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Research Article |
Department of Animal and Poultry Science, University of Guelph, Ontario, Canada N1G 2W1
ABSTRACT
In all vertebrates, GnRH regulates gonadotropin secretion through binding to a specific receptor on the surface of pituitary gonadotropes. At least two forms of GnRH exist within a single species, and several corresponding GnRH receptors (GNRHRs) have been isolated with one form being pituitary specific. In chickens, only one type of widely expressed GNRHR has previously been identified. The objectives of this study were to isolate a chicken pituitary-specific GNRHR and to determine its expression pattern during a reproductive cycle. Using a combined strategy of PCR and rapid amplification of cDNA ends (RACE), a new GNRHR (chicken GNRHR2) and two splice variants were isolated in domestic fowl (Gallus gallus domesticus). Full-length GNRHR2 and one of its splice variant mRNAs were expressed exclusively in the pituitary, whereas mRNA of the other splice variant was expressed in most brain tissues examined. The deduced amino acid sequence of full-length chicken GNRHR2 reveals a seven transmembrane domain protein with 57%–65% homology to nonmammalian GNRHRs. Semiquantitative real-time PCR revealed that mRNA levels of full-length chicken GNRHR2 in the pituitary correlate with the reproductive status of birds, with maximum levels observed during the peak of lay and 4 wk postphotostimulation in females and males, respectively. Furthermore, GnRH stimulation of GH3 cells that were transiently transfected with cDNA that encodes chicken GNRHR2 resulted in a significant increase in inositol phosphate accumulation. In conclusion, we isolated a novel GNRHR and its splice variants in chickens, and spatial and temporal gene expression patterns suggest that this receptor plays an important role in the regulation of reproduction.
chicken, gonadotropin-releasing hormone receptor, hypothalamus, neuroendocrinology, pituitary, pituitary hormones, reproduction
INTRODUCTION
Gonadotropin-releasing hormone (GnRH) is a decapeptide released by the hypothalamus that tightly regulates reproduction in most vertebrates. It acts through binding to specific GnRH receptors (GNRHRs) on the membrane of pituitary gonadotropes, where it stimulates synthesis and release of luteinizing hormone (LH) and follicle stimulating hormone (FSH) [1, 2]. GnRH is also found in the central and peripheral nervous systems and in other tissues, including reproductive organs [3]. GnRH receptors are members of the guanine nucleotide regulatory protein (G) protein-coupled receptor (GPCR) family, which upon activation, produce different second messengers depending on the type of G proteins involved. Coupling to G
q activates phospholipase C, which hydrolyzes phosphatidylinositol 4, 5-bisphosphate (PIP2) to inositol 1, 4, 5-trisphosphate (IP3) and diacylglycerol, whereas coupling to Gs or Gi activates or inhibits adenylyl cyclase, respectively, resulting in changes in cAMP production [4].
In most species, at least two forms of GnRH exist, with GnRH I and GnRH II being the predominant hypothalamic and midbrain form, respectively [5, 6]. In addition, a third form (GnRH III) has also been identified in fish [7]. The existence of more than one GNRHR has been reported in several species [5, 8–12], and in addition to full-length GNRHRs, splice variants have also been cloned [13–16]. Whether these splice variants possess any physiological significance remains to be elucidated. Nonetheless, several studies have suggested that they may interfere with signaling mechanisms of the full-length receptor [13, 15, 17].
In poultry, the hypothalamo-pituitary-gonadal axis is stimulated by increasing the photoperiod (photostimulation) [18], and sexual maturation is characterized by the initiation of egg laying in females and sperm production in males [18, 19]. Although two forms of GnRH have been identified in avian species, only one GNRHR (designated as chicken GNRHR1 in this manuscript) has been characterized to date [20]. It was originally cloned from adult cockerels, and its mRNA was detected in the pituitary and brain as well as in other organs such as testes, spleen, and heart [20]. Based on comparison to other vertebrates, it is likely that additional GNRHRs also exist in avian species, with at least one pituitary-specific form. As a matter of fact, a putative second GNRHR gene was proposed in the recently assembled chicken genome (Ensemble Chicken Genome, http://www.ensembl.org/Gallus_gallus/index.html). In the present study, we report the cloning of full-length cDNAs that encode a new chicken GNRHR and two splice variants. Expression profiles of these receptors in various brain areas and several other tissues from females and males at different reproductive stages were also determined, and the function of the receptor was examined in transiently-transfected GH3 cells. Our results reveal that this novel GNRHR is a pituitary-specific form, and that its expression level correlates with the reproductive state in both females and males.
