central nervous system as CB1 (1, 2) and in peripheral tissues as CB2 (3). The presence of this signaling system was then reported in different nonmammalian vertebrates (4, 5, 6, 7, 8, 9), attracting the neurobiologist attention toward the cannabinoid system phylogeny. A number of evolutionary studies on CB1 sequence alignments performed in both vertebrate and invertebrates (7, 10, 11, 12) have indeed confirmed that CB1 receptors are present in the nervous system of the majority of vertebrates as well as in that of deuterostomian invertebrates. Thus, the presence of the cannabinoid system in animal groups that are so evolutionarily far from mammals could indicate endocannabinoids as modulators of basic physiological activities. Apart from evolutionary speculations, the brain of lower vertebrates has been recognized to possess morphological features to better study the relationships between different neurotransmitter-neuromodulator systems (13). In this respect, the investigation in animal models other than mammals gives the opportunity to analyze comparative and functional aspects of cannabinoid signaling system and better understand the potentiality of these molecules in human health.

The phytocannabinoid, {Delta}9-tetrahydrocannabinol, has been demonstrated to affect reproductive functions in both experimental animals and humans (see the review in Ref. 14). Because CB1 receptors were detected in Leydig cells of the mouse testis and the endocannabinoid anandamide (AEA) suppressed serum LH and testosterone levels in normal but not in CB1 knockout animals, Wenger et al. (15) postulated that CB1 receptors are responsible for the effects of exogenous cannabinoids on reproductive functions at local and central levels.

Recently Gammon et al. (16) reported that hypothalamic immortalized GnRH neurons are capable of synthesizing the two main endocannabinoids, AEA and 2-arachydonoilglycerol, and express CB1 receptors, suggesting that cannabinoids might influence the reproduction through a direct action on GnRH neurons also in vivo.

The amphibian brains are suitable models to study central aspects of reproduction for two main reasons: 1) their laminated structures are the bauplan or archetype (17) of those more elaborated of the higher vertebrates and 2) the annual reproductive cycle of amphibians is characterized in both sexes by the GnRH peptide accumulation in the postreproductive and the GnRH release in the prereproductive periods (18, 19, 20). In the frog, especially Rana esculenta and R. ridibunda, several GnRH molecular forms have been detected in the brain (primarily GnRH-I and GnRH-II) (18, 21, 22) pituitary and gonads, together with GnRH receptors (23, 24, 25, 26). Furthermore, an important involvement of CB1 receptors in the reproductive functions has recently been reported in R. esculenta by Meccariello et al. (27) at both central nervous system and gonadal levels, and fluctuations of CB1 mRNA in different dissected brain areas and testis during the year have also been shown.

In the present paper, neuroanatomical and functional relationships between CB1 and GnRH have been described in the forebrain of R. esculenta (anuran amphibians) by using double immunofluorescence. Opposite fluctuations of CB1 and GnRH-I mRNA have been detected by RT-PCR during the annual sexual cycle. Lastly, cross talk between cannabinergic and GnRH-I neurosecretory system has been demonstrated by in vitro incubations of brains with AEA and a GnRH-I long-acting agonist (GnRHa; buserelin).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
The experiments were performed under the guidelines established by the Italian law for animal welfare.

Animal and tissue collection
Male frogs, R. esculenta, were collected monthly in the neighborhood of Naples (Italy). Animals were anesthetized with MS222 (Sigma-Aldrich, Milan, Italy) and killed by decapitation immediately after capture to minimize stress.

For the immunofluorescence (IFL) procedure, six brains/month, removed from the brain case, were fixed overnight (O/N) in Zamboni fixative or 4% paraformaldehyde, embedded in Killik medium (Bio-Optica, Milan, Italy), and frozen. Cryostat coronal, sagittal, and horizontal sections (12 µm thick) were mounted on 3-aminopropyl-triethoxysilan-coated slides and stored at 4 C until use.

Ten whole brains, testis, ovary, spinal cord, spleen, and pituitary/month were removed and appropriately stored at –80 C for mRNA analysis; 10 brains from July, September, October, November, December, March, and April frog were dissected; prosencephalons were cut in telencephalic and diecephalic areas and stored at –80 C until used for mRNA analysis. Such months are representative of a complete reproductive cycle. In fact, during the annual sexual cycle, GnRH peptide accumulates in the brain in the postreproductive period (May–July), decreases during the resumption period (September–October), and is released during the winter stasis (November–February) and in early spring when reproduction occurs (18, 26).

One frog brain/month and rat brains were used for Western blot analysis. Two Sprague Dawley male rats (Charles River Laboratories, Lecco, Italy) were killed. Brains were removed and stored at –80 C until used for protein extraction and Western blot analysis.

