Download Gonadotropin-Releasing Hormone Genes: Phylogeny

Frontiers in Neuroendocrinology 20, 224–240 (1999)
Article ID frne.1999.0181, available online at http://www.idealibrary.com on
Gonadotropin-Releasing Hormone Genes: Phylogeny, Structure,
and Functions
Russell D. Fernald and Richard B. White
Program in Neuroscience, Stanford University, Stanford, California 94305-2130
Gonadotropin-releasing hormone (GnRH, previously called leutinizing hormonereleasing hormone, LHRH) is the final common signaling molecule used by the brain to
regulate reproduction in all vertebrates. Recently, genes encoding two other GnRH forms
have been discovered. Here we present a phylogenetic analysis that shows that the GnRH
genes fall naturally into three distinct branches, each of which shares not only a
molecular signature but also characteristic expression sites in the brain. The GnRH
genes appear to have arisen through gene duplication from a single ancestral GnRH
whose origin predates vertebrates. Several lines of data support this suggestion, including the fact that all three genes share an identical exonic structure. The existence of three
distinct GnRH families suggests a new, natural nomenclature for the genes, and in
addition, we present a logical proposal for naming the peptide sequences. The two
recently discovered GnRH genes are unusual because they encode decapeptides that are
identical in all the species in which they have been found. The control of gene expression
also differs among the three gene families as might be expected since they have had
separate evolutionary trajectories for perhaps 500 million years. r 1999 Academic Press
INTRODUCTION
Gonadotropin-releasing hormone (GnRH, previously called luteinizinghormone releasing hormone or LHRH) is known and named for its role as the
final common signaling molecule used by the brain to regulate reproduction in
all vertebrates. The GnRH decapeptide is synthesized by neurosecretory cells
in the hypothalamus and secreted into portal vessels, to be transported to the
pituitary gland where it stimulates secretion of luteinizing hormone (LH) and
follicle-stimulating hormone (FSH) from pituitary gonadotrophs. Because of its
central importance in controlling reproduction in all vertebrates, the action of
this GnRH form, called the ‘‘releasing’’ form, is well characterized.
Recently, genes encoding decapeptides slightly different from this GnRH
form have been found in many vertebrates (see 32 for review), including
humans (68). Moreover, these additional genes and the original releasing form
of GnRH are expressed in the body as well as in the brain (e.g., 68). Here we will
review the history of GnRH discoveries and assess what is known about the
genes encoding different GnRH forms. In addition, we will show the phylogenetic relationships, structural similarities, ontogeny, sites of expression, and
0091-3022/99 $30.00
Copyright r 1999 by Academic Press
All rights of reproduction in any form reserved.
224
frne 0181
GnRH GENES: PHYLOGENY, STRUCTURE, AND CONTROL
225
regulation of the GnRH genes. Finally, we will discuss the new insights these
discoveries provide about GnRH.
DISCOVERY AND NOMENCLATURE
Discovery
The releasing form of GnRH was predicted to exist in 1950 (24) but since the
hypothalamus contains minuscule amounts of GnRH, and the peptide has
modified N- and C-termini, it was difficult to isolate. Consequently, its structure was not determined until 20 years later (10, 41). Twelve years after this
proof of its existence, the gene encoding GnRH and its cDNA were described for
human (53) and rat (1). Interestingly, cloning this gene enabled one of the first
demonstrations of the prospect for gene therapy when a GnRH transgene
injected into hypogonadal mice (hpg; 11) embryos was shown to rescue the
phenotype (54). Subsequently, it was discovered that both strands of the DNA
within the GnRH gene are transcribed (2, 8). In 1991, the first cDNA encoding
GnRH in a nonmammalian species was identified in a teleost fish, Haplochromis burtoni, by Bond et al. (9), and shortly thereafter the cDNA encoding a
second form of GnRH was found in the same species (66).
Discovery of multiple GnRH genes was not completely surprising because
indirect evidence had been accumulating since 1987 about multiple GnRH
peptide forms in a single species. These suggestions came from studies in which
GnRH forms were separated using high-pressure liquid chromatography (HPLC)
and were distinguished using peptide antisera (reviewed in 34; 55). However,
this evidence was not always easy to interpret, as evidenced by attempts to
characterize the structure of guinea pig GnRH (cf. 33 vs 29). As noted above, a
second cDNA encoding GnRH was identified in a cichild fish in 1994 (66)
followed by the discovery of a third GnRH cDNA in the same species a year later
(67). Using PCR, several groups were subsequently able to identify more than
one cDNA or gene encoding GnRH peptides in several fish species (4, 7, 14, 20,
37, 51). Since the original discovery in fish, a cDNA encoding a second GnRH
has been found in several placental mammals (tree shrew, 32; humans, 68;
rhesus monkey, 63). The only information to date regarding the spatial distribution in the genome of different GnRH genes is in humans, and at least in this
one species the genes reside on different chromosomes (68).
Nomenclature
An unfortunate outcome of the relatively rapid discovery of new GnRH
peptide sequences and additional GnRH genes is a cumbersome terminology to
identify them. Each new GnRH peptide form was named according to the first
species in which that particular peptide sequence was found. Though initially
convenient and now familiar to the cognescenti, this nomenclature provides
frne 0181
226
FERNALD AND WHITE
neither useful structural nor functional information about the growing number
of known peptides. Recent phylogenetic analysis has provided a more rational
terminology for identifying both GnRH gene and peptide forms.
As more GnRH encoding genes are cloned and their associated peptide
sequences known, there needs to be a common, universal, informative nomenclature for GnRH genes as well as for GnRH peptides. Because two GnRH
genes have now been found in humans, nomenclature recommended by the
Genome Database Nomenclature Committee of March 5, 1997 has been adopted
perforce. Accordingly, the human gene encoding the releasing form of GnRH
(pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly) is now termed GNRH1 (formerly
GnRH or LHRH). The second form of GnRH (pGlu-His-Trp-Ser-His-Gly-Trp-TyrPro-Gly) is known as GNRH2. Correspondingly, GnRH genes in nonhumans
are known as Gnrh1 for the releasing form, Gnrh2 for the second form
(previously called ‘‘chicken II GnRH’’), and Gnrh3 for a third form identified in
several teleost fish but not (yet?) found in humans (previously called salmon
GnRH or sometimes sGnRH).
