BIOLOGY OF REPRODUCTION 60, 1338–1344 (1999)
Evidence for Gonadotropin-Releasing Hormone Peptides in the Ovary and Testis
of Rainbow Trout 1
Kristian R. von Schalburg, Carol M. Warby, and Nancy M. Sherwood2
Department of Biology, University of Victoria, Victoria, British Columbia, Canada V8W 3N5
ABSTRACT
GnRH is usually classified as a neuropeptide that is synthesized in the brain. Recent evidence indicates that GnRH mRNA
is present also in the ovary and testis. However, isolation of the
peptide from testis has not been reported. We used HPLC and
specific RIAs to determine whether the GnRH peptide can be
detected in gonads, the developmental stage at which the peptide is expressed, and the number of molecular forms of GnRH
that are present in the ovary and testis. Extracts of immature
and mature ovarian and testicular tissue were examined from
17- to 21-mo-old rainbow trout (Oncorhynchus mykiss).
For the first time, GnRH peptides were isolated from testis
and identified by HPLC-RIA with specific antisera and by elution
position compared with synthetic standards. GnRH peptides
were also present in the ovary. In addition, multiple forms of
GnRH, including a form not normally detected in the brain of
trout, were shown to be present in the gonads. During development, GnRH peptides were expressed only at specific stages
in the gonads, which may explain the inability to detect and
isolate the GnRH peptides from gonads in earlier studies.
been identified [17], but only sGnRH peptide has been
shown to be present [18]. Goldfish testis has not been studied. There are strong indications from several in vitro physiological studies that GnRH acts as a meiosis-stimulating
factor in the oocytes of rats [19] and in various fresh water
and marine fishes (see [20]). However, definitive evidence
is lacking for any vertebrate to show that one or more forms
of the mRNA are translated into peptides in the testis or
that there is a specific developmental pattern for the expression of individual GnRH peptides (see [21, 22]).
In this paper, the presence of GnRH peptides in the ovary and testis of rainbow trout is demonstrated by HPLC
and RIA. The ovary and testis were examined at various
stages of development in rainbow trout at 17–21 mo of age.
Immunological and chromatographical methods were used
to determine that multiple forms of GnRH are present and
are developmentally regulated in the salmonid ovary and
testis.
MATERIALS AND METHODS
INTRODUCTION
Animals
Ten distinct forms of GnRH have been isolated and identified by primary structure from vertebrate brains. Within
the brains in a single species, two or three identified forms
of GnRH are present [1]. In humans, two forms have been
demonstrated [2, 3], whereas three forms are found in monkeys [4–6]. Likewise two or three forms are present within
individual species in birds, reptiles, amphibians, and fish
[1]. In salmon, the two forms of GnRH are the salmon form
(sGnRH) and a second peptide originally isolated from
chicken and named chicken GnRH-II (cGnRH-II). The primary structure of both peptides was determined for chum
salmon ([7] and J.F.F. Powell, personal communication).
The same two forms of GnRH were detected by a combination of HPLC and RIAs in the brain of both masou salmon [8] and rainbow trout [9]. On the basis of the anatomical
distribution of these two GnRHs from immunocytochemical studies [8–10], it was deduced that sGnRH is the gonadotropin releaser and that cGnRH-II serves a neuromodulatory function in the salmonid brain.
GnRH is expressed not only in the brain but also in the
gonads. Local expression of the mRNA encoding GnRH
was demonstrated in various reproductive tissues including
the ovary and testis in humans [11], monkeys [12], and rats
[13, 14]. A GnRH peptide was reported for porcine ovary,
but the results were not conclusive as blank HPLC injections and assays did not precede tissue injections [15]. In
fish, a transcript encoding sGnRH has been isolated and
sequenced from midshipman ovary and testis [16]. In the
goldfish ovary, both sGnRH and cGnRH-II mRNAs have
Male and female rainbow trout (Oncorhynchus mykiss)
used in this study were raised in an open lake fed by a
natural stream in Sooke, British Columbia (Mountain Trout
Sales). Most trout ovulate and spawn for the first time at 3
yr and then continue to spawn annually. However, 10–20%
of trout mature precociously, beginning at about 1.5 yr of
age, and may spawn at 2 yr, one year ahead of their cohorts.
