Endocrinology Vol. 142, No. 4 1453-1460
Copyright © 2001 by The Endocrine Society
Primary Structure of a Novel Gonadotropin-Releasing Hormone in the Brain of a Teleost, Pejerrey1
Alejandro D. Montaner,
Min Kyu Park,
Wolfgang H. Fischer,
Anthony G. Craig,
John P. Chang,
Gustavo M. Somoza,
Jean E. Rivier and
Nancy M. Sherwood
Instituto de Investigaciones Biomédicas (A.D.M., G.M.S.),
Fundación Pablo Cassará, Saladillo 2452 (C1440FFX), Buenos
Aires, Argentina; Salk Institute (M.K.P., W.H.F., A.C.G., J.E.R.), La
Jolla, California 92037; Department of Biological Sciences (J.P.C.),
University of Alberta, Edmonton, Alberta T6G 2E9, Canada; and
Department of Biology (N.M.S.), University of Victoria, Victoria,
British Columbia, V8W 2Y2, Canada
Address all correspondence and requests for reprints to: Nancy M. Sherwood, Department of Biology, University of Victoria, Victoria, British Columbia V8W 2Y2, Canada. E-mail: nsherwoo{at}uvic.ca
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Abstract
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The neuropeptide GnRH is the major regulator of reproduction in
vertebrates acting as a first signal from the hypothalamus to pituitary
gonadotropes. Three GnRH molecular variants were detected in the brain
of a fish, pejerrey (Odontesthes bonariensis), using
chromatographic and immunological methods. The present study shows that
one form is identical to chicken GnRH-II (sequence analysis and mass
spectrometry) and the second one is immunologically and
chromatographically similar to salmon GnRH. The third form was proven
to be a novel form of GnRH by isolating the peptide from the brain and
determining its primary structure by chemical sequencing and mass
spectrometry. The sequence of the novel pejerrey GnRH is
pGlu-His-Trp-Ser-Phe-Gly-Leu-Ser-Pro-Gly-NH2, which is
different from the known forms of the vertebrate and protochordate GnRH
family. The new form of GnRH is biologically active in releasing
gonadotropin and GH from pituitary cells in an in vitro
assay.
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Introduction
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GnRH is a key neurohormone for reproduction
in all groups of vertebrates. This decapeptide was originally isolated
from mammals as the hypothalamic hormone that regulates the
reproductive system by stimulating the release of gonadotropins from
the anterior pituitary gland. To date, 13 distinct GnRH forms have been
sequenced from vertebrate and protochordate nervous tissue
(1, 2, 3). Evidence based on indirect chromatographic and
immunological techniques suggests the existence of additional GnRH
forms in different groups of vertebrates (4, 5, 6).
All family members are decapeptides with a conserved structure:
the N terminus (pyroglutamyl residue) and C-terminus (amidated glycine)
are conserved, in addition to positions 4 and
9. A mammalian form of GnRH (mGnRH) was
originally isolated from porcine and ovine brains (7, 8). Also, in mammals a novel guinea pig (gp) GnRH was
identified and sequenced from nervous tissue (2). Two
other GnRH forms were isolated first from chicken brains: chicken
GnRH-I (cGnRH-I) and chicken GnRH-II (cGnRH-II) (9, 10, 11).
Additional GnRH peptides have been isolated from brains of salmon
(sGnRH) (12), lamprey (lGnRH-I and lGnRH-III) (13, 14), catfish (cfGnRH) (15), dogfish (dfGnRH)
(16), herring (hrGnRH) (3), and seabream
(sbGnRH) (17). Two more peptides in tunicates (tGnRH-I and
tGnRH-II) were characterized from protochordate nervous tissue
(18).
In all bony fish species examined to date, it is well documented that
at least two GnRH variants coexist in the brain of a single species.
cGnRH-II is universally distributed in bony fish (1). In
addition to cGnRH-II, mGnRH is found in lobe-finned fish
(19), the ancient ray-finned bony fish that evolved before
the teleosts (20, 21), and in teleosts that evolved early
such as Pantodon (22) and eels (23). All
other teleosts studied to date have salmon GnRH with the exception of
catfish, which has cfGnRH (15). The remaining forms of
GnRH in bony fish are the third forms found in addition to cGnRH-II and
sGnRH in some teleosts. The third forms include hrGnRH and sbGnRH.