The identities of the different GnRH peptides encoded by the GnRH genes
are best described by listing the amino acid differences from the human
hypothalamic-releasing form. Thus, the GNRH2 and Gnrh2 genes encode a
peptide identified as 5His5Trp7Tyr8 6GnRH (meaning it has the three substitutions in the three positions noted) in all species in which it has been found.
Correspondingly, GnRH3 genes encode 5Trp7Leu8 6GnRH in all species found to
have a third GnRH form. As described in more detail below, the peptides
encoded by the Gnrh1 genes are expressed primarily in the hypothalamus and
comprise the originally described releasing hormone. Interestingly, the peptides encoded by the Gnrh1 genes are the only ones to date that have been
shown to vary among species. Five of the ten amino acids encoded by Gnrh1
genes are invariant while two other positions show conservative changes
(reviewed in 32).
PHYLOGENETIC RELATIONSHIPS AND THE ORIGIN OF GnRH
The discovery and sequencing of many genes that encode different GnRH
peptides among vertebrate species have made it possible to examine their
relatedness using molecular phylogenetic techniques. These methods use amino
acids or nucleotides as the traits or characters whose comparison form the basis
for constructing phylogenetic relationships. When constructing such a tree,
more cases in which gene sequences are known for a single species will increase
the accuracy of the phylogenetically based predictions.
Based on molecular phylogenetic analysis (Fig. 1), the three known GnRH
genes appear to have arisen from a common ancestral form through gene
duplications prior to the appearance of vertebrates (68). Several methods
(maximum parsimony, maximum likelihood, and neighbor-joining) were used
to test the robustness of this analysis, and each method showed essentially the
same branching patterns. The phylogenetic tree shows the existence of three
frne 0181
GnRH GENES: PHYLOGENY, STRUCTURE, AND CONTROL
227
evolutionarily distinct GnRH groups that are expressed in three distinct
regions of the brain: releasing forms localized to the hypothalamus (GnRH1),
forms previously localized solely to midbrain nuclei (GnRH2), and forms
localized to the telencephalon, to date found only in teleost fish (GnRH3).
Several lines of evidence support the conclusion that the three GnRH groups
identified by this phylogenetic comparison comprise evolutionarily distinct
forms. First, the groupings correspond to GnRH forms with distinct expression
patterns in the brain and thus presumed common biological roles. As described
by Kasten et al. (32), in all species studied the GnRH1 forms appear in the
hypothalamus, the GnRH2 forms appear in the midbrain, and the GnRH3
forms are found in the terminal nerve and olfactory system of the forebrain
telencephalon. This suggests that the phylogenetic groupings can be used to
predict the activities of yet uncharacterized forms once their molecular structure is determined. Second, although only the neighbor-joining tree is shown,
the same groupings were found using other distance-based methods as well as
maximum parsimony and maximum likelihood (not shown). Third, highreliability (e.g., bootstrap) values were generated for the neighbor-joining and
parsimony trees, strongly supporting identification of these groups. Finally, the
branch lengths to each group are relatively long, suggesting an ancient divergence. Furthermore, the branching order within each GnRH gene group matches
the evolutionary branching order of the species (see in particular GnRH1, for
which sequences were available from diverse species).
Although these phylogenetic relationships cannot identify the selective forces
that generated multiple GnRH forms, the analysis does reveal that different
forms of GnRH very likely diverged from one another prior to the divergence of
species represented in the tree. Because the tree is unrooted, the exact order of
the duplications is unclear. However, GnRH1 and GnRH2 include representatives from fish and mammals, so the separation of these two groups must have
occurred prior to the separation of mammalian and fish ancestors. The origin of
GnRH3 is less clear, since, at this time, it has been described only in fish. The
data suggest two alternative hypotheses for the appearance of GnRH3. The
gene duplication leading to GnRH3 may have occurred relatively recently,
within the fish lineage, and thus is restricted to this taxon. Alternatively,
GnRH3 may have resulted from an ancient duplication and either has been lost
in higher vertebrates during evolution or has not yet been discovered. We favor
the latter hypothesis because if the duplication were recent, the GnRH3 forms
would be expected to cluster with one of the other GnRH forms in fish, which
does not occur (68). Perhaps a version of GnRH3 remains to be found in other
vertebrates including humans.
Supporting this latter notion, several lines of evidence indicate that duplication of most of the genome occurred during the transition from simple chordates
to vertebrates (27, 40, 48). Most likely, the multiple GnRH forms are yet
another consequence of these ancient duplications. Recent immunocytochemical work suggests that a chordate ancestor, an ascidian, has at least two
distinct GnRH forms (62). In retrospect, we might have predicted that there
would be several descendants of an ancient GnRH in extant vertebrates.
frne 0181
228
FERNALD AND WHITE
frne 0181
GnRH GENES: PHYLOGENY, STRUCTURE, AND CONTROL
229
In summary, genes encoding GnRH forms fall into three distinct evolutionary
branches. Phylogenetic reconstruction supported by brain distribution of GnRH
gene forms suggests that they arose through gene duplications prior to the rise
of vertebrates and have been adapted to distinct functions over 500 myr of
vertebrate evolution.
GENE STRUCTURE
All GnRH genes share the same basic structure. Within each gene, a given
exon encodes a corresponding preprohormone region as first described for
humans (53). In all cases, the GnRH preprohormone mRNA encodes GnRH and
the GnRH-associated peptide (GAP), separated by a canonical cleavage site.
The preprohormone mRNAs are encoded by four exons (Fig. 2). Exon 1 encodes
the 58-UTR exclusively. Exon 2 encodes the signal peptide, GnRH decapeptide,
the proteolytic cleavage site, and the N-terminus of GAP. Exon 3 encodes the
central portion of GAP and exon 4 encodes the C terminus of GAP along with
the 38-UTR. Among all known GnRH genes, there seem to be two generalities:
(i) exons 2 and 3 are the most similar in length and (ii) the UTR sizes and intron
sizes are the most different. The level of similarity in the coding sequences can
be read from the phylogenetic tree distances (Fig. 1). The greatest differences
within the preprohormone are within the GAP coding sequences. The striking
contrast between conservation of the GnRH coding sequence and lack thereof in
the GAP coding sequence is evidence of differential selective pressure within
the gene. This is evident in cases where the identity and similarity of GnRH
and GAP coding sequences have been compared for mRNAs of different GnRH
genes within a species (e.g., 32, 67, 69).
