of Gonadotropin-Releasing. Hormone. (GnRH). Binding to Pituitary. Receptors in Goldfish. (Carassiusauratus)1. HAMID. R. HABIBI,2'4. RICHARD. E. PETER,4.
BIOLOGY
36, 844-853
OF REPRODUCTION
(1987)
Characterization of Gonadotropin-Releasing to Pituitary Receptors in Goldfish HAMID
R. HABIBI,2’4
RICHARD
JEAN
E. PETER,4
E. RIVIER,5
and
Department University Edmonton, Alberta, The
Clayton
Foundation San
Hormone (GnRH) (Carassiusauratus)1 MIRKA
WYLIE
Binding
SOKOLOWSKA,3
W. VALE5
of Zoology4 of Alberta Canada T6G and
Laboratories Salk Institute
for
The Diego,
California
92037
2E9
Peptide
Biology5
ABSTRACT Goldfish agonist
pituitary
analog
‘251-tGnRH-A
gonadotropin-releasing GnRH
of
teleost
to
a goldfish
pituitary
binding was significantly was a function of tissue Incubation of the pituitary saturable
binding
after slower
tGnRH
after
than
than
the
native
observed
with
tGnRH-A
had
and
provided
the
of
presence
of site.
tGnRH-A, a greater
cold
of
indicating
characterization
of
the
GnRH
enhanced potency of gonadotropin-releasing
attributed pituitary
to a greater GnRH-receptors
Vale et al., degradation
1981), (Koch
GnRH.
presence
The
as well et al.,
the and
acid
the
indicating cold
that
analog
above.
the
The
analog
displaced
binding
is more
bound
23, 1986. 1986. supported
both
peptides than
receptors
the
in goldfish
peptides Catt,
to
the
tGnRH.
and
Fisheries,
ul.
incubation
at
is
of 4#{176}C;
radioligand
per tube. indicated was
two linear unlabeled
effective
in displacing and
sites
same
classes
of
These
results
demonstrated
the
Scatchard
label
analysis
site and in parallel
binding
found
a low-afto that
sites;
however,
the
presence
pituitary.
and replacement ethylamide are
of the glycinamid&#{176} thought to be the main
residue factors
by for
influencing the metabolic stability and enhanced affinity of GnRH-agonist analogs (Conn et a!., 1984). Although some information is available on the
for 1981;
at position
bind native
a superbinding
be resolved into addition of excess
of binding sites: a high-affinity/low-capacity was displaced by tGnRH from both
receptors
structure-activity ous nonmammalian
6
functional
relationships vertebrates
relationship
of GnRH in (Peter, 1986),
between
GnRH
(GtH) release has in mammals (Marian
varithe
receptors been studied et a!., 1981;
Vale et al., 1981; Loumaye et al., 1982) and, to some extent, in chickens (Millar and King, 1983). Teleosts would provide a particularly interesting by
grants
from
the
Natural
and Engineering Research Council of Canada to REP., Heritage Foundation for Medical Research Postdoctoral to H.R.H., and NIH Grant HD 13525 to W.W.V. and J.E.R. 2 Reprint requests. 3Present address: Academy of Agriculture, Department biology Poland.
using
of 1I-tGnRH-A
‘251-GnRH-A,
and gonadotropin extensively only
Accepted October Received July 29, ‘This work was
a 30-mm
and
as to a greater resistance to 1977) compared to native
of a D-amino
after
dissociation curve could were observed after the
in mammals of superactive hormone (GnRH) affinity of (Clayton
achieved
pM
INTRODUCTION
The analogs
was
by Equilibrium
the
that
for
characterized
of 470
at least two classes Bound ‘251-GnRH-A
affinity
were
Pro9-NHEt]-GnRH).
analog, and of 1251-tGnRH-A
tGnRH-A,
Addition
receptors Leu8,
preparation
concentrations
addition
Trp7,
increasing incubation temperature to 22#{176} C. Binding of the with a linear correlation over the range of 0.5-2 pituitary preparation with increasing concentrations of ‘25I-tGnRH-A
membrane
peptide.
