Mar 4, 1988 - to Gonadotropin-Releasing. Hormone in Goldfish1. V. L. TRUDEAU,. R. E. PETER,2 and B. D. SLOLEY. Department of Zoology,. University.
BIOLOGY
OF REPRODUCTION
44, 951-960
Testosterone
and
(1991)
Estradiol
Potentiate
the
to Gonadotropin-Releasing V. L. TRUDEAU,
Department
of Zoology,
Serum
Hormone
R. E. PETER,2
University
Gonadotropin
and
of Alberta,
Response
in Goldfish1
B. D. SLOLEY
Edmonton,
Alberta,
Canada
T6G
2E9
ABSTRACT The
effects
content, the
of gonadal serum
and
seasonal
GTh
months,
respectively.
content,
descence
(late
autumn)
females, did
affect
only.
the
the positive
results
T and
its
actions
GTh
response
action
or
of endogenous
clearly
A in gonad-intact
at any
time whereas
on
the
induced
that
female
and
tg/g).
300
Multiple
T, through
Gonadal
steroids
was
secretion [1, 2] and
aromatization
fish,
mechanisms,
very
little
is known
but
steroid
binding
by autoradiographic species. In platyfish (T) and preoptic
feedback
known
to be
teleost
fish
Many mediated brate
involved
with Specific [4]. In the
about sites
steroid have
control
of GTH
of the actions of androgenic by their localized conversion
brain
by reductase
and
(DHT)
of
of brain
and
January September
‘This ada
Sciences
of Alberta, search
research
#A637l
in areas
the are
secretion
Mrican
by Natural and
A preliminary on Comparative
Sciences
Financial the
Alberta report Endocrinology,
and
support
tion
catfish
for
the
without
effect. inhibitor
39 days),
probably
male
goldfish. LHRH.
V.L.T.
Council from
the
Heritage
Foundation
for
Medical
has
presented
at the
11th
been
Malaga,
Spain,
May
14-20,
in the
control
feedback regulation of GTH in several teleost species replacement
models.
nonaromatizable in teleost
androgen, fish 113, 14],
elevations into the
secretion by clasElevations
in trout. and pituitary
1989.
951
go-
Implantaof intact
stalk
secretion and
comes immature
primarily European
from eels.
studies
of immature
Increased
GTH content occurs following i.p. administration juvenile male and female rainbow trout [17]. The feedback effect of T is dependent on aromatization
Inter-
[10], by T
1 1-keto-testosterone also suppresses
of GTh brain
of
the
salmonids Re-
steroids
GnRH
activity exsupport
and disrupted inhibitory dopaminergic inputs pituitary [16] and caused GTH release. Evidence for the positive effects of gonadal steroids on
GTH
of CanUniversity
gonadal
of brain
aromatase anatomical
GTH release [15], which has been interas further evidence for steroid negative feedback; implantation, however, may have damaged the pi-
pituitary
have
distribution
The
areas, and providing
demonstrating hypothalamus,
stimulate
preted
dis-
[8]. The areas area, anterior
levels caused by gonadectomy in trout [11], and goldfish [12] are suppressible
of antiestrogens
goldfish
to 5a-dih-
Engineering for
of the in fish.
(1 1-1(T) found nadectomy-induced
(i.e. T) are the verte-
activities
GTH
or E2. The
in
pituitary.
gonadectomy/steroid
of blood
1990.
(REP.).
of Zoology,
is acknowledged.
national Symposium tCorrespondence.
funded
or
Tes-
to exogenous
in goldfish preoptic
negative demonstrated
been
sical
1991. 10,
was
grant Dept.
14,
I
aromatase
in mature
responsiveness
the
and
Steroid has
into Received
involvement
pg/animal).
were
the
3 days
to LHPJ-I-A
steroid concentrating considerable overlap,
for involvement GTH secretion
of
[7]. The
aromatase
characterized are
tuitary Accepted
I 1.KT with
every
pituitary
activity
[6,9], hibit
localized
enzymes
E2, respectively
5a-reductase
tg/g
response
telencephalon,
feedback
steroids within
aromatase
and
and
GTH
regressed
with
250
early treat-
serum
the
implanted
(1 1-KT; DHT
the
in sexually
To examine
pretreatment
(0.2
GTH
increase
been high
steroid case of
been
a similar distribution and pituitary. These
in the
of hCG of the
to E2, can
[6].
ydroxytestosterone tribution
regulators
methods in the brains of a variety and goldfish [5], labeled testosterone
estradiol (E2) have area, hypothalamus,
whereas
by 2-day
injections
in potentiation
and have been extensively birds [3]. The gonads! ste-
roids exert their effects by interacting binding sites in the brain and pituitary teleost
blocked
recru-
Steroid
potentiated
were
summer and
in goldfish.
fish.
