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University of Cape Town/Medical Research Council Research Group for Receptor Biology, Institute of Infectious Diseases and Molecular Medicine and Division of Medical Biochemistry (C.A.F., M.C., S.M.), and Department of Medicine (C.A.F.), University of Cape Town Faculty of Health Sciences, Observatory 7925, South Africa; Department of Biological Sciences and Program in Neuroscience (C.-C.C., L.G., R.D.F.), Stanford University, Stanford, California 94305; Department of Molecular Biology and Genetics (K.E.W.), Cornell University, Ithaca, New York 14853; and Department of Molecular and Cell Biology (N.B., N.I.), University of Cape Town, Rondebosch 7700, South Africa
Address all correspondence and requests for reprints to: R. D. Fernald, Department of Biological Sciences and Program in Neuroscience, Stanford University, Stanford, California 94305-2130. E-mail: rfernald{at}stanford.edu.
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Introduction |
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Mammalian GnRH (mGnRH1) was first identified in the mammalian hypothalamus, and additional GnRH forms were discovered in nonmammalian vertebrates including teleost fish, which express three GnRH forms: GnRH1, GnRH2, and GnRH3 (4, 5). All species studied to date, including mammals, have two or three forms of GnRH produced by distinct but phylogenetically related genes (5, 6, 7, 8, 9). The amino acid sequences of GnRH2 and GnRH3 are conserved, but the structure of GnRH1 varies considerably across vertebrate species (5, 6, 8). The presence of two or three GnRH peptides within a single organism raises the possibility that GnRH receptors (GnRH-R) may have coevolved with their ligands.
Multiple GnRH-R types have been reported in mammals, birds, fish, and amphibians (8, 10, 11, 12). GnRH-Rs belong to the G protein-coupled receptor family, characterized by seven hydrophobic transmembrane domains linked by hydrophilic extra- and intracellular loops (8, 13, 14, 15). The first GnRH-R sequences were cloned from mammalian pituitary mRNA (8, 13, 14) and designated type I GnRH-Rs because they regulate reproductive function (16, 17). These receptors exhibit high affinity for mGnRH1 and low affinity for other naturally occurring forms of GnRH. A conserved amino acid motif (Ser-Asp/Glu-Pro, SDP), in extracellular loop 3 (EC3) of mammalian pituitary type I GnRH-Rs, determines their specificity for mGnRH1 (18, 19). Receptors that lack the SDP motif in EC3 are not specific for mGnRH1 (8, 10, 20, 21, 22) (see GnRH-R amino acid alignment, supplemental Fig. A, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). GnRH-Rs with a PEY motif in EC3 (GnRH-R1PEY) were initially classified as a nonmammalian type I GnRH-R, and they were predicted to regulate reproduction in response to GnRH1 (10). GnRH-Rs with a PPS motif were originally proposed to be the receptors for GnRH2 (10), and indeed, mammalian type II GnRH-Rs, which are specific for GnRH2, have a PPS motif (8, 23, 24).
Unfortunately, GnRH-Rs have been named numerically in the order of their discovery, without consideration of functional or evolutionary relationships (11, 25, 26, 27). Several GnRH-R nomenclature systems have been proposed that, although based on phylogenetic relationships, contradict one another (supplemental Table A, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). For example, the Astatotilapia burtoni GnRH-R1SHS described here was initially named GnRH-R2, following Troskie et al. (10) but was renamed as a type I GnRH-R to be consistent with Lethimonier et al. (12). Classification of receptors based on function has been hampered by lack of physiological evidence about regulation of the HPG axis in nonmammalian vertebrates.
We used an African cichlid fish species, A. burtoni, in which reproduction is socially regulated, to identify which GnRH-R regulates reproductive function and to clarify the evolutionary relationship among known receptors. A. burtoni males exhibit one of two distinct social phenotypes: dominant (territorial, T) animals, which are larger, brightly colored, and reproductively capable or subordinate (nonterritorial, NT) males, which are smaller and camouflaged, have regressed gonads, and school with females (28, 29). Shifts in social status produce a corresponding change in the gonads via the HPG axis (29, 30). We have previously shown that [Ser8] GnRH1 is the only form of GnRH found in the pituitary of A. burtoni, and it is up-regulated in T males, implying that it must regulate gonadotropin release (31).
We previously cloned a GnRH-R2PEY from A. burtoni and showed that it has a relatively poor response to [Ser8] GnRH1 in vitro (32) and is expressed in the dorsal-anterior and posterior pituitary (33). We also identified a partial sequence corresponding to EC3 of a second GnRH-R, now designated GnRH-R1SHS (32), which is expressed in the ventral anterior and posterior pituitary and up-regulated in T males compared with NT males (33, 34). We now report the complete cloning and tissue distribution of GnRH-R1SHS and compare the functional activities and regulation of GnRH-R2PEY and GnRH-R1SHS to determine whether expression and structure-function relationships support the identification of GnRH-R1SHS as the receptor regulating reproduction in A. burtoni.