Since animals as phylogenetically distant as fish and humans have essentially identical GnRH gene structures, we expect that GnRH genes yet to be
described will also fit this pattern. The molecular divergence of the 58- and
38-flanking sequences in the different GnRH gene forms, as well as in their
introns, suggests that each of the three branches has had a long period of
independent evolution, consistent with our recent phylogenetic analysis of
GnRH evolution based on cDNA sequences (68).
FIG. 1. Unrooted neighbor-joining phylogenetic tree of cDNAs encoding GnRH. The tree was
generated using the neighbor-joining algorithm and the Hall distance calculation correction
procedure. Evolutionary distances are represented only by the length of the branches and not by
branch angles. Bootstrap values, indicating the number of times a particular set of sequences
groups together when trees are generated from resampled alignments, are indicated for some
important nodes on the tree. An unrooted tree is shown because of the lack of an obvious outgroup.
Some GnRH3 sequences from different species were identical and are grouped together. The scale
bar corresponds to estimated evolutionary distance units as calculated by Protdist in Phylip.
Redrawn from (68).
frne 0181
230
FERNALD AND WHITE
FIG. 2. Schematic diagram showing organization of GnRH genes. The genes encoding GnRH in
the cichlid fish, H. burtoni, are shown. Within each gene, a given exon encodes a corresponding
preprohormone region. Single hatched bars, signal sequence coding region; cross-hatched bars,
GnRH-coding region; open bars, GnRH-associated peptide (GAP) coding region. Horizontal lines
adjacent to exons represent introns. Modified from (69).
ORIGIN OF GnRH EXPRESSING CELLS AND THEIR SITES OF EXPRESSION
The three GnRH genes described above are expressed in cells that have
distinctly different origins, locations, and, presumably, functions. Information
about these topics is best understood for GnRH expression in the brain. In the
adult brain, GnRH1 is expressed in the hypothalamus, GnRH2 in the midbrain–
tegmentum, and GnRH3 in the forebrain telencephalon. The hypothalamic and
telencephalic gene forms (Gnrh1 and Gnrh3) are expressed by cells that arise in
the olfactory placode and migrate into the brain (44, 46, 52, 72). These cells
migrate centrally and take up positions along the ventral surface of the adult
brain in a rostrocaudal continuum from the terminal nerve area to the hypothalamus. The final distribution of these neurons in fish species divides into
two populations. The anterior population of cells expresses the Gnrh3 gene and
the posterior population expresses the Gnrh1 gene (70). Although the original
observations of migration were performed in mouse tissue, using immunocytochemistry (e.g., 52), they have been confirmed using in situ hybridization with
molecular probes (70). The placodal ancestry of these neurons along with their
expression in the terminal nerve suggests that Gnrh3 in the telencephalon
might coordinate sensory input at the time of reproduction (cf. 67), consistent
with the report of Yamamoto et al. (73).
GnRH2 expressing neurons of the brain do not arise from the olfactory
frne 0181
GnRH GENES: PHYLOGENY, STRUCTURE, AND CONTROL
231
placode. This was first shown indirectly by lesioning the placode prior to
development in the salamander (47). Animals with the placode lesioned had
neurons with GnRH immunoreactivity in the midbrain–tegmentum but not in
the telencephalon or hypothalamus. More recently, using gene probes, White
and Fernald (70) demonstrated directly that Gnrh2 is expressed in cells born in
the midbrain ventricle in a teleost fish. The expression of this GnRH gene form
begins shortly after the cell is born, while it is still within the ventricular
germinal zone. All three GnRH genes in this species are expressed by the end of
the first third of development (day 4 of 14). This early onset suggests that
GnRH might play additional roles during development.
At present, very little is known about the ontogeny or type of cells that
express any of the three GnRH forms outside the brain (e.g., 5, 6, 64).
Expression of GnRH Outside the Brain
GnRH1 gene expression has been well studied in the brain of vertebrates, but
only recently has investigation begun of its expression elsewhere in the body.
For example, GnRH1 mRNA has been reported in splenocytes and peripheral
lymphocytes in rats (5, 71) and humans (6, 64). By using reverse transcription–
polymerase chain reaction (RT-PCR) on human tissue, GnRH1 mRNA has been
found in liver, heart, skeletal muscle, kidney, and placenta (e.g., 31). GnRH1
gene expression has been also been reported in the testis of several primates
and rats (15), human ovary (50), and rat prostate (6). Widespread GnRH1
expression was also found in the teleost fish, H. burtoni, by using RT-PCR.
Heart, liver, spleen, kidney, testis, and brain of an adult male all contained
GnRH1 mRNA (69).
Much less is known about the distribution of GnRH2 expression outside the
brain. In humans, White et al. (68) reported widespread distribution of GNRH2
mRNA in the prostate, bone marrow, and kidney, in addition to its expression in
the brain. In goldfish, Lin and Peter (37) demonstrated expression of GnRH2
mRNA in the ovary, and Yu et al. (74) used RT-PCR to detect it in both testis and
ovary. In the cichlid H. burtoni, White and Fernald (69) showed that GnRH2
mRNA is expressed in the testis and brain but not in kidney.
Similarly, there are only a few reports about expression of GnRH3. In the
teleost Porichthys notatus GnRH3 mRNA was found in the testis and ovary
(22). In the cichlid fish H. burtoni, GnRH3 expression was identified in the
testis. All three forms of GnRH have been detected in the testis of H. burtoni,
which suggests that the different GnRH peptides may play distinct roles in
both the brain and the testis. Understanding of the role of multiple GnRH
forms within a single organ will ultimately depend on detailed knowledge of
their specific sites of action, including the distribution of GnRH receptors and
the possible existence of multiple GnRH-receptor types.
frne 0181
232
FERNALD AND WHITE
CONTROL OF GnRH GENE EXPRESSION
Regulation of the GnRH genes is a topic of intense interest, particularly
because it has become possible to compare control of gene expression of several
GnRH genes within a single organism. Such analyses should provide information and insight about how GnRH is regulated at its many sites of action.
