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Laser-Captured Single Digoxigenin-Labeled Neurons of Gonadotropin-Releasing Hormone Types Reveal a Novel G Protein-Coupled Receptor (Gpr54) during Maturation in Cichlid Fish

Department of Physiology, Nippon Medical School, Sendagi, Tokyo 113-8602, Japan

Address all correspondence and requests for reprints to: Ishwar S. Parhar, Department of Physiology, Nippon Medical School, Sendagi, Tokyo 113-8602, Japan. E-mail: ishwar{at}nms.ac.jp.

Abstract

GPR54 is a novel G protein-coupled receptor speculated to be essential for sexual development. However, its role in the regulation of GnRH types is unknown. To address this issue, we cloned GPR54 from the brain of a cichlid fish (tilapia Oreochromis niloticus) and determined its expression in immature and mature males using our newly developed technique: laser-captured microdissection of single digoxigenin-labeled GnRH neurons coupled with real-time quantitative PCR. The tilapia GPR54 cDNA contains an open reading frame of 1131 bp encoding 377 amino acids and exhibits 56% identity to human GPR54. Absolute copies of GnRH1 and GnRH3, not GnRH2, mRNAs were significantly high in mature compared with immature males. At the single-cell level, only in mature males, GnRH1 mRNA levels were inversely related to GPR54 mRNA (P < 0.002). GPR54 was expressed in a significantly high percentage (45.0–60.0%) of mature GnRH1, GnRH2, and GnRH3 neurons and in immature GnRH3 neurons, which had migrated to the vicinity of their final locations in the brain; on the contrary, only 5.0% of immature GnRH1 and GnRH2 neurons had GPR54 transcripts (P < 0.001). Thus, using a novel innovative single-cell gene profiling technique, we provide evidence of the structure of a nonmammalian GPR54, which is highly conserved during evolution and is expressed in GnRH1, GnRH2, and GnRH3 neurons. Furthermore, we propose that the expression of GPR54 is a "stop signal" for GnRH1, GnRH2, and GnRH3 neuronal migration, leading to suppression of cell growth and modulation of GnRH secretion, which is important for normal sexual development.

GPR54 IS A NOVEL G protein-coupled receptor recently cloned from the rat brain (1) and thereafter cloned in mouse (2) and humans (3). The rat GPR54 cDNA encodes for a 396-amino-acid protein, which is widely distributed in the brain with highest expression found in the hypothalamus and amygdala (1). GPR54 has been implicated in pubertal maturation and normal sexual development. Mutation in the GPR54 gene in humans and mice causes hypogonadotropic hypogonadism in which pituitary secretion of FSH and LH are decreased, resulting in delayed puberty, small testes, and impaired reproductive functions (4, 5, 6); mutation of the gene in mice may be corrected with the administration of exogenous GnRH (5). This phenotype resembles Kallmann’s syndrome in humans, which is caused by the failure of GnRH neuronal migration from the olfactory placodes to the hypothalamus (7, 8, 9).

GnRH is now recognized as a family of 16 neuropeptides, and it is well documented that all vertebrate species ranging from fish to humans possess two (hypothalamus, GnRH1; midbrain, GnRH2) or three GnRH types (caudal olfactory bulb, GnRH3) (10). The role of GPR54 in the regulation of GnRH1, GnRH2, and GnRH3 or its presence in GnRH neurons is unknown. To address this issue, we used our newly developed innovative technology, integrating laser-captured microdissection (LCM) of single digoxigenin (DIG)-labeled GnRH neurons coupled with real-time quantitative RT-PCR (RT-Q-RT-PCR), which would greatly facilitate our understanding of the complex interactions that exist within individual GnRH neurons. Tilapia Oreochromis niloticus, a well-characterized teleost model for studying three GnRH types (10), was used to identify the structure of GPR54 cDNA, to detect GPR54 mRNA in three GnRH types, and to analyze using RT-Q-RT-PCR the functional state of GPR54 and GnRH in individual neurons of GnRH1, GnRH2, and GnRH3 in immature and mature male tilapia.