MATERIALS AND METHODS
Animals and Tissue Collection
White Leghorn chickens were raised under an 8-h photoperiod in our poultry research station (Arkell, ON, Canada) until 19 wk of age. They were then photostimulated by an abrupt transfer to a 14-h photoperiod. Before tissue collection, all birds were killed by cervical dislocation. For cloning of the new GNRHR gene, RNA was extracted from the pituitary gland of an adult White Leghorn male. For the study of GNRHR expression levels, five brain tissues (pituitary, diencephalon/mesencephalon, brain stem, cerebrum, and cerebellum) were collected from birds of both sexes. For females, samples were collected at the immature (14 wk old, n = 5), peak of lay (30 wk, n = 5), mid-lay (1 yr old, n = 5), and end of lay (over 1 yr old, n = 5) stages. For males, samples were collected at the immature (14 wk old, n = 5), 4 wk postphotostimulation (23 wk old, n = 5), and mature (14 mo old, n = 5) stages. In addition, hearts, livers, spleens, ovaries, testes, and kidneys were also collected from immature (17 wk old) and mature (30 wk old) females and males. After collection, individual tissues were immediately frozen in liquid nitrogen, and kept at –80°C until RNA isolation. All animal procedures were conducted under the guidelines of the Canadian Council for Animal Care, and were approved by the University of Guelph Animal Care Committee.
Isolation of RNA
Total RNA was extracted using Tri Reagent (Sigma-Aldrich, Inc., St. Louis, MO) according to the manufacturer's instructions, and stored at –80°C until use. Total RNA was treated with DNA-free DNase (Ambion, Austin, TX) to eliminate DNA contamination, and the concentration and purity of RNA were assessed with a GeneQuant RNA/DNA calculator (Pharmacia, Cambridge, England).
Cloning of Chicken GNRHR2 cDNA and Its Splice Variants
Three micrograms of total RNA isolated from the pituitary gland of a mature White Leghorn male were reverse transcribed using an Oligo(dT) primer and SuperscriptII reverse transcriptase (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. The sequence of previously published chicken GNRHR1 (GenBank AJ304414) was compared to that of bullfrog GNRHRs (GenBank AF153914 and AF224277) to identify highly conserved regions. As a result, transmembrane domains (TM) 3 and TM6 were chosen to design forward (GNRHR2-TM3-F) and reverse (GNRHR2-TM6-R) primers (Table 1). Following PCR amplification (95°C, 5 min; 40 cycles of 95°C, 30 sec, 63°C, 1 min, and 72°C, 1 min; and 72°C 10 min), a partial cDNA fragment was obtained. Subsequent sequence analysis revealed a high degree of homology with amphibian GNRHRs. In addition, a shorter cDNA fragment was also obtained, suggesting the existence of a splice variant. The 3' and 5' sequences of full-length cDNA were then isolated by 3' and 5' rapid amplification of cDNA ends (RACE). Briefly, for the 3' end, an adapter was incorporated into the 3' end of the cDNA during the reverse transcription (RT) reaction with SuperscriptII reverse transcriptase and 3RACE-AP primer (Table 1). Subsequently, PCR amplification was performed using gene-specific forward (3RACE-F1) and adapter reverse (3RACE-AUAP) primers (Table 1). For the 5' end, 5' RACE was performed using the BD Smart RACE cDNA amplification kit (BD Biosciences, Clontech, Palo Alto, CA). During first strand cDNA synthesis, a dC-tail was attached to the 5' end of the cDNA, and a series of nested PCR reactions was conducted using an adapter forward primer (UPM) provided with the kit and several gene-specific reverse primers (GNRHR-R2, 5RACE-R1, 5RACE-GSP6, and 5RACE-GSP8; Table 1). Finally, to confirm the integrity of the complete chicken GNRHR2 cDNA, PCR amplifications were performed using pairs of primers designed to amplify from the start codon to the stop codon (GNRHR2-start-F and GNRHR2-stop-R; Table 1), as well as from the beginning of the 5' untranslated region (UTR) to the end of 3' UTR (GNRHR2-5UTR-F and GNRHR2-polyA-R; Table 1). In addition to full-length chicken GNRHR2, these PCR amplifications revealed two additional shorter products. After sequencing and comparison to the recently assembled chicken genome database, it was confirmed that we obtained full-length chicken GNRHR2 and two alternatively spliced variants (GNRHR2_v1 and GNRHR2_v2), all which contain the same 5' UTR and 3' UTR as well as the same start and stop codons.