Chemicals
GnRH-I agonist (GnRHa; buserelin, a gift from Dr. J. Sandow, Hoechst, Frankfurt, Germany); GnRH-I antagonist (GnRH-IAnt; D-pGlu1, D-Phe2, D-Trp3, 6-GnRH; Peninsula Laboratories, Belmont, CA); AEA (Sigma-Aldrich); SR141716A [Rimonabant, a selective CB1 antagonist (28); Sanofi Research, Montpellier, France].

Single IFL
Serial brain sections were alternatively incubated O/N at room temperature (RT) with a polyclonal antirat CB1 receptor (C-terminus, 1:500 dilution), raised in rabbit [kindly provided by Prof. K. Mackie, Indiana University, Indianapolis, IN (29)] or a monoclonal anti-GnRH-I (1:2000 dilution; Sternberg Monoclonals Inc., Baltimore, MD), raised in mouse, primary antibodies. Sections were then processed with an antirabbit IgG antiserum conjugated to cyanin 3 (1:800; Jackson ImmunoResearch Laboratories, West Grove, PA) or a biotinylated antimouse IgG secondary antibody (1:200; Vector Laboratories, Burlingame, CA) and fluorescent avidin D (1:400; Vector Laboratories). Brain sections were then rinsed in PBS, mounted in 1,4-diazabicyclo[2.2.2]octane (Dabco; Sigma), and observed with a fluorescence microscope (Eclipse 80i; Olympus, New Hyde Park, NY).

Standard double IFL
Brain sections were incubated O/N, at RT, in a mixture of rabbit polyclonal anti-CB1 (C terminus; 1:500 dilution) and mouse monoclonal anti-GnRH-I (1:2000 dilution) antisera. After PBS washes, sections were incubated (1 h, at RT) with a biotinylated antimouse IgG secondary antibody (1:200; Vector Laboratories) and then for 1 h antisera, at RT with fluorescent avidin D (1:400; Vector Laboratories) and an antirabbit IgG antiserum conjugated to cyanin 3 (1:800; Jackson ImmunoResearch Laboratories).

Brain sections were then rinsed in PBS, mounted in Dabco (Sigma), and observed with a fluorescence microscope (Olympus Eclipse 80i) or analyzed with an Olympus Fluoview laser-scanning confocal microscope system (LSCM; Olympus).

The specificities of both the anti-CB1 and anti-GnRH antisera were assessed by incubating sections with the antibodies preadsorbed O/N, at RT, with the corresponding immunizing proteins (5 µg/ml). The specificity of the method was evaluated by omitting primary antibodies. Both procedures resulted in the complete absence of tissue immunostainings.

To evaluate the percentage of double-immunolabeled cells in respect to the total GnRH immunopositive septal and preoptic neurons, 25 brain coronal and horizontal sections, belonging to five frogs collected through the year and treated for double IFL, were analyzed with a LSCM. The number of GnRH-I+ and GnRH-I+/CB1+ cells was then determined.

Protein extraction and Western blot analysis
Frog and rat brains were homogenized in ice-cold lysis buffer [10 mM HEPES (pH 7.9), 420 mM NaCl, 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EGTA (pH 8.1), 0.5 mM spermidine, 12% glycerol, 0.5 mM dithiothreitol; 5 ml per 1 g tissue] containing protease inhibitors (0.5 mM phenylmethylsulfonylfluoride, 4 µg/ml leupeptin, 4 µg/ml chymostatin, 4 µg/ml pepstatin A, and 5 µg/ml N-tosyl-L-phenylalanine chloromethyl ketone). Samples were then centrifuged at 10,000 x g at 4 C for 30 min to obtain a clarified lysate. Total protein amounts were quantified using the Lowry method (30).

Sixty micrograms of total proteins, from both frog and rat brains, were resolved by 8% SDS-PAGE and blotted to polyvinyl difluoride membranes (Amersham Pharmacia Biotech, Little Chalfont, UK) for 2.5 h at 280 mA at 4 C. Membranes were then rinsed in PBS (pH 7.6) (20 mM NaH2PO4, 80 mM Na HPO4, 100 mM NaCl) and treated for 2 h with blocking solution [5% nonfat powdered milk in 10 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.25% Tween 20]. Hybridization was performed using anti-C-terminal CB1 antibody (29) diluted 1:1000 in Tris-buffered saline (pH 8.0) and 4% nonfat powdered milk O/N at 4 C. The membranes were washed 3 x 15 min in 10 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.25% Tween 20 and incubated 1 h at RT with a horseradish-peroxidase-conjugated antirabbit IgG antibody (Dako Corp., Copenhagen, Denmark). After an additional series of washes, immunocomplexes were detected using the ECL Western blotting detection system (Amersham Pharmacia Biotech) and Hyperfilm ECL autoradiography film (Amersham Pharmacia Biotech). Filters were then stripped in stripping buffer [62.5 mM Tris (pH 6.8), 2% sodium dodecyl sulfate, 100 mM β-mercaptoethanol] at 60 C for 30 min and reprobed with primary antibody preadsorbed O/N with a large excess (10–6 M) of the respective antigen to check antibody specificity.