Also, there is chromatographic and immunological evidence that there
may be other variants in the brain of some fishes
(24, 25, 26, 27).
The form of GnRH isolated and sequenced from herring has been found
only in that species (3), whereas sbGnRH has been isolated
and sequenced from seabream (17, 28), pacu
(29), and two cichlids (tilapia and the African cichlid,
Haplochromis burtoni) (30, 31). Also, the form
of GnRH found in the pejerrey, Odontesthes bonariensis, was
reported to coexist with sGnRH and cGnRH-II based on RP-HPLC/RIA
studies (6). The pejerrey fish was originally found only
in the inland waters of the Province of Buenos Aires in Argentina.
Later, this fish was introduced into many water bodies around the world
for game fishing and aquaculture (32).
The main objective of this study was to determine the primary structure
of the form of GnRH in pejerrey that had been predicted to be novel by
indirect methods. The structure is characterized by RP-HPLC elution
position in comparison with synthetic standards, cross-reactivity with
antisera, chemical sequencing, and mass spectral analysis. Also,
the biological activity of the novel GnRH is assessed in
vitro.
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Materials and Methods
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Collection of brains
Whole brains (n = 450), some with attached pituitaries,
weighed a total of 51.6 g. The tissue was collected during the
spawning season from both sexes of adult pejerrey (Odontesthes
bonariensis; order: Atheriniformes) in the Province of Buenos
Aires, Argentina (Salada Grande and Chascomús Ponds). The tissues
were removed from freshly killed fish, placed immediately on dry ice,
transported to the laboratory in Buenos Aires, and stored at -70 C.
Frozen brains were then transported on dry ice to the Department of
Biology at the University of Victoria, Canada.
Peptide extraction
Frozen pooled brains and pituitaries were powdered in a Waring
blender with liquid nitrogen. Extraction of peptides was done in an
acetone/1 M HCl mixture (100:3, vol/vol) for 3 h as
previously described (13). The insoluble material was
reextracted in acetone/0.01 M HCl (80:20, vol/vol) for 5
min and refiltered. The combined filtrates were defatted with petroleum
ether (bp 30–60 C) for five successive times using a ratio of 5 parts
filtrate to 1 part petroleum ether (13). Then the final
aqueous phases were concentrated to less than 1 ml using a vacuum
concentrator.
RP-HPLC purification
The brain extract was filtered and applied to a series of 10
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). Sixty fractions of 1 ml were
collected and an aliquot of 5 µl taken for RIA. Immunoreactive GnRH
fractions were pooled, concentrated under vacuum and injected onto a
Supelco (Oakville, Ontario, Canada; Supelcosil
LC-18) analytical column using a Beckman (Mississauga,
Ontario, Canada) 166 model Liquid Chromatograph. The sample was
applied at the beginning of a 10 min isocratic period of 17% ACN in
0.25 M triethylammonium formate TEAF (pH 6.5); then ACN was
increased to 24% over a 7-min period and held isocratically for 43
min. The flow rate was kept at 1 ml/min and 1 ml fractions were
collected (13). Aliquots of 10 µl from each fraction
were used for RIA to determine immunoreactive GnRH (ir-GnRH). Eluted
ir-GnRH fractions were pooled from this step and designated as peak I,
II, or III. The three ir-GnRH peaks were selected for further
purification. Three successive RP-HPLC steps were performed. Column
types, solvents and conditions are summarized in Fig. 1
. Each injection
of the tissue extract was preceded by a blank run, in which the mobile
phase was injected. The blank fractions were radioimmunoassayed under
the same conditions as the samples to ensure that the column was not
contaminated. Fresh standards were chromatographed onto a different
column to compare the elution position of each ir-GnRH peak.
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Figure 1. Steps of GnRH purification from pejerrey brains.