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Patterns of Chicken GNRHR2 Expression and its Splice Variants in Various Brain Tissues
To determine which forms of chicken GNRHR2 are expressed in various tissues from females and males at different reproductive stages, individual cDNA samples were pooled by tissue for each stage, and PCR amplification was performed using primers that amplify all the GNRHR2 variants (R2-instart-F/R2-probe-R1; Table 1).
Quantification of Chicken GNRHRs mRNA in the Pituitary Gland
Although RT-PCR allows qualitative detection of GNRHR mRNA, it cannot be used for quantitative studies. Therefore, chicken GNRHR1, GNRHR2, GNRHR2_v1, and GNRHR2_v2 mRNAs were quantified in individual pituitary glands using real-time PCR, and levels of mRNA were compared among different reproductive stages. Real-time PCR was performed with an ABI prism 7000 thermocycler (Applied Biosystems, CA) using the QuantiTect SYBR Green PCR kit (Qiagen, Valencia, CA). For each reproductive stage, five individual pituitary samples were analyzed, and each sample was analyzed in triplicate. Gene-specific primers (Table 1) were designed to amplify either chicken GNRHR1 (R1-target wt-F2/R1-target wt-R2), GNRHR2 (R2wt-target-F1/R2wt-target-R1), GNRHR2_v1 (R2-spanSV1-F1/R2-spanSV1-R2), or GNRHR2_v2 (R2-spanSV2-F1/R2-spanSV2-R1). To specifically distinguish full-length GNRHR2 from its splice variants, GNRHR2 primers were designed that correspond to the region that is deleted in the splice variants. Additionally, GNRHR2_v1 and GNRHR2_v2 forward primers were designed to span the alternative splice sites (see Fig. 2). Beta-actin cDNA was used as internal control to normalize for minor variations in RNA input or RT efficiency. In order to monitor possible genomic DNA contamination, primers that span an intron of ß-actin (ß-actin-F/ß-actin-R; Table 1) were designed. Two micrograms of total RNA were reverse transcribed in a total volume of 20 µl as described above. Complementary DNA was then diluted 1:4 with double-distilled water (ddH2O), and 1 µl was used for real-time PCR (50°C, 2 min; 95°C, 10 min; 40 cycles of 95°C, 15 sec, 62°C, 30 sec, 72°C, 30 sec). Data were analyzed using the 2–
Ct method as described by Livak and Schmittgen [21]. Average values from immature females and males were used as calibrators for female and male samples, respectively. For comparisons, normalized mRNA levels for each stage were expressed as fold increase over the calibrator.
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Subcloning of Chicken GNRHRs and Their Splice Variants
To obtain the full-length cDNA that encodes the previously identified chicken GNRHR1 [20], RT-PCR was conducted using RNA from the diencephalon of a mature male and primers that span the start and stop codons (GnRHR-F and GnRHR-R; Table 1). As previously reported by Sun et al. [22], two point mutations that result in the substitutions of Gln for Lys(40), and Arg for Gln(338) were found in the cDNA sequence obtained from White Leghorns when compared to that of the Red Jungle Fowl (assembled genome database). These differences are most likely due to polymorphisms. However, it cannot be excluded that these differences result from PCR artifacts. Thus, the cloned cDNA sequence was subjected to two rounds of site-directed mutagenesis (QuickChange Site-Directed Mutagenesis Kit, Stratagene, CA) using two pairs of specific primers (R1wt-GlnLys40-SDM-F/R1wt-GlnLys40-SDM-R for Gln(40), and R1wt-ArgGln338-SDM-F/R1wt-ArgGln338-SDM-R for Arg(338); Table 1) to restore the sequence to that published in the assembled chicken genome sequence database. The resulting "Red Jungle Fowl chicken GNRHR1" cDNA was then used for subsequent inositol phosphate (IP) assays. Coding sequences of the chicken GNRHR2 and its splice variants were also cloned using the start-to-stop primer pair (GnRHR2-start-F/GnRHR2-stop-R; Table 1) as described above. Finally, the newly isolated GNRHR2 variants and GNRHR1 cDNAs were subcloned into the pcDNA 3.1(+) expression vector (Invitrogen, Carlsbad, CA).