Total RNA extraction and cDNA preparation
Total RNA was extracted from frog tissues using Trizol reagent (Invitrogen Life Technologies, Paisley, UK) and was treated for 30 min at 37 C with DNase I (10 U/sample) (Amersham Pharmacia Biotech) to eliminate any contaminations of genomic DNA; total RNA purity and integrity were determined by spectrophotometry at 260/280 nm and electrophoresis. Thereafter, total RNA was reverse transcribed to prepare cDNA. The reverse transcription was carried out using 5 µg total RNA, 0.5 µg oligo-dT(18), 0.5 mM deoxynucleotide triphosphate mix, 5 mM dithiothreitol, 1x first-strand buffer (Invitrogen Life Technologies), 40 U RNase Out (Invitrogen Life Technologies), and 200 U SuperScript-III Rnase H– reverse transcriptase (Invitrogen Life Technologies) in a final volume of 20 µl, following the manufacturer’s instructions.

Expression analysis of GnRH-I and CB1 in frog tissues
cDNA was diluted (1:5) and used for PCR analysis. A frog cDNA fragment of 180 bp was obtained using primers designed upon Xenopus laevis GnRH-I precursor cDNA [sense primer: 5'-TGCCCAGCACTGGTCCT ATG-3'; antisense primer: 5'-TCCTTCCAG CCAGTTCATCA-3'; GenBank accession no. L28040 (20, 31)]. PCR analyses were conducted using 1 µl of diluted cDNA in PCR mix [0.2 mM deoxynucleotide triphosphate, 1x PCR buffer, 1.5 mM MgCl2, 10 pmol forward and reverse specific primers, 1.25 U Taq polymerase (Invitrogen Life Technologies)], using a Thermocycler apparatus (BioRad, Hercules, CA) programmed as follows: 94 C for 1 min, 55 C for 1 min, and 73 C for 1 min, 35 cycles; lastly 72 C for 7 min, one cycle. Amplification products were subcloned in pGEM-T Easy Vector (Promega Corp., Madison, WI). DH5{alpha} high-efficiency competent cells were transformed and recombinant colonies were identified by the blue/white color screening. Plasmidic DNA was extracted by using NucleoBond plasmid extraction kit (Macherey Nagel, Düren, Germany), and insert size was controlled by restriction analysis with EcoRI (Fermentas, St. Leon-Rot, Germany). DNA was then sequenced on both strands by Primm Sequence Service (Primm srl, Naples, Italy). On the basis of the nucleotide sequence, R. esculenta GnRH-I precursor amino acidic sequence was deduced. Nucleotide and the deduced amino acidic sequence of R. esculenta GnRH-I precursor were aligned with other amphibian GnRH-I precursor by LAlign and Clustal W multiple alignments.

The analysis of CB1 receptor mRNA was conducted using primers specific for R. esculenta brain CB1 receptor mRNA (sense primer: 5'-ATTGGGGTAACCAGTGTTCT-3'; antisense primer: 5'-ACCAGGGTCTTTGCTAACCT-3'; amplicon predicted size: 201 bp; GenBank accession no. AM113546) in PCR mix. PCR conditions were: 94 C for 5 min, one cycle; 94 C for 30 sec, 56 C for 30 sec, 72 C for 30 sec, 30 cycles; lastly 72 C for 7 min, one cycle. To normalize the signals, amplification of R. esculenta ribosomal protein P1 mRNA (fp1) was carried out (sense primer: 5'-TACGAGCGTCCATCACACAC-3'; antisense primer: 5'-AGACCAAAGCCCATGTCATC-3'; amplicon predicted size: 356 bp; GenBank accession no. AJ298875). PCR conditions were: 94 C for 5 min, 1 cycle; 94 C for 30 sec, 56 C for 30 sec, 72 C for 30 sec, 22 cycles; lastly 72 C for 7 min, one cycle. Possible contaminations among samples were evaluated using, as negative controls, samples prepared without cDNA.

Finally, 25 µl of PCR amplification mixture were analyzed by electrophoresis on 1.2% agarose gel in 1x Tris-borate buffer and stained with 0.5 µg/ml ethidium bromide.

Treatments with AEA
Thirty-five male frogs were collected in May, anesthetized, and killed by decapitation immediately after capture to minimize stress. Brains were quickly removed, and diencephalons were isolated and rinsed in Krebs Ringer buffer for amphibians (KRB). Five diencephalons were immediately stored at –80 C and used for mRNA extraction as fresh controls. Five diencephalons were incubated in KRB for 1 h. Ten diencephalons were incubated in KRB/AEA at 10–6 M and KRB/AEA at 10–9 M for 1 h (n = 5 diencephalons/time point); 10 diencephalons, previously treated with KRB/SR141716A at 10–5 or 10–8 M for 30 min (n = 5 diencephalons/treatment), were incubated in KRB/AEA at 10–6 M and SR141716A at 10–5 M or KRB/AEA at 10–9 M and SR141716A at 10–8 M for 1 h. Lastly, five diencephalons wee incubated for 1 h with SR141716A at 10–8 M. After the incubation, tissues were stored at –80 C until used for RNA extraction. Anandamide doses were chosen on the basis of dose response experiments described in rat (32).