Materials and Methods

Molecular cloning of GPR54
Total RNA from the brain of juvenile (10 d after hatching) tilapia O. niloticus was extracted with ISOGEN (Nippon Gene, Tokyo, Japan) and the poly(A)⁺ RNAs were purified using Oligotex-dT30 Super (TaKaRa, Tokyo, Japan). First-strand cDNA was synthesized from the poly(A)⁺ RNA using SuperScript III reverse transcriptase (Invitrogen Corp., Carlsbad, CA) and 50 pmol oligo(dT)_21–18 primer (Invitrogen) in a thermal cycler (Gene Amp PCR system 9700; PerkinElmer Applied Biosystems, Foster City, CA). All steps were performed according to the manufacturer’s instructions. To amplify a fragment of tilapia GPR54 cDNA, degenerate PCR primers (GPR1 and GPR2; Table 1) were designed based on conserved sequences of mammalian GPR54 (GenBank accession numbers are as follows: mouse, NM_053244; rat, AF115516; and human, NM_032551). PCRs were performed in a final volume of 20 µl containing GeneAmp 1x PCR buffer (Applied Biosystems), 160 µM of deoxynucleotide triphosphate, 1.0 U DNA polymerase (AmpliTaq Gold, Applied Biosystems), 10 pmol gene-specific primers, and 1 µl first-strand cDNA or the first-round PCR product. Reaction conditions for PCR were 94 C for 10 min; 30 cycles of 94 C for 20 sec, 55 C for 20 sec, and 72 C for 20 sec; and 72 C for 7 min. The PCR products were analyzed by 2.0% agarose gels and the bands of expected size were purified and ligated into pGEM-T Easy vector (Promega, Madison, WI) using the DNA ligation kit version 2 (TaKaRa) according to the manufacturer’s instructions. The plasmid DNA was purified and both strands of the DNA were sequenced with T7 and SP6 primers using an ABI PRISM 310 DNA sequencer and sequencing analysis software (Applied Biosystems). The sequences of the 5' and 3' ends of the cDNAs were obtained using the rapid amplification of cDNA end method. The deduced amino acid sequence of the tilapia GPR54 cDNA was aligned with other known sequences using DDBJ CLUSTAL W SYSTEM (DNA Data Bank of Japan).

Sense and antisense tilapia GnRH1, GnRH2, and GnRH3 riboprobes were synthesized using the pGEM-T easy transcription vector (Promega) constructs containing the GnRH coding region (GenBank accession no. AB101665-7) and linearized with SpeI or NcoI endonuclease (Nippon Gene) as a template for T7 or SP6 RNA polymerase (TOYOBO, Tokyo, Japan). The RNA probes were labeled using DIG-RNA labeling mix (Roche Diagnostics GmbH, Penzberg, Germany).

Twenty-five microliters of hybridization buffer containing 30 ng DIG-labeled GnRH1, GnRH2, and GnRH3 riboprobes were added to each tissue section and incubated in a humidified box at 42 C for 12 h. After hybridization, sections were washed twice in 2x SSC (1x SSC = 0.15 M NaCl, 15 mM sodium citrate) at room temperature for 15 min and in 1x SSC and 0.1x SSC at 55 C for 1 h sequentially. The hybridization signals were detected using anti-DIG conjugated with alkaline phosphatase and visualized with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate substrate solution (Roche). The sections were washed rapidly two times with diethyl pyrocarbonate-H₂O and dehydrated with 70, 95, and 100% ethanol for 30 sec each, incubated in xylene for 5 min, and air-dried. The slides were stored in a desiccating box with silica gel until LCM was performed.

LCM of GnRH neurons
The dehydrated tissue section was overlaid with a thermoplastic membrane mounted on an optically transparent cap (CapSure Macro LCM Caps; Arcturus, Mountain View, CA). Using a Pix Cell II laser capture instrument (Arcturus), DIG-identified GnRH cells were microdissected by focal melting of the membrane through laser activation (laser pulse power, 25–65 mW; laser pulse duration, 1.5 ms; laser spot size, 10–30 µm diameter). A heat-pulled borosilicate glass microcapillary pipette (1.5-mm outer diameter; Harvard Apparatus Ltd., Edenbridge, Kent, UK; micropipette puller, type PE-2, Narishige, Tokyo, Japan) attached to a micromanipulator (Narishige) was used to remove undesirable tissue around the periphery of single GnRH cells. Then, using a negative pressure, single neurons of three GnRH types were harvested from the LCM cap into the micropipette under visual control and subsequently expelled into a sterile 1.5-ml reaction tube containing 50 µl of the lysis buffer and stored at –80 C until total RNA isolation. For unbiased cell sampling, five to six morphologically well-defined cells were harvested at random (1 cell per alternate section) along the rostral-caudal extent of the whole population of each GnRH type in each animal (n = 5 animals per age group). Cells that were located individually were harvested, and only those cells positive for each GnRH type but negative for glial fibrillary acidic protein (GFAP) and free from genomic contamination were used for RT-Q-RT-PCR analysis (n = 4 cells per animal; 20 cells per GnRH type per age group).