Previously most studies of gene regulation were directed at GnRH1 both
because of its importance and because no other genes were known.
Gore and Roberts (19) recently provided an excellent review of hypothalamic
GnRH1 gene regulation in mammals. In contrast, little is known about how the
other two GnRH gene forms are regulated, and comparative analysis of gene
regulation among the three forms of GnRH is in an early stage. Thus the broad
and interesting issues such as differences between in vitro and in vivo GnRH
regulation or between transcriptional and posttranscriptional regulation mechanisms are not yet known except for the GnRH1 gene. We will focus here on what
is known about regulation of the other GnRH genes.
Similar Regulation in Different GnRH Genes?
The discovery that different GnRH peptides are expressed in a variety of
tissues suggests that, as a group, they serve multiple functions, probably
requiring complex gene regulation. Analysis of the 58-flanking sequence in the
three GnRH genes in H. burtoni suggests that this may be the case. Because
each decapeptide is encoded by a different gene, each with a unique 58upstream regulatory sequence, transcriptional regulation of the three GnRH
genes is likely to be independent. Even though some short regulatory motifs are
conserved among the three H. burtoni genes, the proximate 500 bp of upstream
sequence are quite dissimilar. Indeed, the promoter regions of the three genes
are not significantly more similar to one another than are their introns (69).
Promoter sequence comparisons of multiple GnRH genes in closely related
species has only recently become possible with the sequencing of GnRH1 and
GnRH2 genes in two teleosts, H. burtoni (69) and Morone saxatilis (14).
Preliminary comparison of the upstream regions of the Gnrh2 genes in H.
burtoni, an African cichlid, and the striped bass, M. saxatilis, an anadromous
North American species, reveals several regions with remarkable conservation,
in addition to the region near the most proximate promoter. For example, at a
distance more than 2000 bp upstream of the mRNA coding sequence in each
GnRH2 gene, there is a 74% nucleotide identity in a 750-nucleotide overlap.
Apparently there has been a strong selective pressure to maintain this alternative promoter, thus suggesting that it may play an important role in regulation
of Gnrh2 gene expression in these species and perhaps others. Multiple promoter usage has previously been suggested for the GnRH2 gene in humans (68)
and M. saxatilis (14), and this may prove to be widespread within GnRH2
genes.
frne 0181
GnRH GENES: PHYLOGENY, STRUCTURE, AND CONTROL
233
Comparison of Gnrh1 gene upstream sequences in H. burtoni and M. saxatilis shows no region of sequence conservation comparable to that in the GnRH2
genes, although within the proximate 500 bp of the promoter region, there is a
77% nucleotide identity in a 178-nucleotide overlap. It is tempting to speculate
that these gross differences in sequence conservation between the two gene
forms may mirror the relative conservation of the peptide forms encoded by the
genes.
Direct Regulation of GnRH Genes by Steroids?
cDNAs encoding two or even three of the GnRH forms in a single species have
now been characterized in two primates (human, 68; rhesus monkey, 63), one
rodent (tree shrew, 32), and six fish species (African cichlid, 69; African catfish,
7; two species of seabream, 20; striped bass, 14; and goldfish, 38). However,
genomic sequencing that includes the upstream regulatory structures of multiple GnRH gene forms in a single species has been described for only three
species. Two complete GnRH genes have been described for humans (68). In the
striped bass, M. saxatilis, two of the three known GnRH gene forms have been
described (14). Complete genes encoding three distinct GnRH forms have been
characterized in only one species, H. burtoni (69), so this review will focus on
the GnRH genes in this species.
Several putative steroid receptor binding sites are evident in the proximate
500 bp of 58-flanking sequence of each GnRH gene in H. burtoni (69). Specifically, putative glucocorticoid receptor (GR) binding sites (6 in GnRH1 and 10 in
GnRH3) hint at a possible direct role for GR and its ligand cortisol in regulating
these genes. In contrast, GnRH2 has only 2 putative GR sites in this region.
Behavioral, endocrine, and morphological data in H. burtoni are consistent
with GnRH gene regulation by cortisol in hypothalamic GnRH neurons (17).
Though still unknown in fish, a subset of GnRH neurons in the rat hypothalamus contain GR (3). Furthermore, Chandran et al. (12) provided strong evidence for the direct role of GR in GnRH gene regulation in GT1-7 cells.
In contrast to putative GR binding sites, estrogen receptor binding sites were
not found in the 500 bp upstream of any of the three GnRH genes, suggesting
that estradiol may not have a direct effect on GnRH gene regulation in H.
burtoni. Consistent with this, forebrain GnRH neurons in the rainbow trout
(Oncorhynchus mykiss) do not contain immunoreactive estrogen receptor (ER)
(45). In another salmonid, the Atlantic salmon, ER binding sites were found 1.5
kb upstream of the 5Trp7Leu8 6GnRH gene (GnRH3—see 68), and human ER
protein was shown to bind to this sequence (36). However, ER itself has not
been localized to GnRH neurons in that species, so a direct role for estradiol in
the transcriptional regulation of GnRH remains uncertain. Moreover, there is
scant evidence for the presence of ER within GnRH neurons in other vertebrates for which GnRH and ER colocalization has been attempted (e.g., negative results in rat, 25, 56; rhesus macaque, 60; mink, 65; sheep, 26). The
frne 0181
234
FERNALD AND WHITE
well-described role of estrogen in the regulation of GnRH is thus likely to be
indirect, mediated by other cells.
