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Department of Biology, University of Victoria (J.C., J.F.F.P., N.M.S.), Victoria, British Columbia, Canada V8W 2Y2; The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute (M.P., W.H.F., A.G.C., J.E.R.), La Jolla, California 92037; and the Department of Biological Sciences, University of Alberta (J.P.C.), Edmonton, Alberta, Canada T6G 2E9
Address all correspondence and requests for reprints to: Dr. N. M. Sherwood, Department of Biology, University of Victoria, Victoria, British Columbia, Canada V8W 2Y2. E-mail: nsherwoo{at}uvic.ca
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Abstract |
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Herring is the most phylogenetically ancient animal in which three forms of GnRH have been isolated and sequenced. Our evidence suggests that the existence of three GnRHs in the brain of one species 1) is an ancestral condition for teleosts, 2) has the potential for separate regulation of the distinct GnRHs, and 3) may be an evolutionary advantage for refined control of reproduction in different environments.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Studies on the evolution of GnRH indicate that GnRH is present in all vertebrate species studied to date (11) as well as in some invertebrates (12, 13, 14, 15, 16). Twelve different forms of GnRH were initially identified by primary structure or complementary DNA (11, 17). Each identified GnRH peptide has a length of 10 amino acids and identical residues in positions 1, 4, 9, and 10. This conservation of structure is also present in the organization of the GnRH genes. Only one form of GnRH is encoded in each gene or complementary DNA isolated to date (11). The question arises of whether there have been other changes that alter the central GnRH control of reproduction and whether diversification of GnRH peptides is important in this process.
One possibility for change in the control of reproduction is the duplication of GnRH genes with subsequent nucleotide substitutions in the GnRH-encoding region or in the regulatory flanking region. This could result in individual species having more than one GnRH form, each with a distinct function and location within the brain. We know that at least two forms of GnRH are present in the brain in both rhesus monkeys (18, 19, 20) and humans (21, 22). One of these two GnRH forms in the primate species is the most widely distributed and possibly the most ancient form of GnRH in jawed vertebrates (11); it is named chicken GnRH-II (cGnRH-II) because it was first isolated from chicken brains (23). The other GnRH form in primates is mammalian GnRH (mGnRH), which was first isolated from pigs and sheep, but is common to all mammals (24, 25); mGnRH is now considered an ancient form because it is present also in early bony fish (26, 27) and amphibians (28). Other GnRH forms that are not present in mammals include salmon GnRH (sGnRH), which is present in most teleosts studied. cGnRH-I is found only in birds (29) and reptiles (30). These two forms may have arisen as a result of nucleotide substitutions in the mGnRH gene. The mGnRH form disappears as sGnRH (in teleosts) and cGnRH-I (in reptiles) appear in the same brain location as mGnRH does in other species (31). Additional forms of GnRH have been isolated and sequenced from jawless fish (32, 33), cartilaginous fish (34, 35), catfish (36), and the more recent perciform fishes such as seabream (37).
A major problem in understanding the role of GnRH in reproduction is the functional significance of multiple GnRH forms in all vertebrates. One approach to this question is to examine bony fish at one of the earliest points where three forms of GnRH are expressed in the brain rather that the two forms that are detected in tetrapods. The appearance of a novel third form of GnRH in the brain is of great interest.
As a representative of an early evolving teleost group, the herring was selected as an appropriate fish in which to examine these questions. The Pacific herring is an extant representative of a basal teleost group (order: Clupeiformes) that evolved before the euteleosts; the latter group includes fish such as salmon, catfish, and seabream.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Extraction of GnRH
Frozen tissues were powdered in a Waring blender with liquid
nitrogen and extracted in acidified acetone as described previously
(36). After filtration, lipids and most of the acetone were removed by
repeated extractions of the filtrate with petroleum ether; the
remaining acetone was removed from the crude extract with vacuum
centrifugation.