GPR54 expression in GnRH neurons
The harvested single GnRH neuron was digested with 1 µg of proteinase K (Gentra Systems, Minneapolis, MN) and 10 U of Prime ribonuclease inhibitor (Eppendorf, Hamburg, Germany) for an hour at 53 C. The cell lysate was incubated for 1 h at 37 C with 1 U ribonuclease-free deoxyribonuclease I (Promega) to eliminate genomic DNA and was heat denatured at 95 C for 10 min to separate the mRNA from the DIG-labeled riboprobe. Total RNA was extracted from the cell lysate using ISOGEN (Nippon Gene) and Mini RNA Isolation Kit (Zymo Research, Orange, CA) and reverse transcribed to cDNA with 0.1 pmol of random primers (TaKaRa) using 40 U SuperScript III reverse transcriptase (Invitrogen).

To confirm the presence and integrity of GPR54 and GnRH mRNA, the single GnRH neuron’s cDNA was subjected to RT-PCR. PCRs were performed in a final volume of 20 µl containing GeneAmp 1x PCR buffer, 160 µM of deoxynucleotide triphosphate, 1 U DNA polymerase (AmpliTaq Gold, Applied Biosystems), 250 nM gene-specific primers (G1–G6, GPR3 and GPR4, GP1 and GP2; Table 1) and one twentieth of a single neuron’s reverse transcribed cDNA solution. Forward or reverse primers spanned a predicted intron and cDNA sequence on either side of the splice site (G2, G4, G6, GPR3; Table 1). Reaction conditions for PCR were 94 C for 10 min; 50 cycles at 94 C for 15 sec, 60 C for 15 sec, and 72 C for 15 sec; and 72 C for 7 min. Ten microliters of the reaction mixture were run on a 2% agarose gel and visualized with ethidium bromide. To confirm the sequences, some bands were subcloned into pGEM-T Easy vector (Promega) and both strands of the DNA were sequenced with T7 and SP6 promoter primers (Promega) using an ABI PRISM 310 Genetic Analyzer and Sequence Analysis Software (Applied Biosystems). Several controls were included for the RT-PCR: buffer without harvested cells, no reverse transcription, non-GnRH cells, and tilapia genomic DNA for negative control. For GnRH and non-GnRH cells, GFAP primers were also included in the PCR protocol (GP1 and GP2; Table 1). The GenBank accession numbers of the three GnRH types, GFAP, and the newly cloned GPR54 in tilapia are as follows: GnRH1, AB101665; GnRH2, AB101666; GnRH3, AB101667; GFAP, AB109167; and GPR54, AB162143.

RT-Q-RT-PCR for GnRH and GPR54 in GnRH neurons during maturation
RT-Q-RT-PCR was performed in duplicate in 10-µl reaction volumes consisting of 1x TaqMan Universal PCR Master Mix (Applied Biosystems), 300 nM of primers (G7, G8, G10, G11, G13, G14, GPR5, GPR6; Table 1), 200 nM of hybridization probe (G9, G12, G15, GPR7; Table 1), and one twentieth of a single neuron’s reverse transcribed cDNA or absolute standard cDNA using the ABI PRISM 7700 Sequence Detection System (TaqMan PCR; PerkinElmer Applied Biosystems). The PCR conditions were 95 C for 10 min followed by 60 cycles at 95 C for 15 sec and 60 C for 1 min. Hybridization primers and fluorogenic probes for RT-Q-RT-PCR were optimized using the ABI PRISM Primer Express Software (Applied Biosystems). The reverse primers (G8, G11, G14, GPR6; Table 1) spanned an intron and complemented the sequence on either side of the splice site of the gene. For each animal and GnRH type, average mRNA levels per cell were determined and these values were combined to give experimental group means. All values are expressed as the mean ± SEM and statistical comparisons were made between different age groups (n = 5, each group) using Student’s t test or nonparametric ANOVA followed by post hoc Dunn’s multiple comparison test. P < 0.05 was considered statistically significant (see figure legends).

Results and Discussion

Using PCR and the rapid amplification of cDNA end technique, we have cloned and obtained the full-length sequence for a novel receptor in a teleost, designated here as tilapia GPR54. The tilapia GPR54 cDNA contains an open reading frame of 1131 bp encoding 377 amino acids (Figs. 1 and 2; GenBank accession no. AB162143). In the open reading frame, we have identified conserved residues and consensus sequences of seven transmembrane (TM) domains of the rhodopsin superfamily of G protein-coupled receptors (11), which include an asparagine in TM1, an aspartate in TM2 and prolines in TMs 4–7 (Fig. 2). The amino acid sequence of tilapia GPR54 has 54, 55, and 56% identity with mouse, rat, and human GPR54, respectively (1, 2, 3). Phylogenic analysis also showed close sequence identity with rat galanin receptors (GALR1, GALR2, GALR3; 3236%) and RFamide-related peptide receptor types (20%).