Treatments with GnRHa
Thirty male frogs were collected in September, anesthetized, and killed by decapitation immediately after capture to minimize stress. Brains were quickly removed and incubated in KRB or GnRHa 10–6 M in KRB for 1 h (n = 10 brains/treatment). Ten brains, previously incubated for 30 min in KRB containing GnRH-IAnt at 10–5 M, were then incubated in KRB containing GnRHa 10–6 M and GnRH-IAnt 10–5 M for 1 h. After the incubation, brains were dissected and diencephalic areas were stored at –80 C until used for RNA extraction. GnRHa doses have been chosen on the basis of previously published experiments (33).

Statistics
Analysis of mRNA levels was carried out by GELDOC 1.00-UV fluorescent gel documentation system (Bio-Rad). The relative amounts of the signals are expressed as fold increase of the ratio GnRH-I (CB1) mRNA/fp1 mRNA ± SEM. ANOVA followed by Duncan’s test for multigroup comparison was carried out to assess the significance of differences. Observation range was four to six.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
A subset of GnRH-I hypothalamic neurons possess CB1 receptors
To assess the antirat CB1 C terminus antibody specificity, Western blot analysis was conducted on frog brain. As expected (29), a band of high molecular mass (~170 kDa) is observed in frog and also rat brain used as positive control (Fig. 1AGo). Signal strongly attenuates or completely disappears in filters stripped and then incubated with the antiserum previously treated with an excess amount (10–6 M) of the cognate peptide (Fig. 1BGo). Noteworthy is the fact that alignments of the last 73 C terminus amino acidic residues of the frog and rat CB1 protein (GenBank accession no. frog CB1 AM113546; rat CB1 U40395), conducted by LAlign tool, available at Biology Workbench (http://workbench/sdsc.edu/), reveal an identity of 80.8%.


Figure 1
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  		FIG. 1. A, Western blot analysis carried out on rat (lane 1) and frog (lane 2) brains by using an antirat CB1 C terminus antiserum. A high molecular mass band (~170 kDa) is observed in rat and frog brains. B, Signals completely disappear or strongly attenuate using the antiserum previously treated with a large excess (10–6 M) of the corresponding peptide.

 
By using single- and double-IFL techniques, the distributions of CB1 receptor- and GnRH-I-immunoreactivities (IRs) were evaluated in the R. esculenta ventral prosencephalon, namely in the septum of the telencephalon, preoptic area, and infundibular hypothalamus.

The distribution of the immunoreactivities is summarized in a midsagittal view of the frog schematic brain, at the top of Fig. 2Go.


Figure 2
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  		FIG. 2. Top of the figure, Midsagittal view of the frog schematic brain [modified from Ten Donkelaar (47 )]. The colored areas indicate ventral prosencephalic regions, respectively, containing CB1+ (red), GnRH-I+ (green), and CB1+/GnRH-I+ (yellow) cell bodies and nerve fibers. Hy, Hypothalamus; OB, olfactory bulb; OC, optic chiasma; OT, optic tectum; P, pituitary; Pa, pallium; spt, septum. The four vertical and the two horizontal lines indicate the levels, respectively, of the coronal and horizontal sections shown in the photomicrographs below. A, Preoptic area. The periventricular bilateral cluster of CB1 immunoreactive neurons in subependymal position. IIIv, Third ventricle. Horizontal section. B, Consecutive section of A processed for specificity control does not show any immunolabeling. Horizontal section. C, Infundibular hypothalamus. CB1 immunolabeled neurons bordering the infundibular wall. Some neurons show intraventricular processes (arrow). Coronal section. Inf, Infundibulum. D, GnRH-I immunopositive neurons and fibers in the septum of the telencephalon. c, Capillary. Horizontal section. E, Consecutive section of D processed for specificity control does not show any immunolabeling. Horizontal section. F, Cluster of GnRH-I immunopositive neurons in the septum of the telencephalon. Horizontal section. G, Standard double IFL. Four GnRH-I immunoreactive neurons in the ventral septum, three of which (H) are also immunopositive for the anti-CB1 antisera. The GnRH-I immunopositive neuron at the top left (arrow) does not show any CB1-LI-IR. I, Merging of G and H. LSCM. Coronal sections. J, Standard double IFL. GnRH-I fibers and varicosities (green fluorescence) among CB1 neurons (red fluorescence) in the preoptic area. LSCM. Horizontal section. K, Standard double IFL. A septal pyriform neuron colabeled with anti-GnRH-I (green) and anti-CB1 (red) antisera. LSCM. Coronal section. L, Standard double IFL. A detail of the median eminence external zone in which a number of nerve fibers and terminals are colabeled (yellow) with anti-GnRH-I (green) and anti-CB1 (red) antisera. LSCM. Coronal section. Scale bars, 30 µm (A and B); 40 µm (C, D, E, F, J, and L); 20 µm (G, H, and I); 10 µm (K).