Inositol Phosphate Assay
Expression vectors that contain cDNA encoding the chicken GNRHRs were used to assess receptor function in vitro. The protocol for measuring total IP production was adapted from Panchenko et al. [23]. Briefly, GH3 cells (kindly provided by Dr. Ursula Kaiser) cultured in 6-well plates were transiently transfected with 2 µg per well of either chicken GNRHR1, GNRHR2, GNRHR_v1, GNRHR2_v2 cDNAs, or empty pcDNA 3.1 vector (negative control) using Lipofectamine 2000 (Invitrogen). Twenty-four hours after transfection, culture medium was replaced with 1 ml of inositol-free Dulbecco modified Eagle medium (DMEM; MP Biomedicals, Aurora, OH) supplemented with 5% fetal bovine serum (FBS; HyClone, Logan, UT) and 1% penicillin/streptomycin (Invitrogen). After a 2-h incubation, 1 ml of the same medium containing 2 µCi of myo-(2-3H) inositol (MP Biomedicals) was added to each well. Cells were then incubated at 37°C for 15 min, and 10 mM lithium chloride was added to each well. After an overnight incubation at 37°C, cells were stimulated for 45 min with various concentrations of either custom-synthesized chicken GnRH I (cGnRH I, pyro-Glu-His-Trp-Ser-Tyr-Gly-Leu-Gln-Pro-Gly-NH2; Sigma Genosys, Woodlands, TX) or chicken GnRH II (cGnRH II, pyro-Glu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2; American Peptide Company, Sunnyvale, CA). Cells were then lysed for 30 min with 20 mM formic acid on ice twice, and lysates were neutralized to pH 7.5 with 7.5 mM Hepes, 150 mM potassium hydroxide (KOH). After centrifugation at 14 000 x g for 2 min to precipitate cell debris, supernatants were loaded onto Ag-X8 resin anion exchange columns (Bio-Rad Laboratories, Hercules, CA) previously equilibrated with 2 ml 1M sodium hydroxide (NaOH), 2 ml 1M formic acid, and 5 times 5 ml ddH2O. Columns were then washed with 5 ml ddH2O, followed by 5 ml 5 mM borax, 60 mM sodium formate, and total IPs were eluted with 3 ml 0.9 M ammonium formate/0.1 M formic acid. Inositol phosphate accumulation was measured by counting radioactivity in eluates with a scintillation counter. As GNRHR2_v1 and GNRHR2_v2 failed to respond to supraphysiological doses (10–6 M) of either cGnRH I or cGnRH II, dose-response analyses were performed only on full-length GNRHR1 and GNRHR2. All assays were performed in triplicate, and each experiment was repeated three times.
Computational and Statistical Analysis
Messenger RNA levels measured by real-time PCR in individual pituitaries were compared using one-way ANOVA followed by a Tukey multiple comparison test. Inositol phosphate dose response was performed in three separate experiments. Data from each set of experiments were subjected to nonlinear regression analysis, and effective dose 50 (ED50) values were calculated using GraphPad Prism 4.00 software (GraphPad Software, San Diego, CA). Calculated ED50 values were then compared by one-way ANOVA followed by a Tukey multiple comparison test. All statistical analyses were performed using the GraphPad InStat 3.00 software (GraphPad Software). A P < 0.05 was considered significant.
RESULTS
Identification of Chicken GNRHR2
Using primers with sequences that map to domains highly conserved between chicken GNRHR1 and bullfrog GNRHRs, a cDNA fragment that encodes part of a potential chicken GNRHR was isolated by RT-PCR from the pituitary gland of an adult White Leghorn male. The deduced amino acid sequence of the amplified product showed high homology to amphibian and fish Gnrhr (65%71%). Therefore, the full-length cDNA sequence was obtained using a combined strategy of PCR and 5', 3' RACE, and the integrity of this potential chicken GNRHR was further confirmed by amplifying the complete cDNA sequence. Three different cDNAs of 1260 bp (full length GNRHR2), 1065 bp (GNRHR2_v2), and 501 bp (GNRHR2_v1) in length were obtained (Fig. 1). Sequence analysis revealed that the two shorter fragments were produced by alternative splicing, but contained the same 5'and 3' UTRs as well as the same start and stop codons as the full-length receptor (Fig. 2). In addition to the three variants, several RT-PCR products of different sizes were also obtained (Fig. 1). Subsequent sequencing of one of the amplicons revealed that the migration difference most likely results from a secondary structure formed in the cDNA. Such a phenomenon is not uncommon and was previously reported by Cariello et al. [24]. Therefore, we decided to focus on the three predominant products representing the full-length sequence and the two splice variants. However, it cannot be excluded that additional splice variants exist.