suggested the finity/high-capacity
membrane
addition of the rates of dissociation
(GnRH)
[D-Arg6,
reduced after concentration,
at radioligand
to be reversible components;
hormone
(tGnRH-A;
Ambrosowa
6,
30-149
model for such studies, a hypothalamo-hypophysial the control of pituitary through GnRH delivered by nerve fibers (Kaul and
Sciences an Alberta Fellowship
of
Icthyo-
Krakow-Mydlniki,
Also, 844
dopamine
has
because
they do not have portal system, and GtH release is exerted directly to the pars distalis Vollrath, 1974; Ball, 1981).
a potent
GtH
release-inhibitory
GnRH
action teleosts
directly (Peter
on the GtH et al., 1986).
the predominant determined as wood
et a!.,
cells The
GnRH in [Trp7, Leu8]
1983).
This
of
release
in vivo
enzie Pro9
et al.,
tGnRH
in
(Peter
et a!.,
1984).
-NHEt]
The
-GnRH
significantly
more
that receptors
1985)
and
terms
[D-Arg6,
GtH
Trp7,
was
Leu8,
found to
to
tGnRH
receptors as labeled
-teleost
Labeled demonthe also
affinity
in
similar pg of
for
and
sexually
February
of
to those tGnRH-A
regressed
or
in
included
100
early
stages
that of
9.2,
gonadal
approach
and
its superactive
analog
according
to protocols
developed
with were
2% ethanedithiol for deprotection. mediated by dicyclohexylcarbodiimide.
was
the original Ammonolysis overnight at GnRH coupled
Merrifield with liquid 5#{176}C.Both to MBHA
chloromethylated ethylamine was protected [Trp7, and fully protected
Pro9-
1, 1% acetic
=
equilibrated
Couplings tGnRH resin on
radioactivity 1). The
-
M)
with
to
alter area
0.1
the mass
to
the
binding 125
specific
concentrations
I-tGnRH-A.
buffer,
0.02% and
sodium aliquots
the elution fraction was rabbit globulin
act.
of
peak
antiin Figure and was of
carried out on radioimmuno-
I-tGnRH
curves with in-
tGnRH-A was
1100
pCipg’. Membrane
Fish were methansulfonate,
Preparations
killed -
a
was estimated
displacement antibody
unlabeled 125
antibody E., un-
Determination
relationship
of
Sp.
R.
antiserum
tGnRH-A
I-tGnRH-A was a self-displacement from tGnRH-A
(bed
tGnRH-A was indicating iodination
studies.
radioactivity
labeled
M borate
using
shown within the main the highest immunoreactivity
of the basis by
the column
NY) as the second Y. P. and Peter,
binding
receptor
by addition and
determine of each
activity
creasing
Pituitary
(0.1
0.1% gelatin and 1 ml were collected,
binding level by incubating
1300
10 pg chloramine-
a QAE-Sephadex
at 50% obtained
125
et al. (1980); 5 M acetic acid) was
terminated
results). Immunoreactive in only the main peak,
for
assay;
was acid
resin. achieved Leu8 ] super-
15 ml)
125
by radioimmunoassay, serum and goat anti-rabbit
sp. act. routine
by Rivier
assembled on a methyl benzhydrylamine (MBHA), whereas the tGnRH-A was constructed
on
for (Fig.
not
used
were phase
et a!. (1986). In brief, t-Butyloxycarbonyl group used for a-amino protection and trifluoracetic
purified
body; the 1 displayed
[DArg6,
Pro9 -NHEt] -GnRH, tGnRH-A, by using the Merrifield solid
buffer
containing Fractions of
published detected did
GnRH,
acid GnRH
I-Na (dissolved using procedures
using
was
(Arnel Inc., Brooklyn, (validated by Huang,
were
Hormones Teleost
(c
with by
I-Na,
125
reaction
borate
estimated tGnRH-A
recrudescence.
Trp7, Leu8, synthesized
-21.3
=
was labeled buffer)
1 mCi The
pl
counted profile
between
fish
[a] D
acid)
by Piyachaturawata (dissolved in 0.1
with
azide.
in a 1800-! flow-through a simulated natural photoand fed a commercial fish
and
GnRH
1 mm.
pH
July
Peptides
tGnRH-A
iodinated
mainly
1, 1% acetic
=
Teleost GnRH-A 0.5 M phosphate
volume,
performed
(c
perliquid
HPLC. Amino in 4 N meth-
[DArg6,
-38.5
=
Goldfish, Carassius auratus, common or comet variety (mixed sex, ranging 8-13 cm in length) were purchased from Grassyfork Fisheries Co., Martinsville,
were
1984).