Each line indicates the sequential RP-HPLC system and
elution program. The mobile phase at each step was a mixture of A and B
as outlined in the third column. ACN, Acetonitrile; TFA,
trifluoroacetic acid; TEAF, triethylammonium formate; TEAP,
triethylammonium phosphate.
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RIA measurement
Aliquots of 10 µl from fractions collected at each successive
step in the RP-HPLC purification were assayed for ir-GnRH by methods
previously described (13). Two different antisera, GF-6
(1:25,000 final dilution) and PBL 49 (1:150,000 final dilution, kindly
provided by Dr. W.W. Vale), were used in an assay with mGnRH as
radioiodinated hormone and standard. This assay was heterologous for
cGnRH-II, sGnRH, and the novel pejerrey (pj) GnRH. Cross-reactivities
of these antisera were reported in Lescheid et al.
(33) and Montaner et al. (34). The
initial RP-HPLC step (Fig. 2, box B) was
assayed by both GF-6 and PBL 49; each antiserum detected the same three
peaks and the values measured between antisera were comparable. RP-HPLC
purification of GnRH in subsequent steps (Fig. 2, boxes C–E) was
followed using antiserum GF-6.
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Figure 2. Elution of ir-GnRH fractions during
chromatographic purification from pejerrey brains. A, Prepurification:
ACN/TFA (Sep-Pak); B, Step 1: ACN/TEAF, pH 6.5; C, Step 2: ACN/TEAP, pH
2.5; D, Step 3: ACN/TFA, pH 2.0; E, Step 4: ACN/TFA, pH 2.0.
Immunoreactive fractions are indicated by bars.
Different bar graph patterns indicate separate HPLC
purification of the fractions. Graphs C, D, and E are composites of
three HPLC runs. Final step with microbore HPLC is not shown.
Dotted lines indicate ACN concentration of the mobile
phase.
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The RIA detection limit for GtH-II was better than 0.3 ng/ml and for GH
was 2 ng/ml. Basal GtH-II secretion from this study averaged 263
± 58 ng/ml and GH secretion averaged 988 ± 93 ng/ml. Appropriate
dilutions of the samples were made to ensure that the values were
within the useful range of the assays. The RIA for GtH-II had 4.9%
interassay variability as determined by the coefficient of variation
(CV) of GtH-II values at 50% displacement. Within assay variability
was 11% from the same assays as determined by the average CV of
quadruplicate determinations of a 5 ng/ml GtH-II standard in each of
the assays. For the GH assays, the interassay variability is 6.6% and
the intraassay variability is 9.4%, as determined from the average CV
of quadruplicate determinations of a 20 ng/ml GH standard in each
assay.
Characterization of the primary structure
An aliquot of the peptide purified by RP-HPLC on a diphenyl
column was injected into a microbore C-18 column using 0.05% TFA and
ACN for elution. Fractions were collected and analyzed with 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-hydroxy cinnamic acid, allowed
to dry, and rinsed with water before analysis. 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 earlier (35).
This was followed by RP-HPLC separation and sequence analysis by
automated Edman degradation on a PE-ABI Procise 494 protein
sequencer.
Synthesis of synthetic GnRH peptides
The peptides for (pj)GnRH, sGnRH, and cGnRH-II were synthesized
using a solid phase method on a methylbenzhydrylamine resin as
described (36). Boc strategy was used with the following
protecting groups: pyro-Glu(carbobenzoxy), Boc-His(tosyl),
Boc-Ser(benzyl) and Boc-Tyr(2-bromocarbobenzoxy). GnRHs were
deprotected and cleaved from the solid support with hydrofluoric acid.
After purification with reverse-phase HPLC in two solvent systems
(37), the structure was confirmed by mass spectral and
amino acid composition analyses.