fish
and gonadal
(summer
evident
1 1-keto-testosterone
females
and
male
pellets
at 6 h following variations in GSI, spring
regressed
effectively in
silastic
with
in the
ovulation
female
throughout
undergoing
of E2 were
to LHRH-A and
noted
during
and
5 days
in females in sexually
effects
male
for
(GTH)
investigated
serum GTH response animals, seasonal were
Testosterone
positive
gonadotropin
were
goldfish.
important
are
year.
not
found
response
males i.p.
but
to those
.tg/g)
INTRODUCTION
gonadotropin (GTH) studied in mammals
release
the
100
secretion
resulted
levels
GTH
secretion,
in both
GTh
I secretion,
demonstrate male
LH.RH-A
minimal
ip.
tg/g). The saline-injected
females,
pituitary
100),
unplanted
and
GTh
GTh (DHT;
induced
were
stimulated
of the
X
(LHRH-A)
100
levels
cycle,
E2 potentiated
to
of T on (100
stimulation
levels
fish
(spring)
steroid
wt
hormone
(E2;
maximal
mature serum
body
blank-implanted,
effectively
5a-dihydroxytestosterone
androgens
1,4,6-androstatrien-3,17-dione These
Both
In
LHR.H-A
GTH
female
evident;
in sexually
wI/total
or estradiol
assessed.
reproductive
gonad
hormone-releasing
jg/g),
were
fish,
entire
in mediating
potentiated
through
was
levels
serum
the
females
of T aromatization
100
of T or E2 raised
during
(GSI;
Gonad-intact
(T;
and
unstimulated
and post-spawning
Furthermore,
goldfish.
GTh
winter)
and
Implantation
nonaromatizable
the
In blank-implanted
to LHRJI-A
tosterone
of
index
Pro9-Net.1uteinizing
testosterone &g/g LHRH-A serum
and
autumn
not
response
(blank), or 0.1
gonadosomatic
to ID-Ala6,
cycle
no steroid of saline
pituitary
on
response
reproductive
containing i_p. injection
ments
steroids
GTh
pituitary of T in positive since
TRUDEAU
952 this
response
is blocked
androstatrien-3,17-dione treatment in vitro
by the
aromatase
(Am)
[18].
with T enhances 119]. In all of these
inhibitor
In
1,4,6-
addition,
GnRH-induced studies, no
2 mo [20]. GnRH implants levels, but when co-implanted was observed after 1 mo,
roid priming of the release in immature opment pituitary
pituitary trout.
of GTh cells of immature
secretion in plasma
demonstrated [21]. In other
on immature European eels, it has increase in pituitary GTH levels due
been shown to E2 treatment
pendent on dose [22] and tuitary mRNA for alpha-subunit A positive effect of gonads! has
ported gonadal decrease
in the spring blood steroid,
[24, 25]. Following size and blood in blood GTh
ter gonads! roid levels thermore,
in the studies that
the is de-
mediated by stimulation of piglycoprotein hormone [23]. steroids in adult teleost species
yet to be reported. The goldfish is a seasonally
ally spawning nadal size,
si-
for GnRI-I stimulation of GTh Positive effects of E2 on develbeen eels
breeding months. and GTh
cyprinid Seasonal levels
fish
usu-
cycles of gohave been re-
spawning, there is a decrease in steroid levels, concurrent with a levels. During the autumn and win-
recrudescence period, gonads! size and sex steincrease to maximal levels at spawning. Furthe in vivo GTh response to exogenous GnRH
is maximal just prior maximal [11]. Given
to spawning, when gonadal size these observations, we hypothesized
that increasing steroid crudescence period may
levels during have a positive
is
the gonadal effect on GTh
rese-
cretion in the adult goldfish. Since the feedback effect of estrogen could be mediated via conversion to catecholestrogens (CE) in the brain or pituitary [26], we also examined the effects of 2-hydroxy-catechol-estradiol (2-OHE2) and
4-hydroxy-catechol-estradiol
cretion. positive
Evidence effect on
in intact
female
(4-OH-E2)
is presented GnRH-induced and
male
MATERIALS
that both but not
in GTh
se-
I and E2 exert a basal GTh levels
Solid
silastic
year
from
Martinsville, mated to
a commercial
supplier
of goldfish purchased
au-
throughout
(Grassyforks
IN). Prior to experimentation, 17#{176}C and natural-simulated
monton, Alberta; lat. 53.60#{176},long. fed twice daily with trout pellets couver, BC) supplemented with Hagen, Inc., Edmonton, AB).
(Carassius
the
Fisheries;
fish were photoperiods
AID,
accli(Ed-
113.5#{176}).All animals were (Moore-Clark, Inc., VanNutrafin flakes (RoIf C.
or
no
described of steroid. weight. In
(May 1989), sexually mature fish pg/g and 400 g/g I and E2. Silastic
were cap-
sules
250
used
containing
to administer
this
anesthesia, a 2-3 mm
pellets incision
p.g 11-KT
steroid.