A single putative androgen receptor binding site is upstream of the GnRH1
gene in H. burtoni, consistent with the idea that androgen acts directly on
GnRH1 neurons in this species. Gonadal steroid feedback experiments are also
consistent with this hypothesis (59), although these effects could also be
indirect. The possible role of the two progesterone binding sites upstream of
GnRH1 and GnRH3 is unclear, although direct transcriptional regulation of
the GnRH gene by progesterone receptor (PR) has been proposed in other
animals, and Cho et al. (13) have shown that translation effects in vivo appear
to be stimulated by PR. However, in the tilipia Oreochromis niloticus, preliminary studies indicate that progesterone does not influence any of the three
populations of GnRH neurons (49). By using semiquantitative in situ hybridization, they found that progesterone treatment had no effect on mRNA levels for
any of the three GnRH forms. However, castration significantly elevated
GnRH3 mRNA while GnRH1 and GnRH2 mRNA levels remained unchanged,
suggesting that in this species, the terminal nerve GnRH neurons are under
negative gonadal steroid feedback, while the other two GnRH neuron populations are not. In contrast, in H. burtoni, a closely related cichlid fish, cells
expressing GnRH1 are clearly under gonadal steroid regulation: these cells
hypertrophy after castration (59). In other teleosts, hypothalamic GnRH neurons are also affected by gonadal steroids (e.g., 21, 23).
GnRH FUNCTIONS
Among the GnRH peptides, the function of GnRH1 is best understood
because the peptide encoded by this gene is essential for reproduction. Consequently, many features of its regulation are now known (see above and 19).
Recently, the hpg mouse has been used to tease apart the role of GnRH1 in
sexual maturation from its role in reproductive behavior (18).
Both the GnRH2 and the GnRH3 peptides are identical in all species in
which they have been found. Since GnRH2 has been identified in many more
species to date, its complete conservation through 500 myr of evolution suggests that it has been subject to extremely stringent selective pressures. Such
selection might have arisen if GnRH2 serves different functions at different loci
in the body, which is suggested by a growing body of evidence. First, GnRH2 has
been shown to be present in the sympathetic ganglia of amphibia (28), where it
has been shown that GnRH can act as a neuromodulator in spinal cord ganglion
neurons (30). In fish, neurons previously shown to control sperm duct and
oviduct contractility receive input from cells that show GnRH2 immunoreactivity (42). Examples of possible GnRH2 expression outside the midbrain cells
include transient GnRH2 immunoreactivity within mast cells in the habenula
of ring doves following courtship (57, 58). These data have led to the suggestion
that mast cells might be an alternative delivery system for GnRH (57).
Has GnRH2 remained unchanged because of selective pressure from mul-
frne 0181
GnRH GENES: PHYLOGENY, STRUCTURE, AND CONTROL
235
tiple functions? We have speculated elsewhere (68) that GnRH2 may have
originated in the immune system and only later acquired a neuromodulatory
function in the brain. Further characterization of GnRH2 including decisive
functional tests should help clarify whether it serves distinct functions in the
body. This in turn might shed light on what selective forces have been responsible for its remarkable conservation over evolutionary time.
The function of GnRH3 also remains unclear. Lesions of the terminal nerve
Gnrh3 expressing neurons in a fish (Colisa lalia, dwarf gourami; 73) resulted in
a threshold change in courtship initiation. However, virtually no other data are
available about possible functions of this peptide. Interestingly, it has been
known for some time that a subpopulation of axons from the terminal nerve
contain GnRH3 (67) and project to the retina (43), although no function has yet
been ascribed to GnRH3 in the retina.
We have previously noted (67) that each of the sensory, motor, and humoral
systems in teleost fish might utilize distinct but related forms of GnRH peptide:
the terminal nerve form perhaps coordinates the sensory and motivational
systems, the mesencephalic form may modify motor action in the service of
reproductive behavior, and the hypothalamic form causes the release of gonadotropins to facilitate reproduction (67). The report that lesioning the terminal
nerve affected reproductive readiness (73) suggests that GnRH3 expression
might indeed influence reproductive motivation.
The distribution of GnRH peptide types hints at possible functions. However,
the distribution of GnRH receptors, which mediate any effect of GnRH, is
essential for understanding what GnRH forms actually do. However, it is not
yet known whether different forms of GnRH might have different receptor
types. GnRH receptors have been found throughout the body, including the
kidney (31) and prostate (16). Because previous studies have shown that GnRH
receptors bind GnRH2 peptide up to 100 times more effectively than GnRH1
(35), GnRH2 could act through these receptors outside the brain. Recently,
three putative GnRH receptor subtypes were identified, suggesting that there
may be a specific cognate receptor type for each GnRH type (61). These authors
suggest that GnRH receptor types coevolved with the GnRH gene forms,
resulting in a cognate receptor for each GnRH type.
SUMMARY AND FUTURE PREDICTIONS
GnRH was once thought to be a single peptide, acting solely as a brain signal
to the pituitary. However, new discoveries about GnRH have changed our views
of GnRH. Clearly the original gonadotropin-releasing hormone was just the
beginning of the GnRH story. As shown above, there are three GnRH gene
families based on molecular phylogenetic analysis. Each family shares not only
a molecular signature but also a characteristic expression site in the brain.
Surprisingly, the two newly discovered gene families encode decapeptides that
are identical in all species in which they have been found. This is in contrast to
GnRH1, which has substantial variation among species. Although a great deal
frne 0181
236
FERNALD AND WHITE
is known about the hypothalamic GnRH form, exactly where and how the two
recently discovered GnRH forms act remain unknown.
As might be suspected, past predictions about GnRH have had mixed
success. Most notably, GnRH2 was hypothesized to have been lost in placental
mammals (55) and to be the oldest GnRH (39), neither of which now appears to
be true. Still, it seems likely that there are some predictions that could be made
based on the data presented here. First, it seems probable that a third form of
GnRH as described in teleost fish will be found in other vertebrate classes.
Since this form is expressed in the telencephalon and the neurons that express
it share an ontogenetic origin with the releasing form, it may play a role in
reproductive readiness in other species as it appears to do in teleost fish.
Second, the two gene duplications that are proposed to have led to vertebrates
probably produced the multiple GnRH forms. If so, it seems likely that there
will be multiple GnRH receptor types that are also a product of these duplications. Finally, the GnRH genes are closely related to one another but are
evidently not a part of a larger gene family, possibly making them unique
among peptide encoding genes. Does this apparent isolation have any biological
meaning? Perhaps the GnRH genes encode peptides that are unique, making
more urgent the discovery of the roles of GnRH2 and GnRH3.