This true precocious maturation is a normal reproductive
state in which offspring can be produced. Fish were judged
to be precociously mature on the basis of the weight of the
ovaries and testes (see Table 1) and of other defining characteristics. Histology was not done on the gonads. However, the precocious gonads at 17 mo were 2- to 4-fold
greater in weight than those of their nonprecocious counterparts; also the testis was white and smooth, and the ovary
had orange eggs, compared to small, thin translucent organs
in the normal fish. At 18–20 mo, the precocious ovaries
had not changed in weight from 17 mo, but the testes
weight was 8-fold increased. At 21 mo, the precocious ovaries were 44 times the weight of those in normal fish and
contained large eggs.
The fish were exsanguinated for several minutes. The
ovaries, testes, and brains were removed, immediately frozen on dry ice, and subsequently stored at 2808C. Ovaries,
testes, and brains were taken from a total of 109 fish that
were either 17, 19, or 21 mo of age (see Table 1 for details).
Accepted January 20, 1999.
Received October 26, 1998.
1
This study was supported by the following grant: Canadian Medical
Research Council.
2
Correspondence. FAX: 250 721 7120; e-mail: [email protected]
Tissue Extraction
The age, number of organs, and total weight of the tissue
examined are listed in Table 1.
The frozen tissue was crushed to a powder using a cold
mortar and pestle. For larger extractions, a Waring blender
(New Hartford, CT) was used to powder the material. Pep-
1338
1339
GnRH PEPTIDES IN GONADS
TABLE 1. Reproductive data from rainbow trout.
Age
(months)
17
18–20
21
Sex
F
M
F
M
F
M
F
F
M
Reprod. status
normal (immature)
normal (immature)
precocious
precocious
precocious
precocious
normal (immature)
precocious
precocious
No. of
fish
21
19
4
6
8
1
37
2
11
Weight/organ
(mg)
Ovary
Testis
143
GnRH pg/gonada
Brain
230*
192
312
725
244
5600
195
8625
166*
5782
m
—
—
—
—
232
167
—
—
—
c-II
—
—
35
—
106
174
—
—
—
u
—
—
—
—
106
100
—
—
—
GnRH pg/brain
s
c-II
s
—
—
—
—
19
—
—
—
—
918b
945
436*
1088
a
m, mGnRH; c-II, cGnRH-II; u, unknown GnRH; s, sGnRH; —, Not detectable.
* 50 Brains pooled for age mates.
tides were extracted with an acetone-HCl mixture, and soluble lipids were removed with petroleum ether as previously described [23]. The final acetone-water soluble mixture was reduced in a vacuum centrifuge to approximately
2 ml and filtered through a 45-mm filter.
HPLC
The filtrate was loaded onto a Supelcosil LC-18 column
(25.0 cm 3 4.6 mm; 5-mm particle size; Supelco Canada,
Oakville, ON, Canada) with a guard column of the same
material attached. Each filtered extract (2 ml) was loaded
in repeated injections of 600 ml each or less at 2-min intervals onto a 1-ml loop. Initially, an isocratic program of
83% 0.25 M triethylammonium formate (pH 6.5) and 17%
acetonitrile was used over a 10-min period at a flow rate
of 1 ml/min. After 10 min, the percentage of acetonitrile
was elevated to 24% over 7 min and maintained there for
an additional 43 min. Sixty fractions of 1 ml each were
collected in polyallomer tubes; 100 ml was removed from
each fraction, vacuum-dried, reconstituted in PBS with
0.1% gelatin, and assayed for immunoreactive GnRH
(irGnRH).
Injections for each set of extracts were preceded by an
overnight wash and a blank run in which a 600-ml volume
of Milli-Q water (Millipore, Bedford, MA) was injected
onto the column. A blank was injected, and the fractions
were collected and assayed between each application of extract to ensure that the column was free of any contaminating residual GnRH from previous HPLC analyses. Mammalian GnRH (mGnRH) standard had never been previously injected onto this column, also ensuring that the presence of mGnRH was not due to residual synthetic mGnRH
peptide. Synthetic standards were applied to the column
after each set of extracts was assayed. Four GnRH forms—
mGnRH, cGnRH-II, dogfish (df)GnRH, and sGnRH—were
combined at 200 ng each and applied to the HPLC system
as described for the above extracts. The elution positions
of the standards on the chromatograph were confirmed by
absorbance peaks (A 5 280 nm) and a GnRH-specific RIA.