Bioactivity of GnRH peptides
Synthetic peptides for pjGnRH, sGnRH, and cGnRH-II were tested
for release of pituitary hormones in primary cultures of dispersed
goldfish pituitary cells using 2-h static incubations. Goldfish
pituitaries were used because many GnRH forms release goldfish
pituitary hormones (3, 16) and assays for pituitary
hormones are not available for pejerrey. Pituitary cells from both male
and female goldfish were prepared by trypsinization as previously
described (38). Cells were cultured overnight at a density
of 0.25 x 106 cells per ml per well in
24-well culture plates at 28 C under 5% CO2 and
saturated humidity. Culture medium 199 with Earl’s salts was
supplemented with 25 mM HEPES, 2.2 g/l
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). Before the
experiment, cells were washed with 1 ml testing medium (medium 199
containing Hanks’ salts, 0.1% BSA, 2.2 g/liter
NaHCO3, 100,000 U/liter penicillin and 100
mg/liter streptomycin, pH 7.2) and allowed to rest in the incubator for
at least an hour. The cells were then washed again with 1 ml fresh
testing medium and concentrated GnRH solutions added (1 µl per ml) in
distilled deionized water to achieve the final desired concentration.
All treatments were done 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, medium (800 µl) was collected
from each well. All experiments were repeated two times with cells
prepared from sexually regressed and recrudescent stages. All samples
were stored at -20 C until gonadotropin-II (GtH-II) contents were
quantified by RIA validated for measurements of goldfish
gonadotropin-II (LH-like) (39, 40) and GH
(41). The GtH-II assay does not cross-react with mammalian
gonadotropins or with salmonid or carp GtH-II; the GH assay does not
cross-react with goldfish PRL or with other GH or PRL preparations
(39, 40, 41). The sensitivity of the assays as estimated by
90% B/Bo values was lower than 0.16 ng/ml for GtH-II and 2.5 ng/ml for
GH. Results are normalized in that they are expressed as a percentage
of basal release (unstimulated controls) and as pooled data (mean
± SEM). Statistical analyses of hormone responses were
performed using ANOVA followed by Dunnett’s posthoc test. Differences
were considered significant at P < 0.05.
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Results
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Initial analysis
To determine the elution position of cGnRH-II and sGnRH synthetic
standards, these peptides were chromatographed in a different column
from that used for purification in RP-HPLC. The elution position of the
standards was tested by RIA; cGnRH-II eluted in fraction 26 and sGnRH
in fractions 48–49.
Screening of GnRH from brain extract using Sep-Pak cartridges revealed
one extended ir-GnRH area between fractions 10–45 (Fig. 2A
). These
fractions were pooled, concentrated and used for subsequent steps of
purification.
GnRH purification
The first RP-HPLC purification step revealed three main ir-GnRH
peaks (Fig. 2B). The first ir-GnRH peak (peak I, fractions 25–29)
eluted as observed for the synthetic cGnRH-II standard. The second
ir-GnRH peak (II, fractions 31–35) did not coelute with any of the
known GnRH forms. The third ir-GnRH peak (III, fractions 48–52) eluted
in the same position as synthetic sGnRH. All immunoreactive fractions
were pooled for individual peaks and used for subsequent steps of
purification.
In the second RP-HPLC purification step (TEAP step), a single ir-GnRH
peak eluted from each peak of immunoreactive material applied from the
previous run (Fig. 2C). Peak I eluted in fractions 21–25 (as did the
cGnRH-II standard); peak II eluted in fractions 28–31 and peak III
eluted in fractions 38–41 (as did the sGnRH standard). Then the
ir-GnRH fractions from the second HPLC step of purification were
concentrated in a vacuum centrifuge and reinjected into a C-18 column
for the third step of the purification protocol (TFA step). Three
different immunoreactive fractions corresponding to peaks I, II, and
III eluted between fractions 64–68, 41–44, and 48–51 (Fig. 2D).
Each peak was concentrated and subjected to the last step of
purification (Fig. 2E). The three ir-GnRH peaks eluting from the
diphenyl column in positions 29–30, 31–32, and 35–36, were used for
sequence analysis.