E4erimental out
in castor
Under
from 11-KT
oil [28]
tricane
and capsules were in the body wall.
DHT were purchased MO.) and AiD and Inc. (Wilton, N.H.)
were
methanesuphonate implanted i.p. through Testosterone, E2, and
Sigma Chemical was purchased
Co. from
(St. Louis, Steraloids
Protocols
Effect of TorE2 the seasonal
the year, at 5 or injected i.p. with N-ethylamide-LHRH
implantation reproductive
on GTH secretion cycle. At various
10 days following implantation, either saline (0.6% NaC!) or (Syndel Laboratories Inc.,
throughtimes
of
fish were D-A1a6-Pro9Vancouver,
BC LHRH-A; 0.1 pg/g) for assessment of the in vivo GTH release response. The time cOurse of the GTH response to LHRH-A and other GnRH analogues is well established in the
goldfish
thetized h after
[6, 16].
Blood
samples
fish by caudal injection; this
puncture sampling
experiments
(data
preliminary maximal implanted
difference animals
were
taken
from
using 25-gauge time was chosen not
shown)
anes-
needles because
indicated
in response in control was at 6 h. Blood was
6
that
versus allowed
steroidto clot
overnight (at 4#{176}C), and serum was collected by centrifugation and kept frozen (-20#{176}C) until hormone analysis. At the termination of an experiment, fish were killed and total
gonad
and
body
weights
were
somatic index (GSI) was calculated body weight X 100. Pituitaries were injected animals, sonicated in 1 ml frozen
until
hormonal
or E2 release
in vivo,
female varieties g were
1, E2, DHT,
content serum
animals
its actions
were
fish
were
on
levels
LHRH-A implanted
was
examined. with AiD
implantation. following
Effect goldfish. goldfish
Pituitary
To
assess
at several
in the action GTH
I
times
of andro-
of T aromatization secretion,
as described,
nonaromatizable the pituitary (100
A gonadoweight/total from salin#{231}and stored
in saline-injected
determined
pituitary
implanted,
ble androgen (I) or DHT and 11-KT, and
recorded. as gonad removed RIA buffer,
determination. steroid
Involvement of aromatization gens. To examine the involvement
METHODS
Animals comet 15-40
containing
one experiment treated with 100
mediating Common or ratus) weighing
pellets
pellet-implanted during the year.
goldfish.
AND
Treatment
steroid (blank) were manufactured as previously [27], except that elastomere contained 100 mg/g Fish received a steroid dose of 100 g/g body
had no effect on plasma with T, increased plasma suggesting a need for ste-
has also European
Steroid
in vivo
GTh changes
GTh were noted. However, in subsequent experiments, implantation of juvenile rainbow trout with 1-containing lastic capsules resulted in GTH release to the circulation after GTh GTh
ET AL.
with
in
male
and
aromatiza-
androgens [13], i.e. response to exogenous
In another and 300
experiment, males g/g) 2 days prior
responsiveness
was
assessed
were to T 5 days
I treatment.
of catecholestrogens In (75-100
one
experiment, g; purchased
on
GTH sexually
as sexually
secretion
in male
regressing mature
male males
in
March
and
jected
producing
3 times
milt
at hourly
SEX
STEROIDS
when
tested
intervals
with
POTENTIATE
in July) 0.33
GONADOTROPIN
were
pg/g
viously
in-
(3 h) to assess second
the
effects
experiment,
of CE on
males
were
basal
GTh
pretreated
2-OH-E2
OH-E2 or vehicle 1 h prior to LHRH-A (0.1 injection. Treatment with 2-OH-E2 continued tervals jection.
until The
g/g)
blood sampling at 4 h following short-term injection protocol was
similar treatments in the prepubertal male teinizing hormone levels [29]. Furthermore, (data not shown) we found that long-term or 5 p.g/g every 2 days for 3 injections) mately 50% of injected fish. Catecholestrogens chased
from
Sigma
Involvement tuitary GTH
in goldfish
(75-100
g; purchased
producing
the
last
saline, later
[30],
hCG and
steroids hCG
sexually
as sexually
milt
(i.p.) with 0.2 or saline every
when
injection,
tested
fish
pituitary
as previously
in modulating
injection regressing
males
in October)
were
GTh
injected response
with was
regressed
Serum [31].
and by
or 6 h
Rabbit
dilution maximum ples (10-50
pituitary
anti-carp of
1:220
GTH
concentrations RIA
GTh-II 000.