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Animals
A. (Haplochromis) burtoni derived from a wild-caught population were raised in aquaria under conditions similar to their native equatorial habitat in Lake Tanganyika in Africa (pH 8, 28 C) (28). Both male and female fish (3–4 cm length) were used in these experiments. To collect brain tissue, fish were killed via rapid cervical transection. All work was performed in compliance with Stanford University (APLAC) guidelines.
RNA isolation, amplification, sequencing, and analysis of A. burtoni GnRH-Rs
Partial sequences corresponding to the cichlid GnRH-R1SHS were originally amplified using degenerate primers designed to conserved regions of transmembrane TM6 and TM7 (10, 38). We subsequently designed a pair of degenerate primers to conserved amino acid sequences identified by an alignment with amberjack (CAB65407), striped bass (AAF28464) (32), medaka (BAB7056), and European seabass (DLA419594) GnRH-Rs. Sense primer S2 [5'-gtgggcigcica(t/c)(a/t)(g/c)iga(c/t)ggiaa-3'] was designed to the conserved motif WAAHSDGK, and the antisense primer AS1 [5'-gtticc(c/t)tcia(a/g)(g/a)tc(g/a)tcigg(g/a)aa-3'] was designed to the conserved motif FPDDLEGK. These primers were used to amplify a 750-nucleotide product from A. burtoni brain cDNA that was identified as a partial sequence for GnRH-R1SHS. Sequence information from this clone was used to design gene-specific primers for 5' RACE and 3' RACE to clone the full-length GnRH-R1SHS cDNA.
5' RACE and 3' RACE cDNA were prepared according to the manufacturer’s instructions (SMART RACE cDNA Amplification Kit; BD Biosciences, Franklin Lakes, NJ), from total RNA extracted from A. burtoni brain. Antisense primers CH3gsp (5'-tgcaggcaaagtctccggcaagc-3') and CH3ngsp (5'-aatcagcaccctcacgtgggattttcg-3') were used in combination with the 5' universal (UPM) and nested universal (NUP) primers to amplify the 5' end of GnRH-R1 cDNA from the 5' RACE A. burtoni brain cDNA. The 3' end of the cichlid GnRH-R1SHS receptor was amplified using the sense primers 3' gsp1 (5'-gcccgagagcccggatgagaactctg-3') and 3' ngsp1 (5'-gtgattgttctgtctttcatcatctg-3') in combination with the universal (UPM) and nested universal (NUP) from the 3' RACE A. burtoni brain cDNA. Commercial kits (BD Advantage 2 PCR Enzyme System; BD Biosciences) were used according to the manufacturer’s instructions for both 5' and 3' PCR amplification reactions.
Sequence information from the 5' RACE and 3' RACE cDNA clones was used to design a sense and antisense primer that, respectively, included the start and stop codons of the GnRH-R1SHS mRNA transcript, and which also included either an additional BamHI or XhoI site (italic): FLs 5'-gcgcggatccaccatggtggatgggcac-3' and FLas 5'-ggccggctcgagtcataagatgctctcag-3'. The full-length GnRH-R1SHS coding region was amplified from A. burtoni brain cDNA, using this pair of primers and subcloned into a BamHI/XhoI-digested pcDNA3.1(+) expression vector (Invitrogen). Expression plasmids were purified using the PureYield Plasmid Midiprep System according to the manufacturer’s alternative protocol (Promega) and sequenced using T7 and BGH reverse primer (Invitrogen, Carlsbad, CA). The nucleotide sequence of the full-length coding region is in GenBank [AY705931 (GnRH-R1, GnRH-R1SHS)].
Phylogenetic analysis of GnRH-R
We used phylogenetic analysis to situate A. burtoni GnRH-Rs with respect to previously cloned GnRH-Rs. All publicly available (February 14, 2007) vertebrate full-length GnRH-Rs were obtained from GenBank. Multiple GnRH-R sequences were aligned using the predicted sequences of GnRH-R polypeptides (ClustalW program in MEGA3.1) (39). This multiple sequence alignment was used to generate a neighbor-joining tree with bootstrap values using the Jones-Taylor-Thornton matrix. Lamprey GnRH-R was used to root the phylogenetic tree. Full species names and GenBank accession numbers for the receptor cDNAs are listed in the supplemental material (published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org).
Functional assay of GnRH-R1SHS
For functional tests, the A. burtoni GnRH-R1SHS cDNA expression plasmid was transfected into COS-1 cells using diethylaminoethyl dextran, as previously described (40). The ligand structure-function relationships of the putative GnRH-R1SHS were directly compared with A. burtoni GnRH-R2PEY (32) by measuring ligand-stimulated inositol phosphate (IP) production, as described previously (40), except that IP was extracted with formic acid (10 mM). Ligand concentrations ranged from 10–10 to 10–5 M. Data points were determined in duplicate, and EC50 values are the means of three separate experiments.