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The deduced amino acid sequence revealed that the chicken GNRHR2 cDNA encodes a 420-amino acid protein with seven TM, typical of GPCRs (Fig. 3). The amino acid sequence of chicken GNRHR2 was 57%–65% homologous to GNRHRs of amphibians and fish (Fig. 4). Based on the high degree of homology, the new potential chicken GNRHR was designated as GNRHR2, and the sequence was submitted to GenBank (accession number AY895154). As the assembled chicken genome sequence became available, we were further able to confirm the accuracy of the GNRHR2 cDNA sequence. The genomic sequence showed that the GNRHR2 gene is located on chromosome 10 and consists of four exons (Fig. 3). In the case of the splice variants, GNRHR2_v1 cDNA lacks exons II and III, resulting in a deletion of 253 amino acids from part of the extracellular domain (ECD) to part of intracellular loop (ICL) 3, and GNRHR2_v2 cDNA has partial deletion of exon II, resulting in a 65-amino acid deletion from part of the ECD to part of TM1 (Fig. 3).
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Expression of Chicken GNRHR2 is Pituitary-Specific and Correlates with Reproductive State
The previously identified chicken GNRHR1 and its splice variant were found to be widely expressed [20]. Therefore, spatial expression patterns of GNRHR2 and its splice variants were examined in several tissues by RT-PCR. Interestingly, GNRHR2 and GNRHR2_v2 mRNAs were predominantly detected in the pituitary of both females and males from all stages examined, whereas GNRHR2_v1 (501 bp variant) was expressed in several other brain tissues (Fig. 5, A and B). Neither GNRHR2 nor its splice variant mRNAs were detected in the liver, heart, spleen, kidney, ovary, or testes (Fig. 5, C and D). Consistent with the previous report by Sun et al. [20], GNRHR1 and its splice variant mRNAs were detected in all brain tissues examined (data not shown). Since RT-PCR only provides qualitative information, mRNA levels of GNRHR1, GNRHR2, and GNRHR2 variants were quantified in bird pituitary glands at different reproductive stages using real-time PCR (Fig. 6). To avoid cross-amplification of GNRHR2 mRNA and its splice variants, primer pairs were designed to specifically amplify the intended target and cloned GNRHR2 variants were used as controls to assess the specificity of amplification. As shown in Figure 6, levels of GNRHR2 mRNA fluctuated across reproductive stages. In females, mRNA levels significantly increased from the immature to the peak of lay stage, and then gradually decreased toward the end of the laying cycle. Likewise, in males, mRNA levels significantly increased from the immature stage to 4 wk postphotostimulation, and then decreased thereafter. No statistically significant changes were observed in mRNA levels of GNRHR1, GNRHR2_v1, or GNRHR2_v2 in either males or females (Fig. 6).
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Chicken GNRHR2 Possesses Differential Sensitivity to cGnRH I and cGnRH II
To confirm the ability of GNRHR2 and its splice variants to activate intracellular signal transduction pathways, IP production was measured in transiently-transfected GH3 cells stimulated with chicken GnRHs. As shown in Figure 7, stimulation of GNRHR2 with either cGnRH I or cGnRH II resulted in a dose-dependent increase in IP production. On the other hand, no increase was observed in cells transfected with the pcDNA 3.1 empty vector (negative control) or cDNA that encodes GNRHR2_v1 or GNRHR2_v2 (data not shown). As shown in Table 2, both GNRHR1 and GNRHR2 displayed a significantly lower ED50 when stimulated with cGnRH II than with cGnRH I.