as
antiserum
Minimum
were
previously was
used
detection
binding) was approximately p.!) were assayed in duplicate
range
of 0.5-2000
negligible. Serum mined by RIA after
ng/ml
and
deter-
not
transformed
7-16)
=
were
(95%
control five dates
animals
elevation
of
planted
animals
ological
in that
animals, examined
prior
to 1-way
made means,
between Differ-
considered
statis-
both I and E2 levels (Table 1). Sexually
or July
had
limit (0.3 ng/ml); approximately
in October
the
or July
respective (Table
was
T levels
at or be-
at other 2 ng/ml.
times of Sexually
had
they
steroid
time
to be
The
in female
and
of T and
in the
concentration
were
summer
months,
Date
E2 level
Oct27 May
8/89
July
18
*Most
samples
tNP,
experiment
to
blank-im-
E2 were
physi-
ranges
are
throughout
of the year. effects of steroid
goldfish
controls
presented
re-
times
the
autumn
of the year
level
(1988-1989).
female
variations
Data
Testosterone
E2 level
I
level
E2 level
0.6
±
0.2
2.3
±
0.9
7.4
±
0.4
2.0
±
0.7
1.9
±
0.2
2.2
±
0.7
6.0
±
1.1
±
0.1 0.4
10.3 10.5
±
0.2
0.3
±
±
0.1
0.6
±
0.3
±
0.5 2.4 0.4
±
±
0.1 0.3 0.1
±
1.2
1.7 0.6 1.0
±
0.3
1.0 1.0 1.1
0.3
±
in October not
and
performed.
July
were
± ±
0.1 below
±
NP E2 assay
detection
NP
limit
(0.3 ng/ml)
0.6
±
I
0.2
6.5
level ±
0.3
NP
NPt
and were
and
on serum
LHRH-A-injected
3. Seasonal
Estradiol I
Both
GSI
winter
GSI and pituitary GTh con1989. Treatment with either pituitary GTh content at any
implantation and
in Table
at various
respectively.
Treatment
4/88
at or
content were high in May 1988 and deto minimum values in August. Both
increased
line-injected
deteras pre-
Blank
21
E2 levels
relative
1). Levels
months. After spring spawning, tent decreased again in June-July I or E2 did not affect GSI or
The crosswas tested found
spring
parameters
1 ng/ml. All samand withinand
TABLE 1. Serum E2 and T levels (ng/ml) are presented as mean ± SE (n = 7-14).
May
to 2nor-
0.05.
0.05) “NP. experiment not performed.
4.9 16.9 7.9 5.3 5.0 3.9 4.3 10.8 7.5 9.3 5.7
± ± ± ± ± ± ± ± ± ± ±
did
steroid
respectively.
p.g/g dose (see of approximately
levels
not
affect
pituitary
GTh
content
to
7-
(ng/ml) LHRH-A-injected
1.2 4.5 1.6 0.8 0.5 0.5 0.5 2.4 1.4 1.7 1.5
Blank
5.0 ± NP** NP NP NP 5.5 ± 5.1 ± 7.8 ± 8.0 ± 9.0 ± 5.6 ± 2.6 ± treatment
1.0
1.4 0.7 0.5 1.0 1.4 1.0 0.8
on unstimulated
21.3 152.6 21.2 5.3 6.0 20.7 49.5 82.6 61.4 41.0 4.9 9.5
E2
±
8.9
±
380 9.4 0.8 0.6 5.1 10.9 21.9 18.9 9.2 2.0 4.5
± ± ± ± ± ± ± ± ± ±
GIH
levels
<
0.05) of steroid
re-
treat-
animals.
GTh levels in saline-injected animals and did not alter pituitary responsiveness to LHRH-A. There were no differences in pituitary GIH contents for blank-, I-, or DHT-treated 7.3,
9.2’
±
ment on unstimulated GTH levels. ‘Significant (p < 0.05) effect of LHRH-A versus saline-injected blank animals. bsignificant (p < 0.05) enhancement of LHRH-A effect by I versus LHRH-A-injected blank animals. ‘Significant (p < 0.05) enhancement of LHRH-A effect by E2 (400 g/g dose only) versus LHRH-
implanted animals, LHRH-A response was potentiated AID alone at both doses
±
and
±
In July (Table 7), LHRHin blank-implanted fe-
lowing LHRH-A injection in I-implanted experiment, treatment with DHT had
(24.3
LHRH-A 41.0 141.3 939 58.8 86.2
± 1.6 ± 1.4 ± 0.8 ± 1.1 ± 2.0
peated from Table 3 for ““There was no effect (p
secretion. androgens
Cng/ml)
GTH
Saline”
Blank 1 (100) 1 (400) E2 (100) E2 (400)
218b
stimulated GTH levels. ‘Significant (p < 0.05) effect of LHRH-A versus saline-injected blank animals. bSignificant (p < 0.05) enhancement of LHRH-A effect by I versus LHRH-A-injected blank animals.
not shown). duced GTh
955
GOLDFISH
TABLE 6.