 
In anurans, the septum is well developed and forms the ventromedial hemispheric wall bounded dorsally by the medial pallium. In the frog we observed a dense network of CB1-like immunopositive nerve fibers and terminals throughout the pallium and scattered CB1 immunostained cell bodies in the septum. Proceeding caudally toward the rostral preoptic area, a major bilateral cluster of CB1 medium to large (15–20 µm in diameter) pyriform and bipolar perikarya, organized in subependymal rows, together with a dense network of nerve fibers and processes ventrolaterally directed, was encountered (Fig. 2Go, A and J). Tissue sections processed for specificity control procedures did not show any labeling (Fig. 2BGo).

In the infundibular hypothalamus, the CB1 immunostained cell bodies (10–15 µm in diameter), frequently showing intraventricular processes, were distributed through the ventricular wall (Fig. 2CGo), sending their varicose axons ventrally toward the hypothalamic floor and almost projecting to the median eminence of the neurohypophysis.

The amphibian prosencephalic GnRH-I-like (LI)-immunoreactive system generally includes a main group of neurons distributed in the medial and ventral septum of the telencephalon. In the frog, the cell bodies were generally pyriform or bipolar in shape, embedded in a dense network of processes and varicose axons (Fig. 2Go, D, F, and G). A minor group of cells was observed also in the rostral preoptic area. A GnRH-immunopositive fiber tract was seen to run in the ventral hypothalamus directed to the median eminence. No immunopositivities were observed in sections treated for negative controls (Fig. 2EGo).

To investigate morphofunctional relationships between CB1-LI- and GnRH-I-LI-IRs through the septum and preoptic area/hypothalamus, brain sections were incubated with a mixture of the anti-CB1 and anti-GnRH-I primary antibodies, following the standard double IFL procedure.

In both the ventromedial septum of the telencephalon and the rostral preoptic area, a number of CB1-containing cell bodies and terminals together with GnRH-I immunostained nerve cells and fibers were codistributed. Neurons colabeled with both the anti-GnRH-I and the anti CB1 antisera were seen in the septal region and preoptic area (Fig. 2Go, G, H, I, and K), and they roughly correspond to 20% of the GnRH-I immunopositive neurons.

GnRH-positive nerve fibers and varicosities tightly close to CB1-LI-immunopositive neurons (Fig. 2JGo) were also observed.

In the median eminence of the neurohypophysis, fibers and terminals containing CB1-LI-IR were generally more ventral in respect to those containing GnRH-I, although the two immunolabelings were simultaneously shown by a number of them (Fig. 2LGo).

Cloning of R. esculenta GnRH-I and expression analysis in frog tissues
By means of RT-PCR techniques, we were able to clone a R. esculenta GnRH-I precursor cDNA fragment that is 180 bp long and codifies for the decapeptide GnRH-I, a carboxyl-terminal amidation and proteolytical processing site (Gly-Lys-Arg) and a GnRH-associated peptide of 46 amino acidic residues (Fig. 3AGo).


Figure 3
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  		FIG. 3. A, Nucleotide sequence and deduced amino acidic sequence of the partial cDNA encoding the frog GnRH-I precursor. Bold characters indicate GnRH-I; italic characters indicate the conserved processing site; Xenopus laevis primers used for the amplification are underlined. B, Alignments of known anuran amphibian GnRH-I precursor. Fully conserved amino acidic residues are in dark gray boxes; identical amino acidic residues are in black boxes; similar amino acidic residues are in light gray. The percentage identity to R. esculenta precursor is indicated. C, GnRH-I and fp1 mRNA expression in frog tissues. 1, Pituitary; 2, whole brain; 3, spinal cord; 4, testis; 5, ovary; 6, spleen; –, negative control. D, Normalization of the signals observed by RT-PCR. Data are representative of at least three separate experiments and are expressed as fold increase of the minimal GnRH-I to fp1 mRNA ratio observed ± SEM. Asterisk indicates statistically significant differences (P < 0.01).

 
Nucleotide sequence of GnRH-I cDNA was compared with those of other amphibians [the anurans, Xenopus laevis, R. catesbeiana, and R. dybowskii (GenBank accession no. L28040, AF188754, and AF139911) revealing high nucleotide identities (70.5% vs. Xenopus laevis; 95.6% vs. R. catesbeiana; 92.2% vs. R. dybowski)]. Alignments of the amino acidic sequences of R. esculenta GnRH-I precursor and other amphibian GnRH-I precursor are depicted in Fig. 3BGo.