Purification of GnRH
Purification of GnRH was performed by HPLC and RIA in six steps
for the brain extract and in four steps for pituitary extract. A HPLC
pump (model 166, Beckman Coulter, Inc., Palo Alto, CA) was
used for the preparative chromatography. Fractions of 1 ml were
collected from each chromatography step, and aliquots were assayed for
immunoreactive GnRH (irGnRH). The absence of residual contaminating
GnRH on the column was confirmed before purification of GnRH by
collecting and assaying fractions of blank runs.
The brain extract was applied to a column of 10
C18 Sep-Pak cartridges (Waters Corp., Milford, MA) in step 1 and eluted with mobile phases A
[0.5% trifluoroacetic acid (TFA)] and B [0.5% TFA, 80%
acetonitrile (ACN)] in the program shown in Fig. 1A
. In step 2, the immunoreactive
fractions were applied to a C18 HPLC column and
eluted with mobile phases A [0.25 N triethylammonium
formate (TEAF), pH 6.5] and B (100% ACN), as shown in Fig. 1B.
Thereafter, each of the 3 immunoreactive peaks was purified separately,
but in the same sequence. Step 3 was based on the procedure described
by Rivier (38); fractions were applied to a C18
column and eluted with 0.13 M triethylammonium phosphate
(TEAP), pH 2.5 (mobile phase A), and ACN (Fig. 1C). Step 4 used
a C18 column and elution with 0.05% TFA and
0.05% TFA/80% ACN, whereas step 5 used a phenyl column and the same
mobile phases as those in step 4 (Fig. 1, D and E). The final step (not
shown) was microbore HPLC on a Vydac C18 column
using 0.1%TFA and 0.1% TFA/80% ACN.
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; only the earliest eluting peak was purified further using the
methods shown in Fig. 1, C and E. The final step was microbore HPLC as
described for the brain extract.
RIA
During the purification of GnRH herring extracts, 10-µl
aliquots of each 1-ml fraction were assayed for irGnRH using methods
described previously (27). Antiserum GF-4 (1:25,000 final dilution) was
used to detect irGnRH, with mGnRH used as the iodinated tracer and
standard. The GF-4 antiserum has been shown to detect several known
forms of GnRH: mGnRH, 100%; cGnRH-I, 44%; catfish GnRH, 44%; sea
bream GnRH (sbGnRH), 41%; dogfish GnRH, 5%, lamprey GnRH-III, less
than 0.4%; and lamprey GnRH-I, less than 0.03% (19). The
cross-reactivities of GF-4 with peptides used in this paper were
herring GnRH (hrGnRH), 17%; sGnRH, 29%; and cGnRH-II, 7%; the assay
detection limit was [bound/free ratio (B/Bo), 80%] 10 pg. Where
tracer binding was outside the assay limits (B/Bo, <20%), aliquots
were diluted serially, and the value closest to 50% B/Bo was used for
analysis.
Sequencing of peptides
Peptides in the three purified irGnRH components from herring
brain extraction and one purified irGnRH component from herring
pituitaries were sequenced using a model 470A PE Applied Biosystems protein sequencer (Foster City, CA). Sequencing was
initially attempted on 10% of each sample. Failure of this sequencing
indicated a blocked N-terminus. Subsequent sequencing was carried out
on the remaining material after digestion with pyroglutamate
aminopeptidase and microbore HPLC purification, as detailed previously
(39).
Mass spectrometry
The intact molecular mass of hrGnRH was determined on a Bruker
Reflex time of flight instrument using an accelerating voltage of 31 kV
and a reflectron voltage of 30 kV (100-MHz digitizer). The sample was
applied to a thin layer of 
-cyano-4-hydroxycinnamic acid,
allowed to dry, and rinsed with water before analysis. To establish
whether the C-terminus of the peptide was amidated, esterification with
methanolic HCl monitored with matrix-assisted laser desorption
ionization-mass spectrometry was used (40, 41). Control reactions were
carried out with bombesin and bombesin free acid, where a mass shift of
14 Da was observed only for the free acid form.
Synthesis of peptides
The peptides for hrGnRH, sGnRH, cGnRH-II, and sbGnRH were
synthesized using a solid phase method on a methylbenzhydrylamine resin
as previously described (37, 42).