TABLE 4. The effect of I implantation on serum GTH (ng/ ml) in saline-injected (control) and LHRH-A-injected (0.1 g/ g) male fish. Data are presented as mean ± SE (n = 10-
Date
IN
secretion.
sponse sequently
treated
jected animals At both doses, LHRH-A-induced The effects 4-OH-E2
10.
TABLE
GTh
The
effect
p.g/g),
an
of I on in Table
aromatase
the GTh re9. In blank-
stimulated GTh release and this in I-treated males. In contrast, did not affect serum GTH levels and fish
with
Injection
7.
(ng/ml) g/g)
In
300
action shown
I,
did
serum
with
affect
GTH
levels
p.g/g fish
CE did
sub-
in saline-inreduced. of T on
2-OH-E2 are not
and
presented affect
on serum
GTh
GTH
(control) and LHRH-A-injected (0.1 are presented as mean ± SE (n =
8- 12). Serum TABLE 5. The effect of E2 implantation on serum ml) in saline-injected (control) and LHRH-A-injected male fish. Data are presented as mean ± SE (n Serum Date
Treatment
Nov 88 (10 days) August 88 (5 days) “There mulated
Saline”
Blank E2 Blank E2
was
no effect
GTh
(p
GTH
7.6 4.7 3.3 5.9 >
0.05)
± ± ± ±
of steroid
GTH (ng/ (0.1 g/g) 7-8).
Date July
13.9 35.0 8.0 19.4 treatment
89
(ng/ml) LHRH-A
1.9 1.0 0.5 0.6
Treatment
on
Nov
±
2.4’
±
36b
±
4.7
±
53b
“There
unsti-
levels.
‘Significant (p < 0.05) effect of LHRH-A versus saline-injected blank animals. bSignificant (p < 0.05) enhancement of LHRH-A effect by versus LHRH-A-injected blank animals.
89
E2
was
Blank 1 DHT*
4.5 2.6 2.3
Blank 1 11-KI”” DHT
7.8 5.1 4.0 4.1
no effect
stimulated GTH levels. ““Neither 1 1-KT nor DHI
(p>
0.05)
affected
± ± ± ± ± ± ±
LHRH-A-injected
blank
(ng/ml) LHRH-A
1.3 0.8 0.6
9.4 39.3 7.5
1.5 0.6 0.5 0.4
13.4 107.9 6.7 8.2
±
4.5
±
6.8’
±
2.0
±
4.2 12.8’ 1.3 2.1
± ± ±
of steroid
treatment
GTH
in saline-injected
animals nor affected LHRH-A-induced 0.05). ‘Significant (p < 0.05) enhancement I versus
GTH
Saline”
levels GTH
on un-
secretion
of LHRH-A
animals.
re-
and
effect
of 0.33
androgens
GTh
AiD
in male either
of various
the
with
nonsignificantly, potentiating
secretion
in saline-injected female fish. Data
not
pretreated
were slightly, but AID inhibited the GTH secretion. of three injections
on basal
in Table
are
animals
to LHRH-A.
and
effected
(p
> by
956
TRUDEAU
TABLE
8.
The
effect
of various
ET
AL.
androgens
TABLE hourly;
on serum GIH (ng/ml) in saline-injected (control) and LHRH-A-injected (0.1 g/g) male fish (Jan 90). Data are presented as mean ± SE (n
10. The effect of multiple injections 3 injections) of 2-OH-E2 and 4-OH-E2
(ng/ml) in male (n = 10).
goldfish.
Data
are presented
Serum
Treatment
GTH
(ng/ml)
Saline”
Blank 1 11-1(1””
3.6 2.9 2.5
±
DHI””
3.6
“There
was
Treatment”
LHRH-A 19.9 107.9 12.1
±
±
0.8 0.7 0.5
±
0.7
14.4
±
no effect
(p
±
0.05)
>
±
2.8 3.3
±
as a percentage
the
serum
of levels
effect
of injection
GTh
at Time
of hCG
every
serum GTh levels. affect unstimulated
however,
hCG
response
to LHRH-A.
hCG 3.8
±
treatment
treatment; 0.3 and
TABLE
significantly
data
were
ex-
0, no
further
ef-
3 days
over
Gonadal
growth
9.
effect
The
of I alone
Treatment of males serum GTH levels; was
for control and 0.3%, respectively.
the
not
affected
treated
or with
groups
ATD
(100
and 300 g/g) pretreatment on serum GTH (ng/ ml) in saline-injected (control) or LHRH-A-injected male fish in March 1990. Data are presented as mean
±
SE (n
=
Blank Testo AID (100)”” ATD (100) ATD (300)”” AID (300)
Saline”
+
+
T T
GTH by were
and
12.8 12.5 9.2 7.4
GTH
LHRH-A 2.7
46.1
±
±
2.0
140.8
±
133b
±
0.8 1.9 2.2 1.4
54.5 71.1 42.2 61.4
±
13.3 17.7#{176} 9.8 18.4#{176}
± ±
6.8
±
±
±
1.9
91.8
±
was no effect (p> 0.05) of CE treatment or on % of Time 0 values.
on serum
± ± ±
variations
serum
GTh
8.2 7.4 10.6
in GSI levels,
GnRH
[11].
