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Program in Neuroscience and Department of Psychology, Stanford University, Stanford, California 94305-2130; Department of Biochemistry, University of Cape Town (N.I., M.M., R.P.M.), Rondesbosch 7700, South Africa; and Medical Research Council Research Unit for Molecular Reproductive Endocrinology, University of Cape Town Medical School (B.E.T.), Cape Town, South Africa
Address all correspondence and requests for reprints to: Dr. R. D. Fernald, Program in Neuroscience and Department of Psychology, Stanford University, Stanford, California 94305-2130. E-mail: russ{at}psych.stanford.edu
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Abstract |
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Introduction |
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A pituitary GnRH receptor (GnRH-R) complementary DNA (cDNA) was first isolated in the mouse, and its functionality was confirmed via expression in Xenopus oocytes (1, 2). Subsequently, GnRH-Rs have been isolated in many mammalian species (3), and recently full-length clones have been obtained from nonmammalian species, including Xenopus laevis (4), African catfish (5), and two GnRH-R forms in the goldfish (6). These three fish GnRH receptors share approximately 70% amino acid identity and are about 42% identical to the human GnRH-R (6). The genomic DNA of several species contains duplicate forms of GnRH-R (7), but the question of whether multiple GnRH-Rs are typically expressed in diploid species is still unresolved (8).
Previous work from our laboratory has shown that reproductive state is socially regulated in Haplochromis burtoni. Dominant males have larger testes and more mature sperm than nondominant males (9). GnRH-Rs seem a likely site for social control of reproduction. As a first step in testing this hypothesis, we characterized a GnRH-R in H. burtoni.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RNA isolation, amplification, and analysis of GnRH-R
RT and cDNA amplification were performed as described previously
(10) with the following differences. Total RNA was
extracted from the brains of male and female H. burtoni. A
nested PCR protocol was employed, using the following primers based on
conserved regions of both goldfish GnRH-R subtypes (6) and
the catfish GnRH-R (5): FH1
(5'-ATGACITT(C/T)(A/G)TIGTIATGCCI(T/C)T-3') and FH5
(5'-GG(T/C)TG(A/G)AACCA(A/G)TACCA(G/T/A)ATICC-3') for the primary
reaction, and FH3 (5'-AA(C/T)GTIACIGTICA(A/G)TGGTA(C/T)GC-3') and FH4
(5'-GCIA(G/A)IA(G/A)(A/G)TA(A/G)TAIGGIGTCCA(A/G)C-3') for the secondary
reaction. Both reactions were performed in a Rapidcycler (Idaho
Technologies, Idaho Falls, ID) with a 55 C-50 C touchdown
protocol as follows: 15-sec denaturation at 94 C, followed by 35 cycles
of 0-sec denaturation at 94 C, 0-sec annealing (55 C-50 C), and 15-sec
extension at 72 C. These reactions yielded a single product, as
revealed by gel electrophoresis and sequencing.
To obtain the complete cDNA, rapid amplification of 5'- and 3'-cDNA ends (5'- and 3'-RACE) was performed with hypothalamic total RNA using the SMART cDNA Synthesis Kit (CLONTECH Laboratories, Inc., Palo Alto, CA). The RACE products from this cDNA (Marathon kit, CLONTECH Laboratories, Inc.) were sequenced and found to contain the start and end codons. New primers, specific for the 5'- and 3'-untranslated regions of the H. burtoni GnRH-R, were then used to obtain the full-length cDNA.
The nucleotide sequence and predicted peptide sequence were compared with known GnRH-R cDNAs in GenBank (www.ncbi.nlm.nih.gov/BLAST). The sequences were aligned with ALIGN and CLUSTALW (http://dot.imgen.bcm.tmc.edu:9331). Transmembrane domains of the H. burtoni sequence were predicted by SOUSI (http://azusa.proteome.bio.tuat.ac.jp/sosui).
Cloning of GnRH-R cDNA
The full-length PCR product was purified and blunt end-cloned
into pBluescript II (Stratagene, La Jolla, CA). After
ligation, the recombinant plasmid was used to transform competent
Escherichia coli XL1-Blue MRF' cells
(Stratagene). After overnight incubation on Luria Bertoni
broth-ampicillin plates and blue-white color selection, six
transformants were chosen for further analysis. The orientation of the
inserts was confirmed by sequencing with T3 and T7 primers.