A preliminary expression analyses of GnRH-I mRNA was conducted by semiquantitative RT-PCR using cDNA from frog pituitary, brain, spinal cord, testis, ovary, and spleen (Fig. 3CGo). An amplification product of 180 bp was obtained from pituitary, brain and gonads but not from the spinal cord and spleen, used as negative controls. A band of 356 bp was observed for fp1, a housekeeping gene, in all the tissues assayed and used to normalize GnRH-I mRNA expression levels. As expected, GnRH-I mRNA has a higher expression in the brain (Fig. 3DGo; P < 0.01).

CB1 and GnRH-I mRNA expression analysis during the annual sexual cycle
To analyze GnRH-I mRNA expression, we collected brain samples during the annual sexual cycle, from September until July and processed them for semiquantitative RT-PCR (Fig. 4AGo). Then we compared GnRH-I mRNA expression profile with the CB1 mRNA expression profile obtained in the same brain preparations and recently published (27).


Figure 4
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  		FIG. 4. A, GnRH-I mRNA expression in frog brain during the annual sexual cycle (S, September; O, October, etc.). B, Normalization of the signals observed by RT-PCR. Data are representative of at least three separate experiments and are expressed as fold increase of the minimal GnRH-1 to fp1 mRNA ratio observed ± SEM. (a vs. b, P < 0.05; P < 0.01 in all the other cases). Same letters indicate no statistically significant differences. The line on the top represents GnRH-I peptide content observed in brain [data from Fasano et al. (18 ) and Di Matteo et al. (26 )], Gray dotted line, GnRH-I low level; black line, GnRH-I release; dotted line, GnRH-I accumulation. C, CB1 mRNA expression in frog brain during the annual sexual cycle [data from Meccariello et al. (27 ); P < 0.01 at least]. Same letters indicate no statistically significant differences.

 
During the annual sexual cycle, GnRH-I mRNA fluctuates in the whole brain (Fig. 4BGo). Expression of GnRH-I mRNA gradually increases from September until November (September vs. October–November P < 0.01), remains constant in December and February, and reaches the lowest expression level in March (P < 0.01). Afterward, GnRH-I mRNA gradually increases in April–May and peaks in June–July (P < 0.01). The expression profile of GnRH-I mRNA, here described, is the opposite in respect to CB1 receptor mRNA expression profile (Fig. 4CGo) previously reported (27). In particular, CB1 receptor mRNA minimal expression levels in November and June correspond to GnRH-I mRNA expression peak.

Focusing our attention on reproductive functions, we analyzed CB1 receptor and GnRH-I mRNA expression in prosencephalon, the brain region mainly involved in the control of reproductive functions. We chose months representative of the annual reproductive cycle and we carried out an expression analysis on isolated telencephalons and diencephalons (Fig. 5AGo). In both prosencephalic areas, CB1 receptor mRNA expression seems the opposite of that of GnRH-I. In fact, in telencephalon, CB1 receptor mRNA expression peaks in September and March (P < 0.01), whereas GnRH-I mRNA expression peaks in July (June vs. July P < 0.01), November, and April (July and April vs. November P < 0,01) and has minimal values in October, December, and March (P < 0.01) (Fig. 5BGo).


Figure 5
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  		FIG. 5. A, GnRH-1 and CB1 mRNA expression in frog telencephalons (T) and diencephalons (D) during the annual sexual cycle. Normalization of the signals observed by RT-PCR in the telencephalons (B) and in the diencephalons (C) are shown. Data are representative of at least three separate experiments and are expressed as fold increase of the minimal GnRH-I to fp1 (CB1 to fp1) mRNA ratio observed ± SEM. (P < 0.05 at least). Same letters indicate no statistically significant differences.

 
In the diencephalon, CB1 receptor mRNA expression has a spike in December (P < 0.01); significantly higher levels are also detected in November and April (P < 0.01). On the other hand, GnRH-I mRNA expression slightly increases from June to July (P < 0,01), remaining constant until October, and reaching a minimal value just in December (P < 0.01), when CB1 receptor mRNA expression peak occurs.

Treatments with AEA
To assess a possible relationship between CB1 receptor and GnRH-I mRNA expression, diencephalons of frogs collected in May, just prior the GnRH-I mRNA summer spike, were incubated with AEA. After 1 h of incubation with KRB, no statistically significant difference in GnRH-I mRNA basal expression level, evaluated as GnRH-I mRNA expression in untreated diencephalons, was observed. Two doses of AEA, 10–6 and 10–9 M, were tested; low dose of AEA (10–9 M) in vitro is reported to decrease GnRH peptide release from rat medial basal hypothalamus (32). In our experiments, after 1 h incubation, both AEA (10–6 and 10–9 M) decrease GnRH-I mRNA expression as compared with control group (P < 0.01); this effect is counteracted by SR161716A, a specific CB1 receptor antagonists (P < 0.01 vs. AEA treatment groups) (Fig. 6Go). Surprisingly, SR141716A (10–8 M) in combination with AEA (10–9 M) induces GnRH-I mRNA expression above control levels (P < 0.01).