Physiology
To investigate the physiological activity of GnRH peptides, the
ability of synthetic hrGnRH, sGnRH, cGnRH-II, and sbGnRH to stimulate
pituitary hormone release from primary cultures of dispersed goldfish
pituitary cells was examined in 2-h static incubation studies. Goldfish
pituitaries were used because it has been shown that multiple native
GnRH forms stimulate the release of hormones (35) and because assays
are available for goldfish, but not herring, pituitary hormones.
Pituitary cells from both male and female goldfish were prepared by
trypsinization, as previously described (43). Cells were cultured
overnight at a density of 0.25 x 106
cells/ml·well in 24-well culture plates in culture medium (medium 199
with Earle’s salts, supplemented with 25 mM HEPES, 2.2
g/liter NaHCO3, 1% horse serum, 100,000 U/liter
penicillin, and 100 ng/liter streptomycin, pH adjusted to 7.2 with
NaOH; Life Technologies, Inc., Grand Island, NY) at 28 C
under 5% CO2 and saturated humidity. Before the
experiment, cells were washed with 1 ml testing medium (medium 199
containing Hanks’ salts, 0.1% BSA, 2.2 g/l
NaHCO3, 100,000 U/liter penicillin, and 100
mg/liter streptomycin, pH 7.2) and allowed to rest in the incubator for
at least 1 h. The cells were then washed again with 1 ml fresh
testing medium, and concentrated GnRH solutions were added (in
distilled deionized water; 1 µl/ml to achieve the final desired GnRH
concentration). All treatments were performed in triplicate or
quadruplicate. Cells were then returned to the incubator for the
duration of secretion testing (2 h). At the end of the testing period,
800 µl medium were collected from each well. All experiments were
repeated four times (twice each with cells prepared from fish at
sexually regressed and recrudescent stages). All samples were stored at
-20 C until their gonadotropin II contents were quantified by RIA
validated for measurements of goldfish maturational gonadotropin
(GTH-II) (44, 45) and GH (46). Results were expressed as a percentage
of basal release (unstimulated controls), and pooled data (mean ±
SEM) are presented. Statistical analyses were performed
using ANOVA followed by Fisher’s least significant difference test.
Differences were considered significant at P <
0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The fractions with irGnRH
were pooled and carried to step B. HPLC elution of the material in step
B resulted in three distinct peaks of immunoreactive GnRH that were
designated sequentially according to elution time as herring I–III
(Fig. 1B) and were pooled individually. Each pool was carried through
the subsequent purification steps individually (presented as composite
graphs in Fig. 1, C–E) until single absorption peaks were obtained. At
the end of step E, the amounts were 0.12, 0.45, and 1.04 µg herring
I, II, and III irGnRH, respectively.
irGnRH from pituitaries
From the pituitary extract, three peaks of irGnRH eluted in
positions comparable to those of the brain extract (Fig. 1F). Peaks I,
II, and III contained 301, 3, and 68 ng irGnRH, respectively. Only peak
I was purified further.
Sequence and mass spectral analysis
The following three amino acid sequences were deduced from the
combination of pyroglutamyl aminopeptidase digestion and Edman
degradation of the purified immunoreactive components: herring I
(hrGnRH), [pGlu]-His-Trp-Ser-His-Gly-Leu-Ser-Pro-Gly;
herring II (cGnRH-II),
[pGlu]-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly; and herring
III (sGnRH),
[pGlu]-His-Trp-Ser-Tyr-Gly-Trp-Leu-Pro-Gly.