97% pure on analytical after acid hydrolysis
[a]D
Animals
Experiments
et a!.,
was
amino teleost
peptide
diet.
Rivier
in the acetic
acid).
AND METHODS
IN. Fish were maintained aquarium at 16 ± 1#{176}C on period in Alberta, Canada,
HF dilute
anesulfonic acid, gave the expected ratios. Optical rotation was as follows: NHEt]
be
liquid with
Purification high-performance
(HPLC;
at least analysis,
T for
MATERIALS
treated with and extracted
and
in a teleost, ligand. We
has a higher native peptide.
NHEt were of anisole
chromatography were acid
(Mack-
(Peter et al., 1985). we describe the first
tGnRH-A than the
to
of
in vitro
compared
of pituitary GnRH using tGnRH-A
demonstrate pituitary
in
analog
GnRH in vivo in goldfish In the present study, stration goldfish,
goldfish
845
acid prior to lyophiization. formed by preparative
other teleost species et al., 1984; King and have demonstrated
(tGnRH-A) potent
is known
PITUITARY
agonist presence
salmon has been (tGnRH) (Sher-
of tGnRH
be present in a number of (Breton et a!., 1984; Sherwood Millar, 1985). Previous studies bioactivity
IN GOLDFISH
in goldfish and other primary structure of
chum -GnRH
form
RECEPTORS
after a light anesthesia 0.02%), pituitaries
(tricaine removed,
or -
HABIBI
846
ET
AL.
Binding
Studies
Incubations polypropylene
()20
coated
overnight
experiment, pmoles tGnRH-A
15
z
in
(in triplicate) microcentrifuge
the
C,
presence
and
(peptide
displacement
10
-
250
stant 22-29
at
20
30
40
lines)
profile (1-mi column. Fractions were pooled and
mm
fractions) of ‘25I-tGnRH-A 19, 20, 21, and 22 (area used for the receptor assay.
Intact
and
containing
serum 10-ml
1 mM
per
gradual
increase
100
(doubled
buffer
at 250 15-mi
and and
was
resuspended
buffer
initially,
to 5 ml). for 5 mm, polycarbonate
buffer, mm for mination,
unless
otherwise
receptor aliquots
binding of the
an of stated)
in pre-
in the
I-
M unlabeled maximum
assay
volume
of
experiments,
pituitary
pituitary incubated
equivalent with a con-
(3 0,000-40,000 presence and
cpm; absence
the
appropriate and
were washed twice by centrifugation with BSA-free Tris-HCI and assayed content (Bradford, 1976), using BSA
15,000 prepara-
volume per used
of
100
within
containing
the
binding membrane
labeled
peptide
binding
to tissue.
to
the in
for
of
tube and the presence
pellets
in a gamma carried out in
for determination absence of pituitary
nonspecific membrane
BSA, and of excess
determination
of
to
with un-
nonspecific
Analysis
A computerized program (LIGAND)
supernatant tube, diluted at
vessels
into tubes for counting Parallel incubations were
determine pituitary
Data
by aspiration of the supernatant. rinsed twice with 500 pl cold assay
by
homogenate
1 pituitary
studies. For crude membrane
in a (50
followed
The
-
bovine
homogenized homogenizer
X g
in
(equivalent
buffer [Tris]
0.5%
volume) and further centrifuged 30 mm at 4#{176}C. The crude membrane
x g for assay
pl assay
a
ice-cold assay aminomethane
dithiotheritol
of volume
was centrifuged transferred to
tion
ml
albumin EBSA] pH 7.6), glass-teflon (prechilled)
pituitary
10
in a final (one was
incubation
all experiments binding in the
HC!
saturation
at which
other
preparation or as indicated)
transferred spectrometer.
10
of
obtained)
4#{176}C.For
at 4#{176}C, followed pellets were
buffer,
washed (3 times) with (10 mM Tris[hydroxymethyl]
the
For
out
analog in a final volume of 250 p! at 4#{176}C otherwise stated). At appropriate intervals (30 for equilibrium binding studies), incubations termined by centrifugation (15,000 X g) for 5
mm were
FRACTION elution
absence
level of ‘25ItGnRHA fmoles per tube)
.