Transient transfection of COS-1 cells and functional assay
The H. burtoni GnRH-R cDNA was ligated into pcDNA/Amp
vector (Invitrogen, San Diego, CA) and transfected into
COS-1 cells as previously described (6). The functionality
of the putative GnRH-R was tested by measuring inositol phosphate (IP)
production in response to GnRH. The IP assay was performed as described
previously (6). The GnRH agonists tested appear in Table 1
. Among these are the mammalian
releasing form of GnRH (mGnRH1) and the three forms of GnRH found in
H. burtoni: sbGnRH1 (or seabream GnRH) in the hypothalamus,
GnRH2 (or chicken-II GnRH) in the midbrain, and GnRH3 (or salmon GnRH)
in the telencephalon (11). Data points were determined in
duplicate, and the EC50 values represent the mean
of three separate experiments.
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GnRH-R messenger RNA (mRNA) localization
PCR was performed on tissue from adult H. burtoni
(gill, heart, intestine, kidney, liver, muscle, ovary, retina, spleen,
and testes). In addition, brains from six adult fish were partitioned
for RT-PCR analysis: cerebellum, pituitary, optic tectum,
telencephalon, and the remaining brain, including the hypothalamus.
cDNA was synthesized as described above, except 0.5 µg RNA was used
from pituitary and 1.0 µg from cerebellum. Primers specific to the
H. burtoni GnRH-R were designed, Hb5'
(51-TGACAGTGCAGTGGTATGGTG-31) and Hb3'
(51-TCAGAGTCTTCATCCGAGCCTTTG-31), which generated a 483-bp PCR product.
These primers flank an intron in the GnRH-R sequence, so any amplicons
from possible genomic contamination can be eliminated. DNA Positive
controls were performed using primers for glycerol-3-phosphate
dehydrogenase, the ubiquitous housekeeping gene. Every reaction was
performed at least twice. In addition, negative controls were performed
using RNA (without RT) as a template in our amplification protocol.
None of these reactions produced products.
Phylogenetic analysis of GnRH-R
To situate the H. burtoni GnRH-R with respect to
previously cloned GnRH receptors, phylogenetic analysis was used. The
predicted sequences of GnRH-R polypeptides were aligned using ClustalW
1.8 (http://dot.imgen.bcm.tmc.edu:9331/multialign/multialign.html) and
were subsequently converted to the NEXUS format with READSEQ
(http://dot.imgen.bcm.tmc.edu:9331/seq-util/seq-util.html). The trees
were then generated using PAUP1 4.0b3a (13). To test
robustness, trees were generated using neighbor-joining, star
decomposition, heuristic, and branch and bound methods, all of which
produced very similar results. These analyses were performed both on
sequences lacking the relatively unconserved extra- and intracellular
tails and on full-length sequences, both of which yielded similar
relationships in the trees. Full species names and GenBank accession
numbers for the receptor cDNAs are as follows: African catfish,
Clarias gariepinus, O42329; goldfish, Carassius
auratus, IA: AAD20001 and IB: AAD20002; eel, Anguilla
japonica, BAB11961; pig, Sus scrofa, P49922; cow,
Bos taurus, AAC48857; mouse, Mus musculus,
AAB59636; sheep, Ovis aries, AAC37336; rat, Rattus
norvegicus, AAC27349; and human, Homo sapiens, NP
000397. Also, the sequences for possum (Trichosurus
vulpecula) GnRH-R (14) and frog (Xenopus
laevis) type I GnRH-R (4) were used. The following
sequences were unpublished at the time of this writing: perciform
fishes Seriola dumerilii (CAB65407) and striped bass
(Morone saxatilis, AAF28464), a caecilian amphibian
(Typhlonectes natans, AAD49750), horse (Equus
caballus, O18821), and type II from Xenopus (Troskie,
B., N. Illing, and R. Millar, unpublished data). A fragment (33 amino
acids) of a putative type II GnRH-R gene from H. burtoni
genomic DNA was included in the tree for the purpose of illustrating
the evolutionary divergence of the two types (see
Discussion). The sequence for this putative type II GnRH-R
gene has been submitted to GenBank (Accession No. AF356598).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). This sequence shows highest homology
to sequences from other GnRH receptors, followed by sequences from
vasopressin and oxytocin receptors. In particular, the cDNA we found is
most similar to the GnRH-R sequence reported in goldfish and African
catfish (Fig. 1B). Similar to the other teleost GnRH-R receptors, but
in contrast to mammalian GnRH-R forms, the H. burtoni GnRH-R
includes approximately 50 intracellular amino acid residues at the
C-terminus. The intracellular tail, which is lacking in mammals, is
never highly conserved, and the extracellular tail shows at most a
modest conservation.