The accession numbers in SWISS PROT are P81749 (hrGnRH-I), P81750
(hrGnRH-II), and P81751 (hrGnRH-III). Figure 2 shows the matrix-assisted laser
desorption ionization mass spectrum of the untreated hrGnRH in which an
intense species was observed at m/z 1087.9 compared with the calculated
monoisotopic [M+H]+ of 1087.51 Da for the
sequence of hrGnRH (herring I) shown above. Treatment with methanolic
HCl did not result in a mass shift, indicating that the C-terminus was
amidated. We have named the novel structure hrGnRH in accordance with
the convention of naming GnRH molecules after the organism from which
the primary structure is first determined. The deduced sequences and
the elution characteristics during purification of herring II and
herring III corresponded to those previously described for chicken
GnRH-II (23) and salmon GnRH (47), as shown in Fig. 3. Sequencing of the irGnRH form purified
from herring pituitary yielded the same sequence as hrGnRH (herring I)
isolated from the herring brain.
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. sGnRH and cGnRH-II, two GnRH forms
that also are native to the goldfish, stimulated gonadotropin II
release in this as in previous studies (43). Similarly, hrGnRH
increased gonadotropin II release from goldfish pituitary cells. For
these three GnRHs, the lowest concentration of GnRH tested (0.1
nM) significantly elevated gonadotropin II secretion above
basal values and the maximal gonadotropin II responses elicited were
not different from one another. The efficacies of sGnRH and hrGnRH were
also not different throughout the entire dose range examined. On the
other hand, cGnRH-II was consistently more effective than either sGnRH
or hrGnRH in the lower dose range (1–100 nM;
P < 0.05; cGnRH-II vs. sGnRH and hrGnRH).
In contrast to the other GnRHs, sbGnRH failed to stimulate gonadotropin
II secretion throughout the dose range tested (0.1–1000
nM).
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). Also, hrGnRH and sbGnRH elevated GH secretion, and the
maximal GH responses to sGnRH, cGnRH-II, and hrGnRH were similar (Fig. 4B). Both sGnRH and cGnRH-II were effective at the lowest (0.1
nM) dose tested; however, the GH responses were less
sensitive to hrGnRH and sbGnRH stimulation. The minimal effective dose
for hrGnRH was 100 nM, and that for sbGnRH was 1000
nM. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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hrGnRH differs from other known forms primarily in positions 5–8 (Fig. 3), which is consistent with the idea that the C- and N-terminals are
conserved because they are needed for conformation and receptor binding
(48). The histidine in position 5 and the serine in position 8 are
probably responsible for the high hydrophilicity compared with those of
the other forms of GnRH. The forms of GnRH that have the highest
sequence identity (90%) with hrGnRH are sbGnRH, which differs only in
position 5 (Tyr), and catfish GnRH (cfGnRH), which differs in position
8 (Asn). Both sbGnRH and cfGnRH are also hydrophilic. hrGnRH differs
from mGnRH by two residues.
Deduction of separate location and function for GnRH forms
The function of the three forms of GnRH in the herring can be
deduced in part from other studies of teleosts. Published research,
including the use of both specific antisera and in situ
hybridization, shows there are three distinct clusters of
GnRH-containing cell bodies: l) the terminal nerve ganglion (TN) at the
junction between the olfactory bulb and telencephalon, 2) the preoptic
area (POA) just anterior to the hypothalamus, and 3) the midbrain
tegmentum (MT) (49). The TN GnRH fibers project throughout the brain
and spinal cord, but usually not to the pituitary; the POA GnRH cells
project to the pituitary; and the MT GnRH neurons project mainly to
brain areas posterior to the hypothalamus and to the spinal cord (50).
In all of the teleosts studied, only cGnRH-II was found in the MT. In
teleosts such as salmon (or catfish), in which only two forms of GnRH
are detected in the brain, sGnRH (or cfGnRH) was present in both the TN
and POA cluster of neurons (51, 52, 53). In recently evolved teleosts (such
as sea bream, cichlid, or tilapia) that have three forms of GnRH in the
brain, cGnRH-II was in the MT, sGnRH was in the TN, and the new form of
GnRH (sbGnRH) was in the POA (49, 54, 55). It is the new form of GnRH
that is thought to be the principal form that induces gonadotropin
release from the pituitary. This conclusion is based on evidence that
the new form is the most abundant GnRH in the pituitary (41, 56), and
the new form of GnRH is expressed in neurons in the preoptic area of
the brain, with axons terminating in the pituitary. GnRH in the
olfactory region has been suggested to couple sensory information from
the environment with reproduction, although the evidence is based
primarily on the distribution of GnRH neurons; GnRH in the midbrain has
been related to reproductive behavior.