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DISCUSSION
In the present study, full-length cDNAs that encode a novel chicken GNRHR and its splice variants were isolated. In most vertebrates, more than one type of GNRHR has been identified [8–12], and as reported in the bullfrog [11] and the masu salmon [9], one form appears to be pituitary-specific. To date, one GNRHR has been identified in chickens, and it was shown to be expressed in many brain regions as well as other tissues such as the testes, spleen, and heart [20]. This wide range of tissue distribution raises the question of whether a pituitary-specific GNRHR exists in chickens. Using primers that correspond to regions highly conserved in chicken GNRHR1 and bullfrog GNRHRs, we first amplified a partial cDNA from the pituitary gland of a White Leghorn rooster. Subsequently, the full-length cDNA of a novel chicken GNRHR2, including the 5' and 3' UTRs was obtained. An alignment with the deduced amino acid sequence revealed a high degree of homology to amphibian and fish GNRHRs. However, homology to chicken GNRHR1 was lower (53%) than to bullfrog GNRHRs. Interestingly, the highest homology observed was to bullfrog GNRHR1 (65%), a form previously found to be pituitary-specific [11].
Comparison of the cDNA sequence with the assembled chicken genome database revealed that chicken GNRHR2 appears to be composed of four exons and three introns (Fig. 3). It is worth mentioning that the gene structures of chicken GNRHRs differ from their mammalian counterparts, which consist of three exons and two introns [14, 25]. At the amino acid level, several important motifs are present in chicken GNRHR2. For example, a Pro-X-Ser/Tyr motif in extracellular loop (ECL) 3 that was shown to be a determinant of differential ligand selectivity [26] corresponds to Pro-Pro-Ser in chicken GNRHR2. As seen in bullfrog GNRHR2 and 3, Xenopus GNRHR, and catfish GNRHR, chicken GNRHR2 possesses Pro7.31, which is different from the pituitary-specific bullfrog GNRHR1 (it contains Ser-Gln-Ser with Ser7.31) [11, 26]. The positions of Pro and Ser near Glu/Asp7.32 in ECL3 were shown to be important for establishing selectivity between the ligands cGnRH II and mammalian GnRH, with Glu7.32 following Pro7.31 resulting in a decreased sensitivity for cGnRH II [26]. However, neither Glu nor Asp is present in that position in chicken GNRHR2, and it displayed a higher sensitivity to cGnRH II than to cGnRH I. Therefore, there may be other amino acid residues that are critical for ligand selectivity. In addition, a DRxxxI/VxxPL motif in ICL2 (Asp-Arg-Gln-Ala-Ala-Ile-Leu-Arg-Pro-Leu) and a DPxxY motif in TM7 (Asp-Pro-Ile-Thr-Tyr), which are important for receptor activation [27, 28] are also present in chicken GNRHR2.
In the pituitary gland, full-length chicken GNRHR2 mRNA levels correlated with the successive phases of a reproductive cycle in both hens and roosters. In females, mRNA levels increased from the immature stage to the peak of the lay, and gradually decreased toward the end of the laying period. Similarly, in males, mRNA levels increased from the immature stage to the postphotostimulation stage, and then decreased thereafter. These results suggest that this receptor plays an important role during sexual maturation, and is thus probably involved in the regulation of gonadotropin gene expression and secretion. In masu salmon, mRNA levels of a pituitary-specific form of Gnrhr increase prior to precocious maturation in males, and in the prespawning period in females [9], further supporting our hypothesis that this subtype of receptors is involved in sexual maturation and reproduction. Conversely to mammals, LH and FSH reside in two different cell populations in the chicken pituitary [29, 30], suggesting that the two gonadotropins are controlled by different mechanisms. Whether the same GnRH receptors are present on both populations of gonadotropes in chickens is not known. In a recent study, Proudman et al. [31] showed that in vivo stimulation with either cGnRH I or II resulted in a significant increase in LH release in both immature females (17 wk old) and mature males (32 wk old). However, a significant effect on FSH release was only observed with the highest dose of cGnRH II in mature males [31]. Since our study revealed a significant increase in chicken GNRHR2 mRNA in the pituitary only after photostimulation, and found constant levels of GNRHR1 mRNA regardless of reproductive stage, it is possible that GNRHR1 and GNRHR2 differentially control LH and FSH. Further studies will be needed to confirm this hypothesis. In addition to controlling sexual maturation, GnRH can also affect daily ovulatory cycles, and a transient increase in GNRHR1 mRNA was observed with a maximum reached 6 h before ovulation [32]. We did not measure levels of chicken GNRHR2 expression during an ovulatory cycle, but it is not excluded that daily fluctuations in gene expression may occur.