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), as has been done for
the mouse GnRH-R sequence (15). A cloned variant was
missing the sequence from base 196 (TATTG/intron/GTCAG) to base 528
(CACAG/intron/CTATT). The latter position corresponds to an intron
found in GnRH-R genes in humans, rats, mice, sheep, and even a
GnRH-R-like gene from Drosophila melanogaster
(15, 16, 17, 18, 19). This putative conserved intron corroborates
phylogenetic analysis (see below) regarding GnRH-R in H.
burtoni. Another variant, amplified from retinal cDNA in addition
to the wild-type GnRH-R, was missing the sequence from base 710
(CCACC/intron/AGCAG) to base 762 (GCACC/intron/GACAT).
Phylogenetic relationships
The comparison of GnRH-R sequences between H. burtoni
and other teleost fish species (Fig. 1B) suggests that there may be two
distinct types. Specifically, the H. burtoni cDNA sequence
shows higher homology with those of goldfish and African catfish than
to those of S. dumerilii and striped bass, although these
latter species are perciforms like H. burtoni. As previously
described (7), the former sequences are designated type I,
and the latter are similar to the so-called type II sequences. Thus, we
designated our cDNA sequence as encoding the type I GnRH-R in H.
burtoni.
We constructed a phylogenetic tree from the available sequences of
expressed GnRH-R genes to examine the pervasiveness of multiple GnRH-R
types. It is evident from the GnRH-R phylogenetic tree (Fig. 1C) that
the type I and II GnRH-R diverged early in vertebrate evolution and are
not recent duplications or necessarily limited to teleosts. Indeed,
both teleost and amphibian sequences are divided between two branches
of the tree. We have amplified a fragment of a putative type II
receptor gene in H. burtoni, as shown in the phylogenetic
tree. Based on the phylogenetic relationships (Fig. 1C), goldfish IA
and IB receptors (6) appear to be a relatively recent
duplication. In contrast, no second type I receptor has been identified
in the H. burtoni genomic DNA.
Location of GnRH-R in H. burtoni tissue
The location of GnRH-R could suggest its physiological roles.
RT-PCR revealed that the GnRH-R-I mRNA has widespread distribution in
the brain (Fig. 2), in contrast to the
spatially distinct expression of the three endogenous forms of GnRH in
this species (11). GnRH-R-I mRNA is also strongly
expressed in testes, kidney, and retina (Fig. 2). The expression of
this mRNA in both retina, which contains GnRH3, and pituitary, which
contains GnRH1, strongly indicates that this receptor is probably
activated by multiple forms of GnRH in vivo.
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). Furthermore, the receptor does
couple to G proteins in COS-1 cells, as evidenced by the ability of
GnRH agonists to initiate a second messenger cascade involving inositol
phosphate (Fig. 3B).
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) and
potency for IP production (Table 1) than the native GnRH1, mammalian
GnRH1 or GnRH3, as has been shown in other nonmammalian species
(5, 6, 20, 21). The concordance of this receptor’s
characteristics with those of goldfish and catfish (5, 6)
is perhaps to be expected from the high sequence homology among them
(Fig. 1B).
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), we used these analogs to
examine whether a similarity in function could be found (Fig. 4A). Indeed, whereas gfIB showed reduced
IP production for [His8]GnRH vs.
other position 8 analogs, gfIA and the H. burtoni GnRH-R
lack this characteristic. This prompted an examination of the putative
binding regions of the three sequences (Fig. 4B), showing that the
amino acid polarities of H. burtoni and gfIA are, unlike
gfIB, identical in these regions.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The widespread distribution of GnRH-R in both the brain and body confirms and extends data from other teleosts (6, 22, 23). Outside the hypothalamus-pituitary-gonadal axis, the roles of GnRHs and their receptors are largely unknown. For example, the presence of GnRH in the retina and its action on retinal neurons have been known for several years (24, 25, 26, 27, 28), although its behavioral significance remains unknown. The source of retinal GnRH is the contralateral terminal nerve (29, 30, 31), which is found in all groups of vertebrates (32). The widespread distribution of GnRH-R in mammalian brains (33 ; reviewed in Ref. 34) has led to speculation that GnRHs play roles beyond reproduction, including vision, emotion, and memory. What exactly those roles might be remains to be discovered.