In
GTh
the
and
and
serum
levels
pituitary
serum
parallel
present
from
0
7.6 7.2 8.8 GIH
levels
seasonal
tion
of June
the
LHRH-A
1988
when
response
changes
GSI was
atively low. Estradiol was GTh response to LHRH-A descence
from
when [24].
hance LHRH-A-induced mediately following as having
E2 enhanced size
gonadal
was
in potentiating of gonada! 1989.
These
rel-
the recruobserva-
size and serum E2 levels E2 does not further en-
GTh secretion. In the period imspawning season, fish are typified large
atretic
ovaries
and
low
blood
sex steroid levels [24]. In the present study, we found that post-spawning females in June 1988 had moderately sized atretic ovaries, moderately high pituitary GTh content, and no response to LHRH-A. Furthermore, ceived E2 treatment, LHRH-A effectively lease. In contrast, in 1989, fish came several
weeks
earlier
than
in 1988,
when these fish restimulated GTh reinto spawning conand
although
most
ovaries in May, the GSI was still relatively were producing E2 (>1 ng/ml). These fish to LI-Hill-A, and physiological doses of I-
8.9’
“There was no effect (p > 0.05) of steroid treatment on unstimulated GTH levels. “ATD (100 and 300 g/g) alone did not affect (p > 0.05) unstimulated or LHRH-A-induced GTH secretion. ‘Significant (p < 0.05) effect of LHRH-A versus saline-injected blank animals. bSignificant (p < 0.05) enhancement of LHRH-A effect by T versus LHRH-A-injected blank animals. ‘AID blocked the stimulatory effect of I on LHRHA-induced GTH secretion.
re-
intermediate,
gonadal exogenous
the
moderately
size
In sexually
when
1988-May
that when naturally,
increased gonadal
GSIs, LHRH-A was With the excep-
not effective in the period
November
tions suggest are increasing
E2,
were
in gonadal
LHRH-A
increasing
in months
content
previous reports of serum GTh
a period
are
of I and
GTh
experiments,
October-May,
steroid
fish had atretic large and they were responsive
(ng/ml)
±
8.9
±
69.6
±
1.5 1.4 1.9
gressed animals (i.e. summer) with small ineffective in stimulating GTh secretion.
dition
7-10). Serum
Treatment
a 39-
in Table animals
enhanced
GSIs 3.9 ±
81.7
1.5
to exogenous
day period on LHRH-A responsiveness is presented 12. Injection of LHRH-A in saline-treated control resulted in increased with hCG did not
2.0
±
± ±
evident in the present study and confirm [24, 25]. Seasonal variations in the response
fects were noted. In the second experiment, multiple injections of 1 p.g/g 2-OH-E2 did not affect basal or LHRH-A induced Gill secretion (Table 11). The
% lime
±
Seasonal basal
size
when
ng/ml
3 h
DISCUSSION
serum
pressed
SE
±
of steroid
animals.
addition,
9.2 10.1 11.5
“There levels
GTH
0 h
Vehicle 2-OH-E2 4-OH-E2
5.2’ 29.1”
treatment on unstimulated GTH levels. ““Neither 1 1-KI nor DHI affected GTh levels in saline-injected animals and nor affected LHRH-A-induced GIH secretion (p > 0.05). ‘Significant (p < 0.05) effect of LHRH-A versus saline-injected blank animals. 1’Signiflcant (p < 0.05) enhancement of LHRHA effect by I versus LHRH-A-injected blank
In
GTH
as mean
8-14).
=
Serum
levels.
(0.33 g/g on serum
TABLE
11.
The
of multiple injections of GTH (ng/ml) in unstimulated (saline-injected) or LHRH-Ainjected male fish. Data are presented as mean ± SE (n = 10). effect
2-OH-E2 on serum
Serum Treatment”
Saline
Saline
20.7
±
2-OH-E2
17.7
±
G TH (ng/ml) LHRH-A
4.1 2.1
47.2 28.5
± ±
13.2’ 6.0
“There was no effect (p> 0.05) of 2-OH-E2 treatment on GIH levels in saline-or LHRH-A-injected animals. ‘Significant (p < 0.05) effect of LHRH-A versus saline-injected animals.
SEX
TABLE
12.