The deduced amino acid sequence of the full-length chicken GNRHR2 predicts the seven TM characteristic of GPCRs, and the presence of a C-terminal tail that was shown to be responsible for desensitization and rapid internalization of non-mammalian GnRH receptors [33, 34]. Amino acid sequences of the two splice variants (GNRHR2_v1 and GNRHR2_v2) correspond to a 2-TM protein and a partial 7-TM protein, respectively. A 5-TM mutant of a chemokine GPCR with a deletion of TM1–TM2 was previously shown to function as a normal receptor, able to activate intracellular signal transduction pathways. Furthermore, it was also shown to be desensitized and internalized in an agonist dependent manner [35]. On the other hand, a truncated human GNRHR2 consisting of part of TM5 to the C terminus was shown to inhibit signaling of full-length human GNRHR1 in a dose-dependent fashion when co-expressed in COS-7 cells [17]. In that study, the authors suggested that the truncated receptor might impair de novo synthesis of full-length GNRHR1, possibly at the nucleus, endoplasmic reticulum, or Golgi level [17]. Bullfrog GNRHR splice variants have also been shown to inhibit signaling of the full-length receptor, and since they were found in both the plasma membrane and cytoplasm of transfected HeLa cells, they might exert this effect through physical interactions with the full length-receptor [13]. In addition, the ratio between full-length-receptor and splice-variant mRNA levels changes depending on the physiological status of bullfrogs [13]. In our study, neither GNRHR2_v1 nor GNRHR2_v2 responded to GnRH stimulation, suggesting that they each lack the ability to independently participate in signal transduction. However, since a recent study using fluorescence resonance energy transfer showed that both full-length and splice variants of wallaby GNRHR form homodimers and heterodimers [36], it is possible that the splice variants are important as regulators of signal transduction through full-length GNRHR. This is significant, since microaggregation between GNRHRs has been shown to be promoted by the GnRH agonist as a part of normal receptor signal transduction [37].
In summary, the present study reports the cloning of a novel type of chicken GNRHR and its splice variants. The full-length receptor and GNRHR2_v2 are exclusively expressed in the pituitary, whereas GNRHR2_v1 is expressed in the pituitary as well as in other brain tissues. Levels of full-length GNRHR2 mRNA fluctuate with changes in the reproductive status of both females and males, with a minimum level observed in sexually immature animals and a maximum level observed after photostimulation. In vitro, this novel receptor couples to Gq, and stimulation with GnRH results in a dose-dependent increase in IP production. Furthermore, as previously reported for chicken GNRHR1 [22], chicken GNRHR2 shows a significantly higher sensitivity to chicken GnRH II than to chicken GnRH I. In conclusion, the present study suggests an important role for GNRHR2 during sexual maturation and gonadotropin expression.
ACKNOWLEDGMENTS
We would like to thank the staff at Arkell Poultry Research Station for their technical help, Dr. Ursula Kaiser for providing us with GH3 cells, and Dr. Peter Lewis for reviewing the manuscript. We also thank Mr. Zeini for helping with the IP assays.
FOOTNOTES
1 Supported by the Poultry Industry Council; Natural Sciences and Engineering Research Council of Canada (NSERC-CRDPJ 298383-03); Ontario Ministry of Agriculture, Food, and Rural Affairs (OMAFRA project #025958). Chicken GNRHR2 cDNA sequence was deposited to GenBank under the accession number AY895154. 
2 Correspondence: FAX: 519 837 8867; gbedecar{at}uoguelph.ca
Received: 13 December 2005.
First decision: 4 January 2006.
Accepted: 25 July 2006.
REFERENCES
Ct method. Methods 2001 25:402-408[CrossRef][Medline]This article has been cited by other articles:
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  C. A. Flanagan, C.-C. Chen, M. Coetsee, S. Mamputha, K. E. Whitlock, N. Bredenkamp, L. Grosenick, R. D. Fernald, and N. Illing Expression, Structure, Function, and Evolution of Gonadotropin-Releasing Hormone (GnRH) Receptors GnRH-R1SHS and GnRH-R2PEY in the Teleost, Astatotilapia burtoni Endocrinology, October 1, 2007; 148(10): 5060 - 5071. [Abstract] [Full Text] [PDF]
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