Like GnRH (10, 35, 36), GnRH-R mRNA is found in many tissues outside the nervous system. In H. burtoni, the kidney and testes show relatively greater abundance of GnRH-R mRNA. The roles of both GnRH (10, 36) and GnRH-R in the kidney are unknown. Also, in rats GnRH-R mRNA is found in the adrenals, but not the kidney (33), whereas in fish the renal system is inseparable from the kidney, suggesting that the current data reflect a role for the renal system rather than the kidneys. Our positive results with testes were predictable, whereas our negative results with ovaries were surprising, given previous mRNA data on mammals and fish (6, 22, 33, 37). However, previous GnRH-R RT-PCR results have suggested lower abundance in fish ovaries compared with testes (22). Accordingly, we were able to demonstrate the presence of GnRH-R mRNA in the ovaries only through reamplification (data not shown). As GnRH-R mRNA is known to be regulated by estrogens (33, 38, 39, 40, 41), it is possible that GnRH-R mRNA abundance in the ovary depends on reproductive state, which was not strictly monitored in the present study.
H. burtoni expresses three different GnRH peptides in relatively restricted locations (11), suggesting that the specific forms of GnRH may play several different roles. With three distinct ligands, one might expect a priori that there would be distinct receptor types for each. In fact, evidence for duplicate GnRH-Rs has been found in genomic DNA from several species (7). Likewise, analysis of H. burtoni genomic DNA with degenerate primers yielded a novel sequence with high homology to other type II GnRH-R, in addition to the type I H. burtoni GnRH-R characterized here. To date, however, only tetraploid species [goldfish (6) and Xenopus (Troskie, B., N. Illing, and R. Millar, unpublished data)] have been shown to express more than one GnRH receptor. Evidence against the widespread existence of a second form is that radiolabeled GnRH apparently binds to only one high affinity class of sites in pituitary membranes in African catfish (20, 42) and rats (43, 44, 45), as opposed to the two distinguishable binding affinities in goldfish (46). On the other hand, since both type I and type II GnRH-R sequences are expressed in perciform fish, it seems likely that two types of GnRH-R are expressed in individual perciform species, which appear to be diploid (47). Regardless of whether the putative type II gene is expressed, the distribution and binding properties of the H. burtoni type I GnRH-R strongly suggest that all three GnRH forms do act on the type I receptor characterized here. Therefore, it seems unlikely that individual GnRH forms in vivo interact only with specific receptors.
In comparison to the goldfish subtypes IA and IB, the H. burtoni GnRH-R (type I) has a slightly higher similarity to subtype A, but this in itself is insufficient evidence to place it squarely in the subtype A category. Furthermore, there are few differences among these three receptors in terms of relative affinities for GnRH agonists (6). However, the slight differences in sequence and affinity create a natural experiment in transduction efficacy. Whereas goldfish GnRH-R subtype B shows reduced IP production for [His8]GnRH vs. other position 8 analogs, subtype A and the H. burtoni GnRH-R lack this characteristic. Binding affinity, and therefore ligand discrimination and ligand efficacy, have previously been localized to the extracellular ends of the second and seventh helixes (extracellular loops 1 and 3) of GnRH-R (48, 49, 50). In particular, although it has become clear that no one amino acid can be held responsible for ligand binding (5, 7), the polarities and relative positions of amino acid side-chains have repeatedly been shown to be crucial for ligand binding. Following this track, despite the variability among these three sequences for EC1 and EC3, the polarities of the amino acid side-chains are very consistent among the three forms, with the only deviations being in goldfish subtype B. Thus, this small difference in affinities may result from a few differences in amino acid polarities.
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Acknowledgments |
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Footnotes |
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2 Present address: Division of Biology of Growth and Reproduction,
Department of Pediatrics, University of Geneva Medical School, 1211
Geneva, Switzerland.
3 Present address: Medical Research Council Reproductive Biology
Unit, Edinburgh, United Kingdom EH3 9ET.
Received August 30, 2000.
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