For competition binding assays, [His5,D-Tyr6] GnRH1 was radioiodinated as previously described (41). Transfected cells were washed twice with cold PBS and incubated (5 h at 4 C) with 125I-labeled [His5,D-Tyr6] GnRH1 (105 cpm) in the absence (B0) or presence of various concentrations (10–9 to 10–5 M) of unlabeled peptides in 0.5 ml HEPES-buffered DMEM with BSA (0.1%). Unbound 125I-labeled [His5,D-Tyr6]-GnRH was removed by washing twice with PBS, and cell-associated radioactivity was solubilized in 1 ml 1 M NaOH and counted. Nonspecific binding was determined in the presence of excess GnRH2 (10–5 M). Data points were determined in duplicate, and the IC50 values are the means of three separate experiments.
Localization of GnRH-R1SHS mRNA transcripts
GnRH-R1SHS expression was localized using PCR on tissue samples from adult A. burtoni (brain, pituitary gland, retina, gill, heart, intestine, kidney, liver, muscle, ovary, retina, spleen, and testes) and in situ hybridization using antisense receptor-specific probes in the brain and pituitary.
In situ hybridization
In situ hybridization methods are described in detail elsewhere (33), so only essential details are given here. Reproductively active males (n = 3) were killed by rapid cervical transection, and brains were removed and placed in sterile PBS. Tissue was frozen in mounting medium (OCT; Tissue-Tek, Torrance, CA) on dry ice, sectioned coronally, and stored at –80 C until use. Probes for GnRH-R1SHS and GnRH-R2PEY were synthesized, based on GenBank sequences (GnRH-R1SHS, GenBank AY705931; GnRH-R2PEY, AY028476), and plasmids were linearized to generate both sense and antisense templates for each receptor. GnRH-R1SHS and GnRH-R2PEY probes were from 151-1035 bp and from 55–1129 bp, respectively, and were generated by transcribing with SP6 or T7 polymerase in the presence of [35S]UTP (Maxiscript kit; Ambion, Austin, TX). Slides with brain tissue were treated as described previously (33). Photomicrographs were captured digitally (SPOT camera system; Diagnostic Instruments, Sterling Heights, MI) and optimized for clarity (PhotoShop; Adobe, San Jose, CA).
PCR on tissue samples
Tissue from T males (
6 cm; ovaries from sexually mature female 6 cm standard length) was homogenized and RNA was extracted (RNeasy Micro Kit; QIAGEN Inc., Valencia, CA). cDNA was synthesized from total RNA from each tissue sample (3' RACE, SMART kit; Clontech Laboratories Inc., Palo Alto, CA). Sense (5'-attgttctgtctttcatcatctgctgga-3') and antisense (5'-tgctctcagcacgactctcgt-3') primers specific to A. burtoni GnRH-R1SHS generated a 420-bp PCR product, whereas sense (5'-ggctgctcagttccgagtt-3') and antisense (5'-gtgaggaccctctctggtggacatt-3') primers specific to the A. burtoni GnRH-R2PEY generated a 961-bp PCR product from the cDNA template. The GnRH-R2PEY primer pair spans two introns predicted from the conserved GnRH-R genomic structure in fish (12). Positive controls for primers were performed using brain cDNA because in situ hybridization assays have shown both GnRH-R1SHS and GnRH-R2PEY expression in the brain (33). Negative controls were performed using the same procedure as for the experimental group without adding cDNA from any tissue. PCR was performed with a 68–60 C touchdown protocol as follows: 3 min denaturation at 95 C, followed by 16 cycles of 30 sec denaturation at 95 C, 30 sec annealing (68–60 C), and 15 min extension at 72 C. These reactions yielded a single product, as revealed by gel electrophoresis.
Immunocytochemical staining
To localize the somatotropes and gonadotropes, reproductively active fish (T males; n = 2) were killed by rapid cervical transection, and their brains removed and placed in 4% paraformaldehyde in PBS (pH 7.4) overnight and then transferred into a 30% sucrose solution overnight. Fixed tissue was frozen in OCT on dry ice and stored at –80 C. Three series of sections were cut on a cryostat (Microm; Zeiss, Oberkochen, Germany) in coronal or sagittal planes at 14 µm.
Sections were rehydrated (PBS) and incubated in blocking solution (0.3% Triton X-100, 0.2% BSA, 10% normal goat serum diluted in PBS). Slides were incubated separately in three primary antisera [rabbit anti-GH antibody (lot 8502), which labels somatotropes, and rabbit anti-LH antibody (lot 8506) and rabbit anti-FSH antibody (lot 8707), which label gonadotropes in A. burtoni, all generously provided by Dr. A. Takahashi] diluted 1:1000 in blocking solution at 4 C overnight (42). The other set of slides was incubated in blocking solution instead of primary antiserum for the control group. All of the sections were washed in PBS, and the signal was amplified (ABC kit; Vector Laboratories, Burlingame, CA). Sections were incubated in 3',3'-diaminobenzidine (Sigma-Aldrich, St. Louis, MO) to visualize the bound antibody and counterstained with cresyl violet to visualize cell nuclei. The slides were dehydrated in an ethanol/xylene series and coverslipped with Permount medium for microscope viewing.