ACKNOWLEDGMENTS
We thank J. Eisen for collaboration on the phylogeny, A. Greenwood and K. Hoke for helpful
comments on the manuscript, and E. Bennett for invaluable help with the manuscript. This
research was supported by NIH NS 34950 to R.D.F.
REFERENCES
1.
Adelman JP, Mason AJ, Hayflick JS, Seeburg PH. Isolation of the gene and hypothalamic
cDNA for the common precursor of gonadotropin-releasing hormone and prolactin releaseinhibiting factor in human and rat. Proc Natl Acad Sci USA 1986; 83: 179–183.
2. Adelman JP, Bond CT, Douglass J, Herbert E. Two mammalian genes transcribed from
opposite strands of the same DNA locus. Science 1987; 235: 1514–7.
3. Ahima RS, Harlan RE. Glucocorticoid receptors in LHRH neurons. Neuroendocrinology 1992;
56: 845–850.
4. Ashihara M, Suzuki M, Kubokawa K, Yoshiura Y, Kobayashi M, Urano A, Aida K. Two
differing precursor genes for the salmon-type gonadotropin-releasing hormone exist in salmonids. J Mol Endocrinol 1995; 15: 1–9.
5.
Azad N, Emanuele NV, Halloran MM, Tentler J, Kelly MR. Presence of LHRH messenger RNA
in rat spleen lymphocytes. Endocrinology 1991; 128: 1679–1681.
6.
Azad N, Lapaglia N, Abel K, Jurgens KAJ, Kirsteins L, Emanuele NV, Kelley MR, Lawrence
AM, Mohagheghpour N. Immunoactivation enhances the concentration of luteinizing hormonereleasing hormone peptide and its gene expression in human peripheral T-lymphocytes.
Endocrinology 1993; 133: 215–223.
7. Bogerd J, Zandbergen T, Andersson E, Goos H. Isolation, characterization and expression of
cDNAs encoding the catfish-type and chicken-II type gonadotropin-releasing hormone precursors in the African catfish. Eur J Biochem 1994; 222: 541–549.
frne 0181
GnRH GENES: PHYLOGENY, STRUCTURE, AND CONTROL
237
8.
Bond CT, Hayflick JS, Seeburg PH, Adelman JP. The rat gonadotropin-releasing hormone SH
locus: Structure and hypothalamic expression. Mol Endocrinol 1989; 3: 1257–1262.
9.
Bond C, Francis R, Fernald R, Adelman J. Characterization of complementary DNA encoding
the precursor for gonadotropin-releasing hormone and its associated peptide from a teleost
fish. Mol Endocrinol 1991; 5: 931–937.
10.
Burgus R, Butcher M, Amoss M, Ling N, Monahan M, Rivier J, Fellows R, Blackwell R, Vale
W, Guillemin R. Primary structure of ovine hypothalamic lutenizing hormone-releasing factor
(LRF). Proc Natl Acad Sci USA 1972; 69: 278–282.
11.
Cattanach BM, Iddon CA, Charlton HM, Chiappa SA, Fink G. Gonadotropin-releasing
hormone deficiency in a mutant mouse with hypogonadism. Nature 1977; 269: 338–340.
12.
Chandran UR, Attardi B, Friedman R, Zheng ZW, Roberts JL, DeFranco DB. Glucocorticoid
repression of the mouse gonadotropin-releasing hormone gene is mediated by promoter
elements that are recognized by heteromeric complexes containing glucocorticoid receptor. J
Biol Chem 1996; 271: 20412–20420.
13.
Cho BN, Seong JY, Cho H, Kim K. Progesterone stimulates GnRH gene expression in the
hypothalamus of ovariectomized, estrogen treated adult rats. Brain Res 1994; 652: 177–180.
Chow MM, Kight KE, Gothilf Y, Alok D, Stubblefield J, Zohar Y. Multiple GnRHs present in a
teleost species are encoded by separate genes: Analysis of the sbGnRH and GnRH-II genes
from the striped bass, Morone saxatilis. J Mol Endocrinol 1998; 21: 277–289.
Dong KW, Duval P, Zeng Z, Gordon K, Williams RF, Hodgen GD. Multiple transcription start
sites for the GnRH gene in rhesus and cynomolgus monkeys: A non-human primate model for
studying GnRH gene regulation. Mol Cell Endocrinol 1996; 117: 121–130.
Fekete M, Redding TW, Comraru-Schally AM, Pontes JE, Connelly RW, Srkalovic G, Schally
AV. Receptors for luteinizing-hormone-releasing hormone, somatostatin, prolactin, and epidermal growth-factor in rat and human-prostate cancers and in benign prostate hyperplasia.
Prostate 1989; 14: 191–208.
Fox HE, White SA, Kao MHF, Fernald RD. Stress and dominance in a social fish. J Neurosci
1997; 17: 6463–6469.
Gibson MJ, Wu TJ, Miller GM, Silverman AJ. What nature’s knockout teaches us about GnRH
activity: Hypogonadal mice and neuronal grafts. Horm Behav 1997; 31: 212–20.
Gore AC, Roberts JL. Regulation of gonadotropin-releasing hormone gene expression in vivo
and in vitro. Front Neuroendocrinol 1997; 18: 209–245.
Gothilf Y, Munoz-Cueto JA, Sagrillo CA, Chen TT, Kah O, Elizur A, Zohar Y. Three forms of
gonadotropin-releasing hormone in a perciform fish (Sparus aurata): Complementary deoxyribonucleic-acid characterization and brain localization. Biol Reprod 1996; 55: 636–645.
Grober MS, Jackson IMD, Bass A. Gonadal steroids affect LHRH preoptic cell number in
sex-role changing fish. J Neurobiol 1991; 22: 734–741.
Grober MS, Myers TR, Marchaterre MA, Bass AH, Myers DA. Structure, localization, and
molecular phylogeny of a GnRH cDNA from a paracanthopterygian fish, the plainfin midshipman (Porichthys notatus). Gen Comp Endocrinol 1995; 99: 85–99.