In the present study, the highest content of GnRH in the pituitary is the herring form, which is also the newly evolved GnRH form. The level of hrGnRH was 8-fold higher than that of sGnRH and 43-fold higher than that of cGnRH-II in the HPLC fractions after each peak was corrected for relative cross-reactivity in our assay (16.7% for hrGnRH, 6.6% for cGnRH-II, and 29.1% for sGnRH). The isolation and determination of primary structure for hrGnRH from pituitary tissue confirm that this novel form exists in the pituitary and is not an artifact of immunological cross-reactivity. These data suggest that hrGnRH is located in the POA and controls the release of gonadotropin from the pituitary.
hrGnRH is clearly effective in releasing gonadotropin II and GH from fish pituitary cells, with a maximal response similar to that observed for sGnRH and cGnRH-II. hrGnRH was as effective as sGnRH in releasing gonadotropin II. On the other hand, hrGnRH is less potent than both sGnRH and cGnRH-II in increasing GH release. Surprisingly, seabream GnRH, the form responsible for gonadotropin II release in fish that are more recently evolved than herring (e.g. sea bream and cichlids) is ineffective in stimulating gonadotropin II release and is relatively ineffective in stimulating GH release in the goldfish assay. This observation is consistent with the low potency of sbGnRH compared with sGnRH and cGnRH-II when injected in vivo into sea bream; the concentration of gonadotropin II in sequential blood samples was measured after injection of each peptide (57). Thus, a single amino acid difference in position 5 (Tyr in sbGnRH and His in hrGnRH) results in a considerable difference in potency for activating receptors on the goldfish gonadotropes and somatotropes.
Phylogenetic argument for GnRH gene duplication
Herring is the most phylogenetically ancient animal in which three
forms of GnRH have been identified by determination of primary
structure. This evidence suggests that a GnRH gene duplication occurred
in an ancestral fish that gave rise to herring and possibly to the
euteleostean line that evolved after herring. We suggest that the gene
encoding hrGnRH resulted from modification of one copy of a duplicated
GnRH gene. Evolutionary selection of fish with more refined control of
reproduction may be one factor in the success of the teleosts, the
vertebrate group with the largest number of individuals and species
(36% of all vertebrate species) (58).
The novelty of the hrGnRH sequence is important in considering whether a gene duplication with subsequent modification of one copy occurred. The suggestion of duplication rests on both the novel structure and the conservation of GnRH gene organization in which only one form of GnRH is encoded in each gene in the seven forms studied to date (11).
The proof that herring is the first major group of bony fish to have
three forms of GnRH has been provided by an examination of a number of
species that evolved before herring. In bony fish (class:
Osteichthyes), there are four major divisions of bony fish that evolved
before the teleosts. The same two forms of GnRH (mGnRH and cGnRH-II)
have been identified in representative fish (reedfish, sturgeon,
garfish, and amia) in each division by HPLC-RIA methods (27) and by
primary structure for sturgeon (26) (Fig. 5). Thereafter, the teleosts arose as a
monophyletic group and as the most advanced and recent group of bony
fish. The same two forms of GnRH were found by HPLC and RIA, but have
not yet been found by primary structure in the earliest teleosts to
evolve, the eels and one of the bonytongued fish (butterfly fish) (62).
However, the mGnRH form disappeared, and the sGnRH form appeared, most
likely as a substitution within the mGnRH gene. The new combination of
sGnRH and cGnRH-II has been detected by HPLC-RIA methods in a number of
bonytongued fish (featherfins, arawana, and elephantnose) (62).