Regulation of GnRH-R mRNA abundance
Tissue preparation.
To discover whether expression of GnRH-R mRNA was regulated, either as a function of diurnal time or with respect to GnRH ligand expression patterns, animals (n = 24) were housed in separate aquaria with three animals of mixed sex including one T male in each of eight tanks to provide minimal disturbance of other animals in the room when the animals were killed. Tissue was collected from animals in a single tank at 3-h intervals over a 24-h period beginning at 1000 h (fish were maintained on a 12-h light, 12-h dark cycle with dark-to-light transitions at 0900 h). At night, the aquarium room was accessed through a light-tight door, and all tissue-processing steps were performed in complete darkness using minimal infrared illumination. Fish were killed by rapid cervical transection, and their brains and pituitaries were rapidly removed. Brain and pituitary samples were stored at –80 C until use.
Tissue taken at each time point was separately homogenized in chilled TRIzol (Invitrogen) with a Tissue Tearor (Biospec Products, Bartlesville, OK) and kept frozen in TRIzol reagent at –80 C until RNA extraction.
RNA extraction and PCR sample preparation.
Total RNA was extracted from samples following a standard protocol (TRIzol; Invitrogen). RNA was DNase treated to remove genomic DNA contamination (TURBO DNA-free; Ambion), and 1.0 µg total RNA from each tissue sample was reverse transcribed to cDNA (SuperScript II RNase H reverse transcriptase; Invitrogen).
Quantitative real-time PCR of A. burtoni GnRH ligands and receptors.
Primers for quantitative real-time PCR for [Ser8] GnRH1 were 5'-cagacacactgggcaatatg-3' and 5'-ggccacactcgcaaga-3', generating a 128-bp product; for GnRH2, primers were 5'-tggactcctttggcacatcagaga-3' and 5'-ctctggctaaggcatccagaagaa-3', generating a 126-bp product; for GnRH3, 5'-atggatggctaccaggtggaaaga-3' and 5'-tggatttgggcatttgcctcatcg-3', generating a 11- bp product; for GnRH-R1SHS, 5'-tcagtacagcggcgaaag-3' and 5'-gcatctacgggcatcacgat-3', generating a 187-bp product; and for GnRH-R2PEY, 5'-ggctgctcagttccgagtt-3' and 5'-cgcatcaccaccataccact-3', generating a 220-bp product. Three housekeeping genes, actin, 18S rRNA, and tubulin, cloned in A. burtoni were used to control for sample differences in total cDNA content. PCR were performed (iCycler; Bio-Rad, Hercules, CA), and reaction progress in 30-µl reaction volumes was monitored by fluorescence detection at 490 nm during each annealing step. Reactions contained 2x IQ SYBR Green SuperMix (Bio-Rad), 10 µM of each primer, and 1 ng cDNA (RNA equivalent). Reaction conditions were 1 min at 95 C and then 35 cycles of 30 sec at 95 C, 30 sec at 60 C, and 30 sec at 72 C, followed by a melting curve analysis over the temperature range from 95 C to 4 C. All reactions were run in duplicate.
Quantitative real-time PCR data analysis.
Fluorescence readings for each sample were baseline subtracted, and suitable fluorescence thresholds were automatically determined by the MyiQ software. To determine the number of cycles needed to reach threshold, the original fluorescence reading data were analyzed using a special PCR algorithm (43). To calculate the relative mRNA amount, target gene levels were normalized using the geometric mean of housekeeping gene mRNA values.
Statistical analyses
To test the hypothesis that GnRH ligands and/or receptors were expressed with a diurnal rhythm, we analyzed the mRNA samples taken around the clock for rhythmic variation using generalized additive models (44, 45). This technique identifies nonlinear effects that might produce rhythmicity by analyzing the residuals of a scatterplot smoother fit to the data with a back-fitting algorithm. For the generalized additive models and the linear regressions, we took P < 0.05 as the initial significance level and applied a Bonferroni step-down correction to account for family-wise error due to multiple testing. These analyses were computed using R (R:Statistical computing environment; R Foundation, Vienna, Austria).
To identify potential linear relationships between ligands/receptors in a larger data set, we used robust regression (Siegel repeated means) (46) that allowed for the observed nonconstant variance (heteroskedasticity) in the data without requiring data transformation. As a control, we similarly compared the relationships between the ligands/receptors and the Clock 1a gene (GenBank DQ923857). To look for independent sources underlying the data distribution, we used independent component analysis (ICA), which is a recently developed technique (see survey in Ref. 44) related to latent variable and factor analyses. Unlike more traditional approaches, ICA yields unique (except for the sign) solutions to the problem of finding underlying sources of variation by maximizing the information contained in each independent source of interaction (44). The resulting sources may thus be interpreted as latent variables containing information about independent sources of variation in the data. We examined the relationships of each of the ligands/receptors with these latent variables by regressing each of the ligands/receptors on each of the independent sources, again using robust regression. We used a consensus source matrix from 100 solutions arrived at using the maximum-likelihood ICA package (computed using R). It is important to note that because the ICA signs are arbitrary, we can discover whether ligands/receptors relate to sources in the same or opposite directions or not at all (i.e. the signs can be reversed, but not the relative relationships between variables).