FIG. 1. A typical from the QAE-Sephadex indicated by vertical
p!)
of cold _______________ (unless
0
10
BSA.
concentration
was
pl
membrane per tube
5
2.5%
carried (500
3.6-300 fmoles per tube (14-1200 per liter; 5,000-400,000 cpm) of were incubated with membrane preparation
tGnRH-A
C
with
were tubes
pl 30
nonlinear least-square curve-fitting was used for Scatchard analysis
and determination of the best fit through the data points, as applied previously to GnRH binding data (Munson and Rodbard, 1980). A four-parameter logistic curve-fitting program (ALLFIT) was also used for
analysis
(50%
of
effective
curves dose)
protein deterpreparation (15,000 X g) for protein as a standard.
Fresh membrane preparations were used at all times for the radioreceptor assays, as significantly lower total and specific binding levels were obtained using membranes prepared from frozen goldfish pituitaries (results not shown).
and
determination
(DeLean
et a!.,
of
ED50
1978).
RESULTS
Equilibrium
Binding
Time-dependence studied preparation
during
of tGnRH-A of
125
incubation (one pituitary
I-tGnRH-A
binding
with pituitary equivalent per
and 22#{176}Cfor a total period of 180 mm Experiments were carried out in October, goldfish
that
were
sexually
regressed.
was
membrane tube) at 4 (Fig.
2). using
Maximum
GnRH
RECEPTORS
IN
GOLDFISH
847
PITUITARY
#{163}
-J-
12 I-
-j-
0
I-
I-
-S
10
I’.
U 61
-S
a
9
6,
AU.
-I
0
I-
0 I-
TIME
binding of the radioligand was achieved after 30 mm incubation at 4#{176}C, and both total and specific binding were stable for the following 30 mm. Increasing incubation
(pO.05) between total binding or specific binding for the 10-vs. 80-mm preincubations.
-J
I-
condition
at 4#{176}C and condition
0 I-
was for
-S.
0
z D
to
membrane 125
I-tGnRH-A
Binding in May, using goldfish study the correlation concentration binding
and was
deter-
mined in the presence and absence of 10_6 M cold tGnRH-A (Fig. 4). Less than 1% of the total added radioligand was found to bind to the tube as determined
10
Pre-
significantly I-tGnRH-A
binding
tGnRH-A
Experiments were performed were sexually matured,
tGnRH-A
80
10
0
Concentration
pituitary
ITT
r
binding of 22#{176}Cwas
studies.
between
T
T
recrudescing receptors
the
was observed after 30 mm incubation therefore used as standard experimental the
3
specific binding experiment was
sexually membrane
absence
80 mm specific
or
In
the
-6
#{149}lOnj
22#{176}Csignificantly
and specific binding at
out in January, using test the stability of
incubated incubation reduce
to
both Equilibrium
0
6.
(mln)
FIG. 2. Time and temperature dependence of ‘25l-tGnRH-A binding to crude goldfish pituitary membrane preparations (15,000 X g pellet, 30 g protein per pituitary equivalent, October). Data show fraction bound (BIT, percentage of total counts) corrected for nonspecific binding measured in the presence of 10-6 M cold tGnRH-A. The inset shows binding in the absence of cold peptide at 4#{176} and 22#{176}C. Values are mean ± SE of six observations (two experiments, each carried out in triplicate).
the
8
incubation
membrane. binding pituitary
From these of the radioligand membrane was
binding,
as determined
in
the
absence
of
results, maximum in the presence 11% of total counts. in the
presence
pituitary specific of excess Specific
of 106
M of
0.5
1
2
PITUITARY
3
4
5
EQUIVALENT/TUBE
FIG. 4. Equilibrium ‘25l-GnRH-A binding to increasing concentrations of crude goldfish pituitary membrane preparation, equivalent to 0.5 to 5 pituitaries per tube (20-200 g protein per tube, May). Parallel incubations were also carried out in the presence of 10-6 M tGnRH-A and the results used for correction of nonspecific binding. Values are mean ± SEM of three observations. Experiment was carried out in May, using 8- to 12-cm fish.