The
effect
STEROIDS
POTENTIATE
of multiple
GONADOTROPIN
the
hCG
injections (0.2 p.g/g every 3 days for 39 days) on serum GTH (ng/ml) in unstimulated (saline-injected) or LHRH-Ainjected
male
fish
as mean ± SE (n
are presented
Serum Treatment
GTH
Saline”
Saline
7.8
clearly
1.0
27.3
hCG 8.3 ± 0.9 48.9 ± 45l “There was no effect (p> 0.05) of hCG treatment on unstimulated GIH levels. ‘Significant (p < 0.05) effect of LHRH-A versus saline-injected blank animals. bSigniflcant (p < 0.05) enhancement of LHRHA effect by hCG injection versus LHRH-A-injected saline control animals.
but
not
E2-enhanced
this
response.
response.
riod
Given
in different
that
years
fish
the
exhibit
and condition, variable sponses to LHRI-1-A, further
the
highly
and
In
contrast
sponse
to
throughout
ductive cycle. The and E2 is unclear, matization
reason since
to estrogen
post-spawning gonadal
enhanced the
size
and reof GnRH
the
entire
differential demonstrated
is important
for
re-
repro-
response that
the
dur-
GTH
seasonal
for the we have
or
the
secretion
may
binding
proteins.
seasons.
Given
steroid-binding
Pasmanik would not
tissues.
of T I aro-
positive
effect
to clarify
action. Positive
the
basis
effects
for
of sex
eels
[22].
Furthermore,
pituitary
GIH
and
response
to I
be an example of steroid injections of salmon GTH pituitary GTh content [36]. Furthermore, the
in cyprinid steroid
in the
summer
[34].
on aromatase and may be efficiently fectively above
increase control
Given
that
that its effect is present aromatized throughout local
brain
levels.
and
In contrast,
enous serum E2 was low dogenous E2 and gonadal
resulted
in no
hanced
LHRH-A-induced
pituitary
I ef-
E2 concentrations of serum
E2 by
only increased tissue E2 in months when endogwhen enImplanta-
a high dose (400 p.g/g) of E2 serum E2 levels that were apovulatory concentrations [25,32].
change
hance LHRH-A-induced I produced physiological
is dependent
and not during months size were increasing.
tion of female fish with produced nonphysiologica! proximately 2 times normal
ev-
in all seasons, the year and
elevation
hormone implantation may have concentrations to effective levels
This
I action
being
in basal
secretion. serum GTh
secretion
but
In contrast, both I concentrations secretion.
These
data
did
en-
doses of and en-
on
pituitary
levels;
jection
in male
dependence lease
and
pituitary
of
this
may
Indeed, increases
two in trout pitui-
responof 11-KT
steroid
GTh acts
fish. In the present not affect pituitary of I and
increases secretion by resulted
difexGTh
E2 cannot
be
in pituitary Gm. multiple hCG in-
in enhanced
GTH
se-
pituitary seem
system,
successful
exhibited mechanism responsiveness to involve
hCG inOvariwith T
spawning cues (plants and with an ovulation-like surge adult goldfish, there may be a
on endogenous GTH
and
not
brain
rethe
LHRH-A-induced
that
action
also
or E2 respond to environmental increased water temperature) in serum GTh [38]. In the
converted
the
goldfish
demonGTh that
salmonids
affect pituitary lack of effect
and
re-
Testosof pituiEuropean
have
since Kobayashi et al. [30] have demonstrated that jection elevates blood levels of I in male goldfish. ectomized or sexually regressed goldfish implanted
season The
to E2 within
and The
basal,
positive
been
male and female I 1-KT can stimulate
and salmonid treatments did the
[20]
feedback. dramatic
suggests
by steroid-induced increased GTH
have
cretion in response to LHRH-A. This response is presumed to be mediated by stimulation of testicular steroidogenesis,
that the lack of E2 effect in some months is not a result of an inability to respond to estrogen treatment but rather that circulating serum E2 levels are inadequate to elicit an effect. This does not seem to be the case for I since it can be efficiently
content, goldfish
therefore,
explained Mimicking
pituitary
suggest
GTH
in E2 pos-
also stimulates They suggested
in immature androgen
ferently periment,
ident
activity
GTh
in immature
in the
lower
that
the effectivewill be nec-
variation
Evans
positive stimulate
secretion
year;
and is highly
therefore,
decrease experiments
T treatment of salmonids.
has been for T and not vary
that the binding exchange of I
unlikely,
on
I
characteristics
(see Introduction). and accumulation fish [17, 18] and
Crim
strated that long-term lease into the blood
when
observations
suggest enhance
steroids
ported for several fish species terone and E2 induce synthesis tary GTH in immature salmonid
varies
the
possible
interaction
a seasonal
tary GTH accumulation [18, 37] siveness to GnRH in trout [37].
throughout
The
or
greater
to the pres-
be related
these
Its seem
of I on WRH-A-induced GTH secretion. Goldfish brain aromatase activity [34] is maximal during the spawning season. Similarly, albeit to a lesser degree, pituitary aromatase also slightly
other
an effect
globulin
and Callard [35] limit but would
E2 in neural
essary itive
pe-
of GTh secretion cycle is warranted.