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Phylogenetic analysis
Phylogenetic analysis of vertebrate GnRH-Rs identified two families of GnRH-Rs (a and b), each containing two subfamilies, which are distinguished by conserved motifs in EC3 (Fig. 1
and supplemental Fig. A). The a1 family includes GnRH-Rs from all nonmammalian vertebrate taxa, including fish, reptiles, birds, and amphibians that are characterized by a conserved TPEYVH motif in EC3. All mammalian GnRH-Rs, known to regulate reproduction, form a distinct subfamily a2, characterized by a consensus VSDPVN motif in EC3 and the absence of a C-terminal tail (supplemental Fig. A). With less bootstrap support, family b GnRH-Rs can be split into two subfamilies that are also characterized by different but related motifs in EC3. Teleost and amphibian receptors in subfamily b2 have a VSHSLT motif, whereas GnRH-Rs from amphibians and some mammals belong to subfamily b1, characterized by the VPPSLS motif. It should be noted that although the GnRH-R2PPS from chicken appears to be an exception, its phylogenetic inclusion in subfamily b2 has a very low bootstrap value. Phylogenetic analysis with alternative algorithms (e.g. minimal evolution) yielded very similar topology (data not shown). These and other data (8, 10, 12) showing that GnRH-R families can be distinguished by conserved amino acid motifs in EC3 allow an unambiguous classification system for vertebrate GnRH-Rs. The phylogenetic tree shows that the two types of GnRH-Rs found in teleosts, GnRH-RSHS and GnRH-RPEY, diverged early in vertebrate evolution and are neither recent duplications nor limited to teleosts.
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and Table 1). We found that GnRH-R1SHS had relatively low affinity for the three forms of GnRH that are expressed in A. burtoni. IC50 values ranged from 53.5 nM for GnRH2, which had highest affinity for GnRH-R1SHS, to 20,300 nM for [Ser8] GnRH1 (Table 1 and Fig. 2). Consistent with our previous report, GnRH-R2PEY exhibited similar low affinities for all peptides, and peptides had the same rank order of IC50 values in cells expressing either GnRH-R1SHS or GnRH-R2PEY (GnRH2 < GnRH3 < mGnRH1 < [Ser8] GnRH1; Table 1 and Fig. 2).
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and Fig. 2). In contrast, only GnRH2 exhibited high potency at the GnRH-R2PEY (EC50 0.208 ± 0.034 nM) (Table 1 and Fig. 2).
In vitro analyses show that the A. burtoni GnRH-Rs have similar affinities for each of the GnRH forms in A. burtoni. However, comparison of IC50 and EC50 values indicates that the two receptors differ in their efficiency in coupling binding of different GnRH peptides to intracellular signaling. This is evident from the similar potencies of GnRH2 and GnRH3 (EC50 values 8.11 ± 2.63 and 11.0 ± 2.5 nM) at the GnRH-R1SHS, even though GnRH3 has considerably lower affinity (IC50, 1143 ± 578 nM) at this receptor than GnRH2 (IC50, 53.5 ± 24.6 nM). To quantify these observations, we have calculated an efficiency quotient (IC50/EC50, Table 1). Clearly, the GnRH-R1SHS signaling response is particularly sensitive to GnRH3 and poorly responsive to GnRH2, whereas the GnRH-R2PEY is highly responsive to GnRH2 (Table 1). It is notable that neither receptor is highly responsive to [Ser8] GnRH1.
Tissue distribution and localization of A. burtoni GnRH-R1SHS and GnRH-R2PEY
We mapped the distribution of both GnRH-Rs in A. burtoni using RT-PCR for peripheral tissue and in situ hybridization for expression in the brain. GnRH-R1SHS is significantly more widespread in the body than is GnRH-R2PEY, being expressed in muscle, intestine, liver, and heart in addition to brain, retina, pituitary, gill, kidney, testes, and ovary where both forms are expressed (Fig. 3).
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) with each receptor type mapped using in situ hybridization (Fig. 5). The organization of the teleost pituitary requires that it be sectioned parasagittally to see these relationships clearly. In sagittal sections of the pituitary, the distributions of cells containing GH (Fig. 4A) or LH (Fig. 4B) and a schematic representation (Fig. 4D) are shown. GH- and LH-containing cells are in nonoverlapping regions of the pituitary. Distribution of FSH-containing cells was similar to that of LH-containing cells (not shown). Figure 4D shows the location of the plane of the coronal section through the pituitary used in Fig. 5. Based on in situ hybridization, GnRH-R1SHS is localized in a crescent around the ventral border of the pituitary, and GnRH-R2PEY is located dorsally, directly adjacent to the attachment of the pituitary to the brain, confirming previous results for A. burtoni (33). Comparing the in situ hybridization results with those of GH and LH distribution in the pituitary of A. burtoni, it is clear that the distribution of the GnRH-R1SHS correlates with gonadotropes and GnRH-R2PEY with somatotropes (Fig. 6).