Halpern-Sebold LR, Schreibman MP, Margolis-Nunno H. Differences between early-and-latematuring genotypes of the platyfish Xiphophorus maculatis in the morphometry of their
immunoreactive LHRH containing cells: A developmental study. J Exp Zool 1986; 240:
245–258.
Harris GW. Oestrous rhythm: Pseudopregnancy and the pituitary stalk in the rat. J Physiol
1950; 111: 347–360.
Herbison AE, Theodosis DT. Localization of oestrogen receptors in preoptic area neurons
containing neurotensin but not tyrosine hydroxylase, cholecystokinin or luteinizing hormonereleasing hormone in the male and female rat. Neuroscience 1992; 50: 283–298.
Herbison AE, Robinson JE, Skinner DC. Distribution of estrogen receptor-immunoreactive
cells in the preoptic area of the ewe: Co-localization with glutamic acid decarboxylase but not
luteinizing hormone-releasing hormone. Neuroendocrinology 1993; 57: 751–759.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
frne 0181
238
FERNALD AND WHITE
27.
Holland PWH, Garcia-Fernandez J, Williams NA, Sidow A. Gene duplications and the origins
of vertebrate development. Dev Suppl 1994; 125–133.
28.
Jan YN, Jan LY, Brownfield MS. Peptidergic transmitters in synaptic boutons of sympathetic
ganglia. Nature 1980; 288: 380–382.
29.
Jimenez-Liñan M, Rubin BS, King JC. Examination of guinea pig luteinizing hormonereleasing hormone gene reveals a unique decapeptide and existence of two transcripts in the
brain. Endocrinology 1997; 138: 4123–30.
30.
Jones SW, Adams PR, Brownstein MJ, Rivier JE. Teleost luteinizing hormone-releasing
hormone: Action on bullfrog sympathetic ganglia is consistent with role as neurotransmitter.
J Neurosci 1984; 4: 420–429.
31.
Kakar SS, Jennes L. Expression of gonadotropin-releasing hormone and gonadotropinreleasing hormone receptor mRNAs in various non-reproductive human tissues. Cancer Lett
1995; 98: 57–62.
32.
Kasten TL, White SA, Norton TT, Bond CT, Adelman JP, Fernald RD. Characterization of two
new preproGnRH mRNAs in the tree shrew: First direct evidence for mesencephalic GnRH
gene expression in a placental mammal. Gen Comp Endocrinol 1996; 104: 7–19.
33.
Kelsall R, Coe IR, Sherwood NM. Phylogeny and ontogeny of gonadotropin-releasing hormone: Comparison of guinea pig, rat, and a protochordate. Gen Comp Endocrinol 1990; 78:
479–94.
34.
King J, Millar R. Genealogy of the GnRH family. Prog Comp Endocrinol 1990; 54–59.
35.
King JA, Millar RP. Gonadotropin-releasing hormones. In: Pang PK, Schriebmen MP, Eds.
Vertebrate Endocrinology: Fundamental and Biomedical Implications. Vol 4, Part B. New
York: Academic Press, 1991: 1–31.
36.
Klungland H, Andersen O, Kisen G, Alestrom P, Tora L. Estrogen receptor binds to the salmon
GnRH gene in a region with long palindromic sequences. Mol Cell Endocrinol 1993; 95:
147–154.
37.
Lin XW, Peter RE. Expression of salmon gonadotropin-releasing hormone (GnRH) and
chicken GnRH-II precursor messenger ribonucleic acids in the brain and ovary of goldfish.
Gen Comp Endocrinol 1996; 101: 282–296.
38.
Lin XW, Peter RE. Cloning and expression pattern of a second [His5Trp7Tyr8]gonadotropinreleasing hormone (chicken GnRH-H-II) mRNA in goldfish: Evidence for two distinct genes.
Gen Comp Endocrinol 1997; 107: 262–72.
39.
Lovejoy DA, Fischer WH, Ngamvongchon S, Craig AG, Nahorniak CS, Peter RE, Rivier JE,
Sherwood NM. Distinct sequence of gonadotropin-releasing hormone (GnRH) in dogfish brain
provides insight into GnRH evolution. Proc Natl Acad Sci USA 1992; 89: 6373–7.
40.
Lundin LG. Evolution of the vertebrate genome as reflected i nparalogous chromosomal
regions in man and the house mouse. Genomics 1993; 16: 1–19.
41.
Matsuo H, Baba Y, Nair RMG, Arimura A, Schally AV. Structure of the porcine LH- and
FSH-releasing hormone. I. The proposed amino acid sequence. Biochem Biophys Res Commun
1971; 43: 1334–1339.
42.
Miller KE, Kriebel RM. Peptidergic innervation of caudal neurosecretory neurons. Gen Comp
Endocrinol 1986; 64: 396–400.
43.
Munz H, Stumpf W, Jennes L. LHRH systems in the brain of platyfish. Brain Res 1981; 221:
1–13.
44.
Muske L. Evolution of gonadotropin-releasing hormone (GnRH) neuronal systems. Brain
Behav Evol 1993; 42: 215–230.
45.
Navas JM, Anglade I, Bailhache T, Pakdel F, Breton B, Jego P, Kah O. Do gonadotrophinreleasing hormone neurons express estrogen receptors in the rainbow trout? A double
immunohistochemical study. J Comp Neurol 1995; 363: 461–474.
46.
Norgren RB, Gao C. LHRH neuronal subtypes have multiple origins in chickens. Dev Brain
Res 1994; 165: 735–738.
frne 0181
GnRH GENES: PHYLOGENY, STRUCTURE, AND CONTROL
239
47.
Northcutt RG, Muske LE. Multiple embryonic origins of gonadotropin-releasing hormone
(GnRH) immunoreactive neurons. Dev Brain Res 1994; 78: 279–290.
48.
Ohno S. Patterns in genome evolution. Curr Opin Genet Dev 1993; 3: 911–914.
49.