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Evolutionary advantage for separate control of expression for each
GnRH form
In herring we can deduce the brain locations of the three forms of
GnRH: sGnRH is in the TN region, hrGnRH is in the POA, and cGnRH-II is
in the MT. The evidence is based on the definitive identification of
hrGnRH in the pituitary at 8- to 43-fold the content of the other
forms. In teleosts the axons of POA-GnRH neurons are distributed to the
pituitary. Expression of distinct GnRH forms in separate locations
implies that at least some regions in the regulatory 5'-flanking region
of each of the three genes are different.
Separate control of expression of the three GnRHs within the brain would offer an evolutionary advantage. The TN GnRH neurons connect the olfactory region with the retina and other parts of the brain. Some of these neurons synapse onto the POA neurons. The MT neurons with cGnRH-II may influence reproductive behavior, and many of their axons enter the spinal cord. Recent evidence shows that sGnRH and cGnRH-II can alter spawning behavior in goldfish (66).
Separate control of the three GnRHs is important when each form has a different location and a different function. All three GnRHs can release gonadotropin II if used in an in vitro assay, but in vivo only the GnRH form in the POA neurons is delivered in abundance to the pituitary. There are several possibilities for the advantage of separate control of the new form of GnRH, which releases gonadotropin II.
First, ovulation in herring can be separated from spawning by 1 or 2 months. When spawning does occur, it is rapid and almost simultaneous for all males and females in the school of herring. This mass spawning appears to be under the control of a male pheromone in the milt (67), and thus it may be a considerable advantage for all females in the school to hold the ovulated eggs until the best conditions for spawning have been detected. Separate control of hrGnRH for ovulation and of sGnRH and cGnRH-II for sensory detection or behavior in spawning would be useful.
Second, many prolific and phylogenetically recent fish with three forms of GnRH release their eggs in batches, either daily or periodically, over a prolonged period of time. The advantage would be to avoid "putting all the eggs in one basket," that is in a single environmental context. For example, sea bream release batches of eggs daily for several months, and tilapia spawn many times a year. The advantage of the third GnRH (sbGnRH) found in both of these fish is that it has a low potency, as measured in sea bream (57), which may result in ovulation of only fully mature eggs. If sbGnRH were more potent, immature eggs might be released, and batch spawning lost.
Evolutionary pressure may have selected fish with three forms of GnRH provided that each form has a distinct location and/or function. Initially, the duplicated form of the GnRH gene would be a neutral change. At the point where the GnRH sequence changed in one gene and where regulation of expression was distinct from the original gene, then selection could occur. The refined control of reproduction may be a considerable advantage in fish in which spawning occurs many times over a prolonged period of time or spawning is delayed to match optimal environmental conditions. Increased survival of the offspring is likely to occur in both types of fish.
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Acknowledgments |
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Footnotes |
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2 Present address: Syndel International, Inc., 9211 Shaughnessy
Street, Vancouver, British Columbia, Canada V6P 6R5.
Received August 3, 1999.
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References |
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 L. Wang, J. Bogerd, H. S. Choi, J. Y. Seong, J. M. Soh, S. Y. Chun, M. Blomenröhr, B. E. Troskie, R. P. Millar, W. H. Yu, et al. Three distinct types of GnRH receptor characterized in the bullfrog PNAS, December 14, 2000; (2000) 11508498. [Abstract] [Full Text]
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T. Madigou, E. Mañanos-Sanchez, S. Hulshof, I. Anglade, S. Zanuy, and O. Kah Cloning, Tissue Distribution, and Central Expression of the Gonadotropin-Releasing Hormone Receptor in the Rainbow Trout (Oncorhynchus mykiss) Biol Reprod, December 1, 2000; 63(6): 1857 - 1866. [Abstract] [Full Text]
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L. Wang, J. Bogerd, H. S. Choi, J. Y. Seong, J. M. Soh, S. Y. Chun, M. Blomenrohr, B. E. Troskie, R. P. Millar, W. H. Yu, et al. Three distinct types of GnRH receptor characterized in the bullfrog PNAS, January 2, 2001; 98(1): 361 - 366. [Abstract] [Full Text] [PDF]
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