View larger version (55K): [in this window] [in a new window]   FIG. 1. Nucleotide and deduced amino acid sequence of GPR54 cDNA in tilapia. The numbers on the right refer to the nucleotide sequence. Predicted transmembrane regions (TM1–TM7) are underlined. The stop codon is marked by an asterisk. The predicted exon-intron junctions are marked by arrowheads.

View larger version (103K): [in this window] [in a new window]   FIG. 2. An alignment of amino acid sequences of the tilapia GPR54 with mammalian GPR54. Conserved amino acid residues among GPR54 sequences are shaded. Predicted transmembrane regions are boxed.

View larger version (80K): [in this window] [in a new window]   FIG. 3. A, A schematic illustration showing location of GnRH1, GnRH2, and GnRH3 (black dots) in a sagittal brain section. B–D, Photomicrographs of DIG-labeled GnRH1, GnRH2, and GnRH3 neurons; B, preoptic neurons (POA, GnRH1); C, midbrain neurons (MB, GnRH2); D, terminal nerve ganglia at the caudal-most olfactory bulbs (TN, GnRH3). E and G, DIG-labeled GnRH1 and GnRH2 neurons before LCM and, F and H, after LCM. I and J, Microdissected cells being harvested from LCM cap using micropipette attached to a micromanipulator. Scale bar, 20 µm.

View larger version (36K): [in this window] [in a new window]   FIG. 4. Composite gel showing amplicons of GPR54 in GnRH1, GnRH2, and GnRH3 neurons taken from mature males (lanes 1–10), without reverse transcriptase (lanes 11–15); and non-GnRH cells surrounding GnRH1, GnRH2, and GnRH3 neurons (lanes 16–20). M, Marker, DNA 100-bp size ladder; NC, tilapia genomic DNA as negative control; PC, whole brain cDNA as positive control for PCR. The arrowheads show RT-PCR product of GPR54 at 216 bp.

View larger version (23K): [in this window] [in a new window]   FIG. 5. Histograms showing copies of GnRH1, GnRH2, and GnRH3 mRNA in (A) GPR54-positive GnRH cells and (B) GPR54-negative GnRH cells. The average copies of mRNA transcripts per cell deduced from the total number of individual cells for each GnRH type are given in parentheses. Statistical analysis on an animal basis (n = 5, each group) showed significantly more GnRH transcripts in GnRH1 and GnRH3 neurons of mature (M, white bars) compared with immature males (IM, dark bars) (P < 0.05, Student’s t test).

View larger version (20K): [in this window] [in a new window]   FIG. 6. Scatter diagram showing copies of GnRH and GPR54 mRNA transcripts in individual GnRH1, GnRH2, and GnRH3 neurons of mature (white circles) and immature (dark circles) males. Each data point represents a single cell (n = 20 cells, each group). Immature GnRH1 and GnRH2 neurons were almost devoid of GPR54 mRNA transcripts, whereas most neurons of mature GnRH types and immature GnRH3 had GPR54 mRNA transcripts. Linear regression statistics performed in mature males (white circles) showed inverse relationship between GnRH1 and GPR54 transcripts (P < 0.002, Spearman’s rank correlation). Regression analysis in immature males (dark circles), which was performed only for GnRH3 vs. GPR54, showed no correlation.

View larger version (14K): [in this window] [in a new window]   FIG. 7. Histograms showing the frequency of detection of GPR54 transcripts in immature (IM, dark bars) and mature male (M, white bars) GnRH neurons. Statistical analysis on an animal basis showed a significantly high percentage of GPR54-positive mature GnRH1, GnRH2, and GnRH3 and, immature GnRH3 compared with immature GnRH1 and GnRH2 neurons. *, P < 0.001; nonparametric ANOVA (P = 0.003) followed by post hoc Dunn’s multiple comparison test; n = 5, each group.

Acknowledgments

We thank Dr. R. Kiyama for providing us the laser capture facility and for his valuable discussions and Ms. Y. Aita for her technical assistance.

Footnotes

This study was supported in part by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan, nos. 14580777 (to I.S.P.) and 4370025 (to Y.S.).

Abbreviations: DIG, Digoxigenin; GFAP, glial fibrillary acidic protein; LCM, laser-captured microdissection; RT-Q-RT-PCR, real-time quantitative RT-PCR; TM, transmembrane.

Received March 26, 2004.

Accepted for publication May 14, 2004.

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