RIA
Aliquots of 100 ml from each fraction collected for each
HPLC run were dried and assayed for irGnRH by methods
previously described [24]. The reconstituted extracts were
assayed using various antisera and 125I-labeled synthetic
GnRH tracers in a competitive RIA. The three assay systems are as follows: 1) mGnRH standard, GF-6 antibody
at a final dilution of 1:25 000, and mGnRH 125I tracer; 2)
mGnRH standard, B-7 antibody at 1:10 000, and mGnRH
125I tracer; and 3) cGnRH-II standard, 7CR-10 antibody at
1:37 500, and cGnRH-II 125I tracer. Serial dilutions were
done on brain fractions if values of irGnRH were less than
B/B0 5 20%; the value closest to 50% B/B0 was used to
estimate the quantity of irGnRH present.
Antisera GF-6, B-7, and 7CR-10 were raised in rabbits
in our laboratory against sGnRH, mGnRH, and dfGnRH,
respectively. GF-6 cross-reacts with a number of forms of
GnRH. It detects mGnRH (100% cross-reactivity with
mGnRH tracer), seabream (sb)GnRH (94.1%), sGnRH
(23.7%), and cGnRH-II (10.5%) [5]. B-7 detects mGnRH
(100% cross-reactivity with mGnRH tracer) [4]. Antibody
7CR-10 detects cGnRH-II (100% cross-reactivity with a
cGnRH-II tracer), sGnRH (84.8%), dfGnRH (25.0%), and
lamprey GnRH-III (12.6%) [4]. The limits of detection (B/
B0 5 80%) were 2–12 pg for GF-6, 1–16 pg for 7CR-10,
and 3–6 pg for B-7.
RESULTS
Ovaries and Testes from Immature Normal Trout at
17 Months
The GF-6 antibody, which detects many forms of GnRH,
did not detect any peaks indicative of irGnRH-like material
in the extracts of immature normal ovaries or testes (Fig.
1).
Ovaries and Testes from Precocious Trout at 17 Months
GF-6 detected a small amount of immunoreactive material (0.075 ng) in fractions 24–25 of the extract from the
precocious ovaries (not shown) but not in precocious testes.
With antiserum 7CR-10, which has a higher cross-reactivity than GF-6 for cGnRH-II, 0.282 ng (total in two fractions) of irGnRH was found in fractions 24–25 in the extract from the precocious ovaries (Fig. 1), but GnRH was
not detected in the testes. Synthetic cGnRH-II standard
eluted in position 24 under the same HPLC conditions. The
mGnRH-specific antibody, B-7, did not detect mGnRH immunoreactive material in the extract from the precocious
ovaries at this stage (not shown).
Ovaries and Testes from Precocious Trout at 18 to
20 Months
In precociously mature ovaries, the GF-6 antibody detected four peaks indicative of GnRH-like immunoreactivity: mGnRH (fractions 18–20), cGnRH-II (fractions 24–
26), an unknown GnRH (fractions 30–32), and sGnRH
1340
VON SCHALBURG ET AL.
FIG. 1. Immunoreactive GnRH in gonads from 17-mo-old (May) rainbow trout. Normal immature ovary (top left) and testis (top right) extract assayed
with antiserum GF-6. Precocious ovary (bottom left) and precocious testis (bottom right) extract assayed with antisera GF-6 or 7CR-10. HPLC elution
position (min) and the amount of GnRH immunoreactivity (nanograms per fraction) detected by antisera GF-6 or 7CR-10 are shown. The arrow above
HPLC elution profiles shown for the precocious ovaries represents the detection of cGnRH-II by antiserum 7CR-10 as well as where the synthetic form
elutes under the same HPLC conditions.