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). Although previous reports suggested GnRH-R mRNA levels change with a diurnal rhythm (49), we found no statistically significant nonlinearity as would be consistent with a diurnal rhythm in either GnRH-R1SHS or GnRH-R2PEY expression.
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Comparison of vertebrate GnRH-R expression and responsiveness to different GnRH forms
The physiological role(s) of GnRH-Rs have been assessed by expression in appropriate cells of the pituitary, measurement of expression after physiological perturbations, and measurement of high affinity for GnRH1. To determine whether data in other species support the identification of GnRH-R1SHS as the reproductive receptor in A. burtoni, despite its low affinity, we reviewed prior research. We asked whether receptor amino acid sequence correlates with ligand responsiveness and with evidence of expression in gonadotropes or responses to perturbation of the HPG axis in nonmammalian vertebrates. We found no correlation of expression of either SHS- or PEY-type GnRH-Rs with reproductive function (supplemental Table B, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org).
Because there are limited data on ligand-binding affinities of most vertebrate GnRH-Rs, we compared EC50 values. However, absolute EC50 values measured in recombinant signaling assay systems are influenced by variables such as receptor expression levels and coupling efficiencies that vary among systems. Because all nonmammalian GnRH-Rs are highly responsive to GnRH2, we compared signaling responses stimulated by various GnRH forms relative to the response stimulated by GnRH2 to calculate a ligand response index (Table 2). We included all vertebrate GnRH-Rs that fit the four defined receptor types, SDP, PEY, SHS, and PPS, for which we could find dose-response data for GnRH2 and at least one other GnRH form (Table 2). The comparison shows that ligand response is similar within each receptor type, and there is a strong correlation of receptor sequence with ligand-stimulated response relative to the response to GnRH2 (see Table 2). As expected, the mammalian, SDP-type GnRH-Rs are highly responsive to mGnRH1 relative to GnRH2 (mean EC50 ratio 0.15, Table 2), whereas PPS-type receptors, the classic type II GnRH2 receptors, show high responsiveness to GnRH2 (mean ratio 239, Table 2). Of the GnRH-R types that occur in teleosts, the PEY-type receptors are highly selective for GnRH2 and very poorly responsive to all other GnRH forms (mean EC50 ratios 140-2900, Table 2). It is notable that chicken and bullfrog PEY-type receptors are exceptions, exhibiting good responses to GnRH1. In contrast, teleost SHS-type receptors show smaller differentials in peptide-stimulated responses (mean EC50 ratios 3.3–58, Table 2), meaning they are much less selective for GnRH2 and suggesting that a receptor of this type may be the GnRH1 receptor in teleosts.
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Discussion |
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To identify which GnRH-R regulates reproduction in A. burtoni, we cloned the GnRH-R1SHS receptor, known to be up-regulated in reproductively active T males, and measured its phylogenetic and functional characteristics. We found that GnRH-R1SHS is less closely related than the GnRH-R2PEY to the SDP-type receptors, which are clearly established as reproductive receptors in mammals. Interestingly, neither cichlid GnRH-R has high-affinity interaction with [Ser8] GnRH1, but GnRH-RSHS expression colocalizes with gonadotropes, whereas GnRH-RPEY does not.
Phylogenetic relationships of jawed vertebrate GnRH-Rs
Phylogenetic analysis identified four GnRH-R subfamilies with characteristic motifs in EC3. We have defined GnRH-R types according to their EC3 motif, indicated by a three-letter superscript, to reduce confusion of multiple contradictory nomenclature systems for GnRH-Rs (supplemental Table A) and poor correlation of assigned receptor names with receptor type. This unambiguous classification scheme placing GnRH-Rs into four types, SDP, PEY, PPS, and SHS using the EC3 motif, is based solely on receptor amino acid sequence. It has major advantages in that it includes all but a few closely related SHS- and PPS-type receptors (Fig. 1 and supplemental Fig. A) and cannot be confused with GenBank assigned receptor names.