Figure 6
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  		FIG. 6. Effects of AEA (10–6 and 10–9 M) alone or in combination with SR141716A (10–5 and 10–8 M) on GnRH-I mRNA expression in diencephalons (n = 5) collected from May animals. –, Negative control; C0, untreated diencephalons of May frogs (fresh control); C, diencephalons incubated with KRB for 1 h (control group). The signals observed by RT-PCR were normalized against the housekeeping gene fp1 mRNA expression. Data are representative of at least three separate experiments and are expressed as fold increase of the minimal GnRH-I to fp1 mRNA ratio observed ± SEM (P <0.01). Same letters indicate no statistically significant differences.

 
Possible endogenous cannabinoid effect during the treatment was assayed by incubations with SR141716A (10–8 M) alone. Without any AEA administration, GnRH-I mRNA synthesis in SR141716A (10–8 M) treatment is at basal levels (Fig. 7Go).


Figure 7
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  		FIG. 7. Effects of SR141716A 10–8 M on GnRH-I mRNA expression in diencephalons (n = 5) collected from May animals. –, Negative control; C0, untreated diencephalons of May frogs (fresh control); C, diencephalons incubated with KRB for 1 h (control group). The signals observed by RT-PCR were normalized against the housekeeping gene fp1 mRNA expression. Data are representative of at least three separate experiments and are expressed as fold increase of the minimal GnRH-I to fp1 mRNA ratio observed ± SEM.

 
Treatments with GnRHa
In September, when low levels of GnRH peptide are detected in frog brain (21, 26), we assayed the possibility of an inverse relationship with CB1 mRNA expression, incubating whole brains with GnRHa (10–6 M).

After 1 h incubation with GnRHa, CB1 mRNA expression increases as compared with control group (P < 0.01 vs. control group); by contrast, this treatment strongly decreases GnRH-I mRNA expression (P < 0.01 vs. control group). These effects are counteracted by GnRHAnt (P < 0.01 vs. GnRHa treatment) (Fig. 8Go).


Figure 8
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  		FIG. 8. A, Effects of buserelin (GnRHa, 10–6 M) on CB1 and GnRH-I mRNA expression in diencephalons (n = 5) collected from September animals. B, The signals observed by RT-PCR were normalized against the housekeeping gene fp1 mRNA expression. Data are representative of at least three separate experiments and are expressed as fold increase of the minimal GnRH-I to fp1 mRNA ratio observed ± SEM (P < 0.01). C, Control group; G, GnRHa-treated group; G+A, group treated with GnRHa (10–6 M) and GnRH-IAnt (10–5 M). Asterisk and letter indicate statistically significant differences.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In recent years, the involvement of the endocannabinoid system in the control of reproductive functions has been postulated in mammals of both sexes (see the reviews in Refs. 34 and 35). Recently a CB1 receptor has been cloned in R. esculenta (36), and its involvement in the control of reproductive functions in males emerged (27, 37). In fact, CB1 activation has been suggested to inhibit sperm motility (37). Although in the frog brain fluctuations of CB1 receptor mRNA have been reported during the annual sexual cycle (27), a direct involvement of the cannabinergic system in the control of reproductive functions at central level is still lacking. Furthermore, although it is known that AEA decreases GnRH release in rats (32) and that immortalized GnRH neurons are capable to both synthesize endocannabinoids and express CB1 receptors (16), as far as we are aware, reports investigating the functional anatomy linking endocannabinoids and GnRH activity are still absent. We took advantage of a submammalian vertebrate species to clarify this point.

In the present study, by using double-labeling IFL technique, we looked for morphofunctional relationships among CB1-LI- and GnRH-I-LI-IRs in the septum, preoptic area, and hypothalamus, brain areas controlling reproduction in all vertebrates.

A primary antibody directed against the rat CB1 receptor C terminus was used. By comparing the amino acidic identity between the rat epitope and the frog CB1 C terminus amino acidic residues, a high amino acidic identity has been evidenced. Furthermore, Western blot analysis was performed to verify the anti-CB1 antibody specificity. A high-molecular-weight band in both frog and rat brain, used as positive control, is observed. As reported by Wager-Miller et al. (29), in rat such antibody specifically recognizes a high molecular weight protein accounted as a dimeric form of the receptor.

Our double IFL results show a close contiguity of the GnRH-I and CB1 signaling systems. Indeed, we found the presence of CB1 receptors in a subpopulation of the septal and preoptic GnRH-I neurons. They roughly correspond to a 20% of the GnRH-I immunopositive neurons, which seemingly project their axons to the vascular zone of the median eminence.