(fractions 39–41) (Fig. 2, upper graph). In our HPLC program, mGnRH standard consistently eluted at position 20,
the cGnRH-II standard at position 25, the dfGnRH standard
at positions 31/32, and the sGnRH standard at positions 39/
40. However, it is not clear whether the GnRH-like immunoreactivity that eluted in fractions 30–32 corresponds
to dfGnRH or if it is a novel form. There was a total of
3.7 ng of immunoreactive mGnRH compared to only 308
pg immunoreactive sGnRH. The amount of immunoreactive cGnRH-II and of the unknown GnRH form were
roughly the same (1.7 ng, total in three fractions). Synthetic
mGnRH, cGnRH-II, and sGnRH eluted in positions corresponding to the native material under the same HPLC conditions.
Although testes from only one precocious animal was
used, analysis of the precocious testis extract showed three
immunoreactive peaks. Two peaks corresponded to elution
positions of synthetic mGnRH and cGnRH-II (Fig. 2, upper
graph). The third peak in fractions 30 and 31 (201 pg) could
represent a novel GnRH form. Immunoreactive mGnRHlike (333 pg) and cGnRH-II-like (348 pg) material was detected with GF-6 in fractions 19–21 and 24–25, respectively (Fig. 2). sGnRH-like immunoreactivity was not detected in the precocious testis extract.
All the HPLC values have been converted to picogram
per organ for comparison purposes (Table 1). The total
amount of immunoreactive mGnRH, cGnRH-II, unknown
GnRH, and sGnRH detected in each precocious ovary was
232, 106, 106, and 19 pg, respectively. In each precocious
testis the total amount of immunoreactive mGnRH, c-
GnRH-II, and unknown GnRH detected was 167, 174, and
100 pg, respectively.
The presence of mGnRH-like immunoreactivity in the
precocious ovaries (3.5 ng, total in 16 ovaries) and testes
(195 pg in 2 testes) was confirmed by the immunoreactive
peaks, primarily in fractions 18 and 19, using the mGnRHspecific antiserum, B-7 (Fig. 2, lower graphs).
Ovaries and Testes from Immature or Precocious Trout at
21 Months
GF-6 antiserum did not detect any peaks of irGnRH-like
material in the extracts of the normal immature ovaries or
in precocious ovaries or testes from 21-mo-old fish (Fig.
3). Also, each fraction of the extract from the precocious
ovaries was reassayed with 7CR-10, but irGnRH material
was not detected.
Brains from 17- or 21-Month-Old Rainbow Trout
Both cGnRH-II-like (fractions 24–28) and sGnRH-like
(fractions 38–40) immunoreactivity were detected in brain
extracts from 17- and 21-mo-old fish (Figs. 4 and 5). Peaks
corresponding to immunoreactive mGnRH were not detected with GF-6 in brain extracts (see fractions 18–20;
Figs. 4 and 5). However, a small amount of irGnRH that
could correspond to the unknown form observed in the ovaries and testes is present in position 30–31 (Figs. 4 and 5).
The irGnRH detected in position 33–35 has previously been
analyzed and determined to be sGnRH (J.F.F. Powell, personal communication).
GnRH PEPTIDES IN GONADS
1341
FIG. 2. Immunoreactive GnRH in gonads from 18- to 20-mo-old (June–August) rainbow trout. Top) Precocious ovary and testis extracts assayed with
antiserum GF-6. Bottom) Precocious ovary and testis extracts assayed with antiserum B-7. HPLC elution position (min) and the amount of GnRH
immunoreactivity (nanograms per fraction) detected by antisera GF-6 and B-7 are shown. In the upper figures, arrows above HPLC elution profiles
represent the different forms of GnRH detected by antiserum GF-6 as well as where the synthetic forms elute under the same HPLC conditions. m,
mGnRH; c-II, cGnRH-II; df, dfGnRH; s, sGnRH. In the lower figures, the arrows above HPLC elution profiles represent the detection of mGnRH by
antiserum B-7 as well as where the synthetic form elutes under the same HPLC conditions.