Sequence and mass spectral analysis
The retention of the second ir-GnRH fraction on RP-HPLC was
different from that of any known form of GnRH. Initial attempts to
analyze the peptide by chemical sequence analysis failed due to a
blocked N terminus. When the peptide was reanalyzed after treatment
with pyroglutamyl aminopeptidase, the following sequence was obtained:
His-Trp-Ser-Phe-Gly-Leu-Ser-Pro-Gly. The intact molecule mass of the
purified peptide as determined by MALD mass spectrometry was 1097.8
(M+H+) (Fig. 3), which is in good
agreement with the theoretical mass of 1097.52 kDa for the sequence of
pGlu-His-Trp-Ser-Phe-Gly-Leu-Ser-Pro-Gly-NH2. The
isolated native peptide was found to elute at the same percentage of
ACN as the identical synthetic peptide.
The purified ir-GnRH fraction I was identified as cGnRH-II by protein
sequencing and mass spectrometry. The amino acid sequence of the
pyroglutamyl aminopeptidase-treated GnRH peak was determined to be
His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly. The monoisotopic molecule mass
measured by electrospray was 1236.6 (data not shown), comparable to the
cGnRH-II theoretical mass of 1236.53 kDa.
The ir-GnRH peak III showed the same immunological characteristics and
retention time as sGnRH in all RP-HPLC systems used. However, the
amount was not enough to determine the sequence.
Bioactivity of peptides: GtH-II and GH release
The release of GtH-II and GH from goldfish pituitary cells after
the addition of synthetic forms of pjGnRH, cGnRH-II or sGnRH is shown
in Fig. 4. Similar to cGnRH-II and sGnRH,
pjGnRH released GtH-II and GH from dispersed goldfish pituitary cells
in vitro. The lowest dose at which GnRH resulted in a
significant release of GtH-II compared with the control was
10-11
M for cGnRH-II and
10-10
M for sGnRH and pjGnRH. The maximal GtH-II
response was not different when the three GnRHs were compared.
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Figure 4. Comparison of cGnRH-II, pjGnRH, and sGnRH to
stimulate the release of (A) gonadotropin-II (GtH-II) and (B) GH from
dispersed goldfish pituitary cells. GtH-II and GH secretions are
expressed as a percentage of the basal mean ± SEM.
For GnRH peptides, doses resulting in responses that are not different
from one another are identified by the same underscore (by ANOVA
followed by Dunnett’s posthoc test with significant differences at
P < 0.05).
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cGnRH-II and sGnRH induced release of GH to the medium as in previous
studies (3, 38). Also, pjGnRH induced GH release. The
minimal effective dose for GH release was
10-10 M for
all three GnRHs. The maximal response for pjGnRH was at
10-7 M.
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Discussion
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The present results prove that there are three forms of GnRH in
the pejerrey brain and that one form is a novel GnRH. The primary
structure of the novel form was determined to be:
pGlu-His-Trp-Ser-Phe-Gly-Leu-Ser-Pro-Gly-NH2.
Previous indirect evidence based on chromatographic and immunological
data had suggested that three forms might be present (6).
The novel form in the present paper is termed pejerrey (pj)GnRH in
keeping with the tradition that GnRH peptides are named for the species
in which they are first described. Analysis of the primary structure
for pjGnRH, especially the modified terminal residues, show that pjGnRH
is a member of the GnRH family (Fig. 5),
but is the first GnRH form to have Phe in position 5.
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Figure 5. Comparison of the known GnRH structures with
mGnRH. Boxed residues indicate amino acids changes.
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Proof of the structural identity of the pjGnRH amidated decapeptide
included: Edman degradation to determine the sequence of the GnRH
fragment (2, 3, 4, 5, 6, 7, 8, 9, 10), specific cleavage with pyroglutamyl
aminopeptidase to identify the NH2-terminal
pyroglutamyl acid and mass spectrometry to determine the intact
molecule mass. The primary structure for cGnRH-II isolated from
pejerrey brain was established with the same techniques; sGnRH was
identified by chromatographical and immunological methods as its
elution position is distinct and insufficient material was
available.