Parhar IS, Soga T, Ishikawa Y, Nagahama Y, Sakuma Y. Neurons synthesizing gonadotropinreleasing hormone mRNA subtypes have multiple developmental origins in the medaka. J
Comp Neurol 1998; 401: 217–226.
50. Peng C, Fan NC, Ligier M, Vaananen J, Leung PC. Expression and regulation of gonadotropinreleasing hormone (GnRH) and GnRH receptor messenger ribonucleic acids in human
granulosa-luteal cells. Endocrinology 1994; 135: 1740–1746.
51.
Penlington MC, Williams MA, Sumpter JP, Rand-Weaver M, Hoole D, Arme C. Isolation and
characterization of mRNA encoding the salmon- and chicken-II type gonadotropin-releasing
hormones in the teleost fish Rutilus rutilus (Cyprinidae). J Mol Endocrinol 1997; 19: 337–346.
52.
Schwanzel-Fukuda M, Pfaff D. Origin of luteinizing hormone-releasing hormone neurons.
Nature 1989; 338: 161–163.
53.
Seeburg PH, Adelman JP. Characterization of cDNA for precursor of human luteinizing
hormone releasing hormone. Nature 1984; 311: 666–668.
54.
Seeburg P, Mason A, Stewart T, Nikolics K. The mammalian GnRH gene and its pivotal role in
reproduction. Rec Prog Horm Res 1987; 43: 69–98.
55.
Sherwood N, Lovejoy D, Coe I. Origin of mammalian gonadotropin-releasing hormones.
Endocrinology 1993; 14: 241–254.
56.
Shivers BD, Harlan HE, Morell JI, Pfaff DW. Absence of oestradiol concentration in cell nuclei
of LHRH immunoreactive neurons. Nature 1983; 304: 345–347.
57.
Silver R, Ramos CL, Silverman AJ. Sexual behavior triggers the appearance of non-neuronal
cells containing gonadotropin-releasing hormone-like immunoreactivity. J Neuroendocrinol
1992; 4: 207–210.
58.
Silverman AJ, Millar RP, King JA, Zhuang X, Silver R. Mast-cells with gonadotropinreleasing hormone-like immunoreactivity in the brain of doves. Proc Natl Acad Sci USA 1994;
91: 3694–3699.
59.
Soma KK, Francis RC, Wingfield JC, Fernald RD. Androgen regulation of hypothalamic
neurons containing gonadotropin-releasing hormone in a cichlid fish: Integration with social
cues. Horm Behav 1996; 30: 216–226.
60.
Sullivan KA, Witkin JW, Ferin M, Silverman AJ. Gonadotropin-releasing hormone neurons in
the rhesus macaque are not immunoreactive for the estrogen receptor. Brain Res 1995; 685:
198–200.
61.
Troskie B, Illing N, Rumbak E, Sun Y-M, Hapgood J, Conklin D, Millar R. Identification of
three putative GnRH receptor subtypes in vertebrates. Gen Comp Endocrinol 1998; 112:
296–302.
62.
Tsutsui H, Yamamamoto N, Ito H, Oka Y. GnRH-immunoreactive neuronal system in the
presumptive ancestral chordate, Ciona intestinalis (Ascidian). Gen Comp Endocrinol 1998;
112: 426–432.
63.
Urbanski HF, White RB, Fernald RD, Kohama SG, Densmore VS. Regional expression of
mRNA encoding a second form of gonadotropin-releasing hormone in the macaque brain.
Endocrinology 1999; 140: 1945–1948.
64.
Varma S, Sabharwal P, Malarkey WB. Human splenocytes secrete LHRH, which inhibits
lymphocyte proliferation. Prog Neuroendocrinimmunol 1992; 5: 187–191.
65.
Warembourg M, Leroy D, Peytevin J, Martinet L. Estrogen receptor and progesterone
receptor-immunoreactive cells are not colocalized with gonadotropin-releasing hormone in
the brain of the female mink (Mustela vison). Cell Tissue Res 1998; 291: 33–41.
66.
White SA, Bond CT, Francis RC, Kasten TL, Fernald RD, Adelman JP. A second gene for
GnRH: cDNA and patterns of expression. Proc Natl Acad Sci USA 1994; 91: 1423–1427.
frne 0181
240
FERNALD AND WHITE
67.
White SA, Kasten T, Bond C, Adelman J, Fernald R. Three gonadotropin-releasing hormone
genes in one organism suggest novel roles for an ancient peptide. Proc Natl Acad Sci USA
1995; 92: 8363–8367.
68. White RB, Eisen JA, Kasten TL, Fernald RD. Second gene for gonadotropin-releasing
hormone in humans. Proc Natl Acad Sci USA 1998; 95: 305–309.
69.
White R, Fernald R. Genomic structure and expression sites of three gonadotropin-releasing
hormone genes in one species. Gen Comp Endocrinol 1998; 112: 17–25.
70. White R, Fernald R. Ontogeny of gonadotropin-releasing hormone (GnRH) gene expression
reveals distinct origin for GnRH-containing neurons in the midbrain. Gen Comp Endocrinol
1998; 112: 322–329.
71.
Wilson TM, Yu-Lee L-Y, Kelley MR. Coordinate gene expression of luteinizing hormonereleasing hormone (LHRH) and the LHRH-receptor after prolactin stimulation in the rat Nb2
T-cell line: Implications for a role in immunomodulation and cell cycle gene expression. Mol
Endocrinol 1995; 9: 44–53.
72.
Wray S, Grant P, Gainer H. Evidence that cells expressing lutienizing hormone-releasing
hormone mRNA in the mouse are derived from progenitor cells in the olfactory placode. Proc
Natl Acad Sci USA 1989; 86: 8132–8136.
73. Yamamoto N, Oka Y, Kawashima S. Lesions of gonadotropin-releasing hormone-immunoreactive terminal nerve cells: Effects on the reproductive behavior of male dwarf gouramis.
Neuroendocrinology 1997; 65: 403–412.
74. Yu K-L, He M-L, Chik C-C, Lin X-W, Chang JP, Peter RE. mRNA expression of gonadotropinreleasing hormones (GnRHs) and GnRH receptor in goldfish. Gen Comp Endocrinol 1998; 112:
303–311.
frne 0181