DISCUSSION
Evidence is provided here for the first time that GnRH
peptide is present in both the ovary and testis. In addition,
the GnRH protein is shown to be present at a specific maturational stage, which is in precociously mature ovaries and
testes of fish that are 18–20 mo old. These fish are ad-
vanced by 1 yr and within a few months of spawning. The
amounts of GnRH per organ were similar for the ovary and
testis except for sGnRH; all amounts were more than 14fold above the detection limit of the assay. The present
results are supported by our previous use of polymerase
(PCR) amplification to show that sGnRH mRNA transcripts
FIG. 3. Immunoreactive GnRH in 21-mo-old (September) normal immature ovary or precocious ovary and testis extracts from rainbow trout after
HPLC elution. HPLC elution position (min) and the amount of GnRH immunoreactivity (nanograms per fraction) detected by antisera GF-6 or 7CR-10
are shown. GnRH was not detected with either antiserum.
1342
VON SCHALBURG ET AL.
FIG. 4. Immunoreactive GnRH in 17-mo-old (May) rainbow trout brain
extracts showing HPLC positions and amounts of immunoreactivity. HPLC
elution position (min) and the amount of GnRH immunoreactivity (nanograms per fraction) detected by antisera GF-6 (top) or 7CR-10 (bottom)
are shown. Arrows above HPLC elution profiles represent the different
forms of GnRH detected by either antiserum GF-6 or 7CR-10 as well as
where the synthetic forms elute under the same HPLC conditions. m,
mGnRH; c-II, cGnRH-II; df, dfGnRH; s, sGnRH.
are present in the ovary and testis of rainbow trout [25].
These transcripts were identical to those in the brain except
that extended 59 untranslated regions were present in the
gonadal transcripts.
The demonstration of the local presence of GnRH decapeptide in these tissues has been reported for the goldfish
ovary [18], but not for testis. The first report of a GnRHlike factor extracted from rat follicular fluid capable of releasing FSH and LH from rat pituitary cells in vitro was
made in 1981 [26], but the authors reported later that they
could not repeat the result [27]. Other HPLC studies detected GnRH-like activity in ovarian extracts from various
species, but the protein products were shown not to be
GnRH [28, 29]. Similar reports of GnRH-like immunoactivity in testicular tissue (see [30]) or ovarian tissue [15]
have been inconclusive. Pati and Habibi clearly showed that
sGnRH was present in the goldfish ovary, but they did not
identify any other GnRH forms [18].
This is the first report showing that mGnRH is present
in the ovary and testis, but is not present in the brain, at
least not at 17 or 21 mo of age (Figs. 4 and 5). In addition,
small amounts of irGnRH-like material in the brain and
gonads are detected in positions 30–32, which could represent the expression of an unknown (possibly novel) form
of GnRH. Clearly, cGnRH-II and sGnRH are the dominant
FIG. 5. Immunoreactive GnRH in 21-mo-old (September) rainbow trout
brain extracts showing HPLC positions and amounts of immunoreactivity.
HPLC elution position (min) and the amount of GnRH immunoreactivity
(nanograms per fraction) detected by antisera GF-6 (top) or 7CR-10 (bottom) are shown. Arrows above HPLC elution profiles represent the different forms of GnRH detected by either antiserum GF-6 or 7CR-10 as
well as where the synthetic forms elute under the same HPLC conditions.
m, mGnRH; c-II, cGnRH-II; df, dfGnRH; s, sGnRH.
forms of GnRH in the salmonid brain, in which they are
expressed in large quantities compared to the amounts in
the gonads. Nevertheless, our results show that different
forms of GnRH (m, c-II, s, and unknown GnRH in females
and all but sGnRH in males) are synthesized at specific
stages during ovarian and testicular cell maturation.
Previously we showed the expression of two different
sGnRH-encoding mRNAs and genes in both the ovary and
testis of rainbow trout [25]. Isolation and characterization
of neither mGnRH- nor cGnRH-II-encoding transcripts
have been reported to date. In contrast to the previous
study, which demonstrated expression of sGnRH mRNA in
precociously mature testes from 18 to 20 mo of age, the
present HPLC/RIA study found that the sGnRH peptide
was not detectable, at least not at the time the tissue was
collected (20 July). Whether this was due to a small sample
number or to fluctuations in the levels and/or forms of
GnRH remains to be clarified. Furthermore, detection of
cDNA for sGnRH confirms that the gene is transcribed. The
difference between these two results may be one of difference in sensitivity of the two techniques: PCR amplification
is sensitive compared to RIA, for which the threshold of
detection for the peptide is only 3–7 pg.