To better assess such morphofunctional data, comparison between GnRH-I precursor expression and CB1 receptor has been carried out during the annual sexual cycle of R. esculenta. Besides mammals, in which GnRH secreting neurons are quite scattered in the brain and the GnRH peptide pulse is quite fast, in this experimental model, as in several submammalian vertebrates (38), GnRH peptide slowly accumulates in the brain during the annual sexual cycle and is slowly released during the winter stasis (18, 26) to sustain the gonadotropin discharge responsible for the beginning of a new reproductive wave (39). In this paper, by molecular cloning we obtained a partial GnRH-I precursor cDNA. Expression analysis, carried out in a set of frog tissues, confirms GnRH-I mRNA presence in brain and in gonads (23) and the absence of the transcript in the spinal cord (26). Interestingly, the detection of a scanty GnRH-I mRNA expression in pituitary is also consistent with data reported in fish and mammals (40, 41). The expression of GnRH-I precursor mRNA in the brain is modulated in a stage-specific manner during the annual reproductive cycle of the frog and matches the protein profile previously described in this species (18, 26). In fact, the GnRH-I mRNA increase observed between March and April (reproductive period) and its higher expression levels detected in May–July (postreproductive periods) are related to GnRH-I peptide accumulation in the brain; then when GnRH-I peptide decreases in the brain (September–November; resumption) and it is finally released (November–March; winter stasis) to sustain gonadotropin discharge, GnRH-I mRNA remains quite constant (November–February) and reaches a minimal value in March. Interestingly, GnRH-I mRNA expression during the annual reproductive cycle is the opposite as compared with the expression profile of CB1 receptor mRNA previously analyzed in this experimental model (27). The present paper clearly demonstrates that this kind of expression profile also persists during the annual reproductive cycle in the telencephalon and diencephalon, key areas in the control of reproductive function and release of GnRH-I peptide.

Recently the involvement of the endocannabinoids in the control of reproductive function has been suggested. Besides the local effect on sperm cells motility and acrosome reaction and the activity in the control of embryo implantation (42, 43, 44), negative effect on neuroendocrine regulation of pituitary hormone secretion has been reported (45). Whether endocannabinoids inhibit GnRH release into the hypothalamic-pituitary portal vessels through a direct effect on GnRH secreting neurons or indirectly through the regulation of other neurotransmitters is still unclear. Immortalized GT1 cells are able to produce endocannabinoids and possess CB1 receptors (16); by contrast, GnRH-secreting neurons do not present a significant amount of CB1 receptors but are able to secrete endocannabinoids and are closely located to a neuronal population that express CB1 (16). Treatments of frog diencephalons, carried out in this study, clearly demonstrate that in this experimental model AEA is able to inhibit GnRH-I mRNA expression; this effect is quite strong after 1 h incubation using low doses of AEA (10–9 M). Also, in female rats, the same AEA dose decreases GnRH release from medial basal hypothalamus (32). It is interesting to note that SR141716A at 10–8 M, in combination with AEA, induces GnRH-I mRNA synthesis above control levels. We have no explanation for this phenomenon. On the other hand, both AEA and SR141716A are reported to affect the activity of vanilloid receptor (TRPV1) (46). Furthermore, we also checked for a putative endogenous endocannabinoid activity performing incubations with SR141716A (10–8 M) alone. This treatment does not influence GnRH-I mRNA expression. Of course, further experiments are necessary to clarify the above described SR141716A effects.

To assess how the cannabinergic system may affect GnRH-I circuitry, whole brains have been incubated with a specific GnRH-I agonist, buserelin, and effects on both diencephalic CB1 receptor mRNA expression level and GnRH-I mRNA synthesis have been evaluated. Interestingly, the cross talk between cannabinergic and GnRH neurosecretory system once again emerged: in fact, buserelin inhibits the synthesis of GnRH-I mRNA and induces the transcription of CB1 receptor mRNA, a negative modulator of GnRH neuron activity. Therefore, GnRH-I peptide levels may regulate their own concentrations through CB1 activity. In this respect, feedback control over GnRH function may be exerted trough the activation of the endocannabinoid system, with a consequent up-regulation of the CB1 receptors.

Taken together, our results indicate an anatomical functionality between endogenous cannabinoids and GnRH-I mediated by CB1 and suggest that the control exerted by this signaling system on the GnRH neurons, projecting to the median eminence, could be of significance in term of fine-tuning of the gonadotropic pituitary functions.


   Acknowledgments
 
The authors thank Professor Ken Mackie (University of Indiana, Indianapolis, IN) who kindly provided the anti-CB1 antiserum.


   Footnotes
 
This work was supported by the Italian Ministry of the University (Grant COFIN 2005/2005052784, to R.P; Grant COFIN 2003/2003059955-002 and ex-60%/2005/2006, to M.F.F.) and Regione Campania (Grant L.R. N.5/2005 to R.M.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online January 24, 2008

Abbreviations: AEA, Anandamide; CB, cannabinoid; GnRHa, GnRH-I long-acting agonist; GnRH-IAnt, GnRH-I antagonist; IFL, immunofluorescence; IR, immunoreactivity; KRB, Krebs Ringer buffer for amphibians; LSCM, laser-scanning confocal microscope; O/N, overnight; RT, room temperature.

Received October 2, 2007.

Accepted for publication January 15, 2008.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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