It is likely that the four GnRH-R subfamilies identified (a1, a2, b1, and b2, corresponding to types PEY, SDP, PPS, and SHS) arose through gene duplication, an important process in evolution. Duplications allow new gene functions to evolve (50, 51), and there is good evidence in yeast that duplicated genes can provide functional compensation against null mutations (52). In this case, GnRH-RSHS and GnRH-RPEY show similar affinity for [Ser8] GnRH1, but they are clearly not functionally redundant, because their expression patterns are divergent. GnRH-R subfamilies can be understood in terms of the two rounds of genome duplication that occurred early in the vertebrate lineage (known as the 2R hypothesis) (53). Because this hypothesis would predict the presence of four GnRH-Rs in all vertebrates, the phylogenetic tree predicts that GnRH-R genes have been lost independently, on multiple occasions from different branches of the vertebrate lineages. For example, SDP-type (family a2) GnRH-Rs have been identified only in mammals and thus must have been lost independently in fish, amphibians, and reptiles. Similarly, PEY-type (family a1) GnRH-Rs are not found in mammals. Amphibians have GnRH-Rs in both subfamilies of family b (PPS-type and P/SQS variations of the SHS type), whereas all fish GnRH-Rs in family b are SHS type and all bird and mammalian receptors are PPS type. Although GnRH-RPPS receptors have been identified in amphibians, chicken, and nonhuman primates, this subtype is absent from the mouse and rat genome sequence data bases. The human GnRH-RIIPPS lacks a start codon, is truncated at the second transmembrane segment, and is nonfunctional (54, 55, 56). Similarly, a sheep GnRH-RIIPPS ortholog is also nonfunctional (57). Thus, this receptor has been independently lost in rodents and has become a pseudogene in humans and sheep.
Teleost fish have undergone an additional genome duplication since their divergence from tetrapods (58, 59), so it is not unexpected that there are multiple GnRH-Rs of a single receptor type in the Ostariophysi lineage including goldfish (GnRH-RAPEY and GnRH-RBPEY) (38), catfish (GnRH-R1PDY and GnRH-R2PEY) (25), and zebrafish (GnRH-R1PEY, GnRH-R3PEY, GnRH-R2SHS, and GnRH-R4SHS) (12). Similarly, in the Neoteleostei lineage multiple GnRH-Rs are present in pufferfish (GnRH-R1/III-1SHS, GnRH-R1/III-2SHA, and GnRH-R1/III-3SHS) and medaka (GnRH-R1SHS and GnRH-R3SHS).
In the case of A. burtoni, the different expression patterns of the GnRH-R genes suggest that these receptors are in the process of diverging in function. Only GnRH-R2PEY is colocalized on all three groups of neurons that produce GnRH, apparently specialized for feedback control of GnRH production (33). Moreover, GnRH-R1SHS colocalizes with gonadotropes, whereas GnRH-R2PEY colocalizes with somatotropes, revealing different roles in the pituitary. When detailed data about receptor localization and function are available from other species, it will become clear whether these specializations are unique to teleosts and whether evolution has taken different directions in different species.
Role of GnRH-R1SHS as a functional receptor for [Ser8] GnRH1
We have previously shown, in A. burtoni, that [Ser8] GnRH1 is the only GnRH peptide found in the pituitary and that it is up-regulated in reproductively active T males. The low affinity of both A. burtoni receptors for [Ser8] GnRH1 indicates that high concentrations of [Ser8] GnRH1 (in the micromolar range) would be required in the pituitary to activate either receptor. Superficially, this might suggest that neither receptor is the target of [Ser8] GnRH1 but that some other receptor with a higher affinity for [Ser8]-GnRH1 may exist. However, all of the nonmammalian GnRH-Rs that have been functionally analyzed have the highest affinity for GnRH2, and all fish receptors are poorly responsive to GnRH1 forms (11, 12, 25, 27, 38) (Table 2). Available information from zebrafish and pufferfish genomes does not reveal additional GnRH-Rs, making it unlikely that receptors with high similarity to currently known GnRH receptors and high affinity for GnRH1 exist.
Although [Ser8] GnRH1 had low potency in stimulating IP production in our recombinant system, the efficiency of [Ser8] GnRH1-stimulated activation of fish G proteins is not known. High-affinity GnRH-R binding has been reported in fish pituitary tissue (60, 61, 62, 63) using synthetic, high-affinity ligands, before the discovery of the teleost forms of GnRH1 (see Ref. 12). However, consistent with results in recombinant systems, catfish GnRH1 was found to have low potency in stimulating gonadotropin release and had very low affinity in GnRH-R binding assays performed with catfish pituitary membranes (60). This suggests the known receptors may regulate reproduction despite their low affinity for the biologically relevant ligands, raising the possibility that synchronous release of GnRH in the pituitary may lead to transiently elevated levels triggering the receptors.
Consistent with their known functions in mammals, all SDP-type GnRH-Rs are highly selective for mGnRH1, and all PPS-type receptors are highly selective for GnRH2 (Table 2). This suggests that PPS-type GnRH-Rs may have a conserved function in amphibians and in nonhuman primates. As in A. burtoni, all SHS-type GnRH-Rs exhibit relatively low ligand selectivity, with similar responses to different GnRH peptides, suggesting that SHS-type receptors may have similar functions in teleost and amphibian species. In contrast, all PEY-type GnRH-Rs from fish species are highly selective for GnRH2, suggesting that GnRH2 may be their major physiological ligand. However, PEY-type receptors from chicken and bullfrog are much less selective for GnRH2 and are quite responsive to GnRH1 ([Gln8] GnRH1 or mGnRH1, respectively) suggesting that the functions of PEY-type GnRH-Rs may differ between teleosts and other vertebrates. Indeed, there is physiological evidence that the GnRH-R1PEY regulates reproduction in chickens, because its expression is up-regulated in the pituitaries of castrated cockerels (64). However, these data are not consistent with a recent study in which expression of GnRH-R2SHS was shown to increase with reproductive status in male and female chickens (65).