Rainbow trout were chosen because, unlike other salmonids in the same genus (Oncorhynchus), rainbow trout
mature early at 3 yr; they do not die after spawning (iter-
GnRH PEPTIDES IN GONADS
oparous); and they are readily available because they remain in fresh water throughout their lives. In the second
year of life, up to 20% of the population may have welldeveloped reproductive tissue. The precocious testes examined were very large compared to those of their immature counterparts (see Table 1). The ovaries examined were
considered to be precocious because they had well-defined
features indicating they were maturing (visible eggs, orange
coloration, larger size).
Our HPLC/RIA data from extracts of immature normal
and precociously mature gonads show that the GnRH peptides are present in a stage-specific period, i.e. GnRH peptides were evident in precocious gonads at 18–20 mo, but
were not present even in large precociously ripe gonads at
21 mo. The first indication of GnRH protein was the presence of cGnRH-II in the precocious ovary at 17 mo (35
pg/organ). The greatest expression of GnRH peptide in this
study was for mGnRH at 232 pg/organ, followed by 106
pg/organ for both cGnRH-II and the unknown GnRH in
pooled extracts from 18- to 20-mo-old precocious ovaries.
These contents were considerably less than in the brain but
were easily detectable (Table 1). Only in precocious ovaries
from 18- to 20-mo-old fish was sGnRH detected (19 pg/
organ). The extract from the 19-mo-old precocious testis
showed the presence of similar amounts of mGnRH, cGnRH-II, and the novel GnRH as found for the 18- to 20mo-old pooled ovaries (see Table 1). If there is a requirement for GnRH at stages just before ovulation in the salmonid follicles, these tissues need to be analyzed later in
the year (October or November), because GnRH was not
present in any tissues examined in September.
Synthesis of the mRNAs that encode both GnRH and its
receptor in ovarian and testicular cells points to a direct
physiological role for this factor. It is unlikely that secreted
hypothalamic GnRH acts peripherally on the gonads because of the low concentration and short half-life in the
blood (see [14]). The present results suggest that GnRH is
not present as a protein in detectable amounts except in
specific stages, which may offer a clue to the function of
gonadal GnRH.
The present experiments do not indicate the function of
GnRH in the gonads of rainbow trout. However, others
have used rats to show that there may be a function during
early maturation. In immature granulosa cells, for example,
exogenous GnRH inhibits FSH-stimulated steroidogenesis
and FSH/LH receptor production [22, 31]. This FSH-suppressing action has been demonstrated to profoundly disrupt granulosa cell maturation in vitro [32]. GnRH also
inhibits gonadotropin-mediated progesterone biosynthesis
in the ovary and androgen biosynthesis in the testis. This
GnRH-suppressing action has been shown to affect the synthesis of LH receptors and steroidogenic enzymes [31], as
well as enzymes involved in cAMP production and signal
transduction [22, 32, 33]. The present experiments in trout
indicate that caution should be used in interpreting results
from rat studies. In rats a number of effects have been reported for the gonads after the addition of GnRH in vitro.
However, these effects may not be physiological if the rat
gonad does not normally express GnRH peptide at that
stage of development.
We show that GnRH peptide is present only at a time
that coincides with oocyte and sperm maturation. In mature
granulosa cells, GnRH is associated with stimulatory effects on meiotic maturation, oocyte cleavage, ovulation,
and luteinization [19, 22, 32]. The precociously mature gonads in the present study are most likely at the stage at
1343
which the oocytes and sperm are undergoing maturation
and will be released in 4–5 months at spawning. Stimulation of steroidogenesis is also evident in cultured adult rat
Leydig cells after short-term exposure (4 h) to GnRH [34].
In the ovary, GnRH may modulate follicular development
by selection of particular follicles for ovulation while promoting atresia in follicles more sensitive to early GnRH
inhibition [21, 32].
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