Teleost fishes are known to lack a functional hypothalamo-hypophysial
portal system and to have pituitary cells that are directly innervated
by GnRH nerve terminals (42). In a previous paper
(6), we showed the presence of only one ir-GnRH peak
(having pjGnRH characteristics) in pejerrey pituitary extracts. This
fact strengthens the idea that pjGnRH has a hypophysiotropic role. In
the present study, all three forms of GnRH were shown to release both
GtH-II and GH from fish pituitary cells in culture. The purpose of the
assay was to establish that the novel pjGnRH form is biologically
active and to determine its relative potency. For release of GtH-II,
the ED50 estimate of potency for cGnRH-II is
shifted one order of magnitude to the left relative to that for sGnRH
and pjGnRH. For GH secretion, the ED50 estimates
for the three GnRHs did not differ greatly (less than 0.3 orders of
magnitude difference between the three). The goldfish assay system has
become the standard one to test fish GnRH bioactivity because so few
teleost assays (and none for pejerrey) exist for pituitary
hormones.
It is important to consider the effects of negative controls in the
goldfish assay system because all three pejerrey GnRHs released
both GtH-II and GH. Previous results indicate that the goldfish GtH-II
and GH cell secretion assay to various neuroendocrine factors is
selective and specific. For example, a related naturally occurring GnRH
peptide (seabream GnRH) did not stimulate GtH-II release but was
effective in stimulating GH secretion (3). Similarly, GnRH
analogs that differentially inhibit the ability of sGnRH to stimulate
GtH-II and GH secretion from the goldfish pituitary have been
identified (43). In addition, dopamine stimulates GH
secretion, but inhibits GtH-II release, whereas somatostatin inhibits
GH, but not GtH-II secretions (44). Taken together, these
and other data (including the demonstrated presence of GnRH receptors
on goldfish gonadotropes and somatotropes (45), strongly
suggest that the ability of pjGnRH to elevate GtH-II and GH secretion
from goldfish pituitary cells in this study is due to specific effects
on the pituitary cells.
In pejerrey fish, ir-GnRH fibers reach all three areas of the
adenohypophysis: rostral pars distalis, proximal pars distalis, and
pars intermedia as revealed by immunocytochemistry (46).
The specific form of GnRH in these fibers could not be determined by
immunocytochemistry, but these fibers were in close association with
cells expressing PRL, GtHs, GH, and somatolactin (SL). In addition, the
presence of GnRH receptors was reported in cells expressing GtH, GH,
PRL, and SL using a combination of autoradiography and
immunohistochemistry on dispersed pejerrey pituitary cells
(47). These data suggest that GnRH may play a broad role
in regulating pituitary cell function in this as in other teleost
fishes (30).
In considering evolutionary changes in the structure of the GnRH
molecule, there is a general agreement that two genomic duplications
occurred between protochordates and the ancestral bony fish. A third
duplication may have occurred early in teleost evolution
(48). One of these ancestral forms became the highly
conserved cGnRH-II, whereas other forms gave rise to further GnRH
peptides (1, 49). In this context, knowledge of the
primary structure of pjGnRH, allows us to modify the scheme for the
evolution of GnRH (1, 3) within teleost fish (Fig. 6). This scheme takes into consideration
the decapeptide sequence, the distribution of the GnRH forms among the
bony fishes, the distinct location of different GnRH forms in the brain
(50), and the minimum number of amino acids changes.
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Figure 6. Proposed evolutionary scheme showing GnRH
multiplicity in teleost fish. All GnRH forms were identified by primary
structure. Dotted lines indicate possible changes or
represent unknown data. CII, cGnRH-II; M, mGnRH; S, sGnRH; SB, sbGnRH;
CF, cfGnRH; H, hrGnRH; PJ, pjGnRH. See text for references.