Which GnRH-R regulates reproduction in A. burtoni?
Several lines of evidence suggest that the GnRH-R1SHS receptor regulates reproduction in A. burtoni. First, GnRH-R1SHS receptor mRNA transcripts are up-regulated in the pituitaries of T males, whereas GnRH-R2PEY receptor mRNA transcripts are not (34). Second, GnRH-R1SHS mRNA transcripts specifically localize with expression of gonadotropes, whereas GnRH-R2PEY transcripts localize with somatotropes. Our results using in situ hybridization contrast with results using receptor immunocytochemistry in the closely related teleost O. niloticus (42). In O. niloticus, antibodies that would be predicted to recognize the GnRH-R1SHS (AB111356) reacted with somatotrope cells, whereas antibodies that would be predicted to recognize GnRH-R2PEY (AB111357) reacted with gonadotropes (42). Because both species are cichlids, this difference is likely to result from the different techniques used. The antibodies were raised against short peptides corresponding to the EC3 sequences of GnRH-Rs. These peptide sequences can be readily identified in diverse proteins by a BLAST search, so it is possible that the antibodies recognize the peptide sequences of other proteins besides GnRH-Rs (57). Thus, although comparing GnRH-R expression patterns in different species (supplemental Table B) does not yield a consensus on whether GnRH-RPEY or GnRH-RSHS receptors regulate release of gonadotropins in nonmammalian vertebrates, our additional expression data confirm that GnRH-R1SHS is the receptor expressed in the gonadotropes of A. burtoni.
Coregulation of GnRH ligand and receptor expression
The widespread distribution of GnRH-Rs (66, 67) has led to speculation that different forms of GnRH may play roles beyond reproduction. Chen and Fernald (33) recently showed that the receptor types differ in their brain distribution in A. burtoni and only GnRH-R2PEY colocalized with the three GnRH-producing cell types in the brain, suggesting direct feedback control of the GnRH production. The coregulation of receptors with GnRH suggests that the GnRH system has some common regulatory underpinnings. The presence of multiple GnRH forms across all vertebrates suggests they might share some functional role in reproduction with GnRH1.
GnRH-R evolution and the usefulness of the EC3 motif classification system
The poor correlation of GnRH-R sequence with a role in reproduction across species (supplemental Table B) makes it impossible to predict which GnRH-R will regulate reproductive function in any particular species. The absence of SHS-type GnRH-Rs in higher vertebrates precludes their role as the receptor responsible for reproduction in these species. Different families of duplicated receptor genes regulate reproductive function in mammals (family a, SDP-type) compared with fish (family b, SHS-type). There is insufficient evidence in other taxa (birds, reptiles, and amphibians) to conclude which receptor is used or when selection for one receptor rather than another might have occurred. Thus, it makes sense to define GnRH-R types by genotype, which, perforce, correlates with EC3 amino acid motif and with ligand selectivity, rather than using a classification system based on physiological function in the face of insufficient and conflicting information.
In summary, we cloned the GnRH-R1SHS receptor, which is up-regulated in reproductively active A. burtoni, and showed that it is coexpressed with LH in the pituitary. Like all teleost GnRH-Rs, it binds and responds poorly to GnRH1. Although this remains puzzling, it suggests that synchronous release of GnRH1 into the pituitary allows delivery of sufficient ligand to activate the receptor. The absence of the SHS-type GnRH-R in many taxa and the poor correlation of GnRH-R sequence with a role in reproduction across species, suggest that different families of duplicated GnRH-R genes have been selected to regulate reproduction during vertebrate evolution.
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Acknowledgments |
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Footnotes |
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Present address for C.A.F.: School of Physiology, University of the Witwatersrand, Parktown, South Africa.
Present address for K.E.W.: Centro de Neurociencia, Universidad de Valparaiso, Valparaiso, Chile.
Disclosure Summary: The authors have nothing to disclose.
First Published Online June 26, 2007
Abbreviations: EC3, Extracellular loop 3; GnRH-R, GnRH receptor; HPG, hypothalamic-pituitary-gonadal; ICA, independent component analysis; IP, inositol phosphate; mGnRH1, mammalian GnRH1; NT, nonterritorial; T, territorial.
Received October 17, 2006.
Accepted for publication June 15, 2007.
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), [Ser8] GnRH1 (
), GnRH2 (
), or GnRH3 (
) for competition binding assays or (C and D) labeled with [3H]myoinositol and incubated with increasing concentrations of peptides in the presence of LiCl for peptide-stimulated IP production.
) and GnRH-R2PEY (
) receptor types relative to the GH cells (horizontal lines) and LH cells (vertical lines). Scale bar, 100 µm. Abbreviations are as in Fig. 4