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The origin of pjGnRH could be due to nucleotide substitutions in either
hrGnRH or sbGnRH because both forms of GnRH: 1) evolved before pjGnRH;
2) are a third form in the brain; and 3) have the same location and
function in the brain. Both herring and pejerrey express cGnRH-II,
sGnRH and a third form that is the dominant form in the pituitary. The
data suggest that hrGnRH and pjGnRH are the GnRH peptides expressed by
preoptic neurons that send axons to the pituitary gland (3, 6). However, the origin of hrGnRH is not yet clear. If hrGnRH is
the first of the third forms of GnRH, then hrGnRH could have evolved by
a gene duplication from either mGnRH (2 amino acid differences) or
sGnRH (3 amino acid substitutions). In this context, both mGnRH and
sGnRH were present in early teleosts (bonytongue fish and eels) that
evolved before the herring, although these two peptides never appear
together in the same species. The origin of the third form may be tied
to the genome duplication that is thought to have occurred in early
teleosts (48). RP-HPLC and RIA evidence suggest that the
third form of GnRH was present already in some species of bonytongue
fish (22), but direct proof is needed.
The other possible origin of pjGnRH is from the GnRH molecule that was
first identified in sea bream (sbGnRH), but is present also in fish
that evolved long before sea bream and pejerrey, i.e.
pacú (29) and sábalo (27) (order
Characiformes). The pjGnRH differs in only one amino acid from sbGnRH
(position 5), but the distribution of sbGnRH in different fish species
appears to be much broader than that of pjGnRH in studies to date. The
distribution of sbGnRH in teleosts has been determined by several
methods. Sequence determination was used to identify sbGnRH not only in
pacú (29), but also in three perciform species, sea
bream (17, 28), tilapia (30), and the African
cichlid, Haplochromis burtoni (31, 50). Also,
indirect chromatographic and immunological data give evidence that
sbGnRH is the third molecular form in addition to cGnRH-II and sGnRH in
other fish in the orders of Perciformes (24, 25, 26, 51),
Pleuronectiformes (52), Characiformes (27),
and Gonorynchiformes (25). In all cases, although not
sequenced, this GnRH form followed a similar chromatographic pattern to
that of sbGnRH, suggesting a wide distribution of this GnRH variant in
teleost fishes. These distribution data imply a nucleotide substitution
occurred only in the stem line leading to the order (Atheriniformes) in
which pejerrey is found.
Although the neuroanatomical distribution of pjGnRH has not been
clearly demonstrated as yet, it can be considered the releaser of
pituitary hormones because it is the only GnRH form found in pituitary
extracts of pejerrey (6). Our evidence shows that the
newly described pjGnRH form is present in pejerrey brain together with
cGnRH-II and sGnRH. pjGnRH is likely to have emerged from sbGnRH
through a substitution in position 5 (Phe instead of Tyr) or less
likely from herring GnRH (Phe instead of His in position 5). The
phylogenetic position of pejerrey in an order of teleosts thought to
have evolved between orders of fish that have sbGnRH, adds weight to
the argument that pjGnRH was due to a substitution in the sbGnRH
gene.
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Note Added in Proof
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It has come to our attention that a cDNA isolated from medaka
encodes a form of GnRH with deduced amino acids identical to the
pejerrey form reported here as a peptide. Medaka and pejerrey are in
different orders of fish. The reference is Okubo K, Amano M, Yoshiura
Y, Suetake H, Aida K 2000 A novel form of gonadotropin-releasing
hormone in the medaka, Oryzias latipes. Biochem Biophys Res
Commun 276:298–303.
|
Acknowledgments
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We thank Carol Warby and Dan O’Neill for their technical help
with the RP-HPLC and RIA. We are also in debt to Lic. Marcela Alvarez
(Dirección de Pesca, Ministerio de Asuntos Agrarios de la
Provincia de Buenos Aires, Argentina), Dr. Leandro Miranda
(UNSAM-INTECH, Argentina), Dr. Jorge M. Affanni (INEUCI-CONICET) and
Lic. Alberto Espinach Ros (Instituto Nacional de Investigación y
Desarrollo Pesquero, Argentina).
|
Footnotes
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1 This work was supported by the Medical Research Council of Canada,
ANPCYT (Argentina, PICT 01–04424) and Fundación Antorchas
(Argentina) as well as the Foundation for Medical Research (to W.H.F.
and A.G.C.). 
Received October 9, 2000.
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Top
Abstract
Introduction