E2, T effectively
to LHRH-A
post-
responsive enhanced
pituitary GTH contents, investigation of the role
and gonada! steroids in the control ing this period of the reproductive
uptake in the
One
of E2 to have
the steroid binding protein would ness of steroid treatments. Further 1989,
variable
sexes
fact that
unstable, protein
In June
during
steroid
of tissue
E2, resulting
experiments.
failure
GTH
of serum
between
spawning fish had small ovaries and were not to LHRH-A; however, both I and E2 effectively this
the
affinity
I over
of a T and E2 binding globulin in goldfish serum described [35]. It has a high affinity that is similar E2 and the number of serum binding sites does
3.6’
±
the
favors
of I in our for
957
GOLDFISH
Perhaps
enhanced
ence
LHRH-A
±
[34].
explanation
(ng/ml)
IN
mechanisms
effectiveness
8-10).
=
goldfish
binding
1989. Data
in October
RELEASE
gonadal
steroids
therefore
ensuring
ovulation
during
by this species. and site of action accumulation
the of I and
in the goldfish
to prime
adequate short
re-
spawning
E2 to increase
is unknown,
of pituitary
the
GTH
GTH.
but
does
In other
TRUDEAU
958 vertebrate reduce mals,
species, enhance
or
estradiol
sex the
can
sponsiveness
by
steroids can GTh response
either
increase
direct
[39, 40], whereas tuitary to inhibit
action
or
on
androgens gonadotropin
have
act
at the pituitary to to GnRH. In mam-
ceptor crease
number during
decrease
species.
We
pituitary
GnRI-I
re-
action [41,42].
on
the piIn com-
and E2 can act LHRH-stimulated
di-
GTh release [43]. In adult female trout, E2 can also act directly at the pituitary to enhance GnRH-stimulated GTh secretion [44]. In goldfish, it is not known if gonads! steroids have a direct positive action on the pituitary. This seems likely, Peter,
however, unpublished
since
24 h incubation)
study (Trudeau found that both
E2 (100
nM
pituitary Very
fragments to GnRH in vitro. little is known about steroid
GIL-I
for
in a preliminary data), we have
secretion
in other
nadectomized
ranid
[46]. group
In a single demonstrated
E2 suppressed positive These
that
comparisons
relative importance androgen action Gonadectomy roid replacement
control
vertebrates. treatment
GnRH GnRH
with
variations; varied difficult.
11-Icr
or
In the DHT)
a
GTh secretion in vivo. on GTh secretion may however, greatly
Regardless,
steroids that
versus an intact for this difference It is possible account steroid relation
that
for the treatment between
alterations
in other reduces receptor
vertebrate
[11].
tor number are sponse to GnRH sex
steroids
in goldfish,
and
accompanied in vivo [11]. GnRH
pituitary
receptor
the
The
year,
responsiveness
models
reason numbers
[48,49].
increases
In mice,
increases pito rats where
in GnRH
recep-
increased GTh the interaction has to
secretion
[50].
In gold-
to
gonads!
function
imal.
In vivo
domperidone
not reveal reproductive
any
the
DA
antagonists
pimozide
[53] is greatest in sexually mature that the DA inhibitory tone is greatest (i.e.
sex
steroid
dose
differences cycle. This
secretion)
responses
is max-
[53],
in drug potency suggests that factors
however, throughout other than
in the DA inhibitory tone are responsible for of DA antagonist action. Gonads! steroids may GTH secretion by altering catecholamine metab-
olism in the brain, since E2 modulates amine oxidase (MAO) in goldfish [55]. Since steroid treatment effects were
[54] on
not
GnRH,
known.
are mediated pituitary, to vestigators
not evaluated in these relevance of E2 regulation We
by their estrogens
are
hypothalamic monoand Indian catfish pituitary hormone
propose
radation
and
been GnRH
reof tested re-
hence
Our
data
studies [54, 55], the of hypothalamic MAO
currently
examining
conversion, within and subsequently that
CE
echol-O-methyl-transferase
secretion following there is a good corand pituitary GnRH
binding
GTh
of catecholamines and gonadal steroids in the of GTH secretion. Using the male African catfish as one research group has proposed [26,56] that the feedback effects of androgen on Gill secretion
receptor
by an Although
in response
when did the
and
is not
whereas E2 treatment levels; this is in contrast
Castration-induced
also
numbers.
teraction control a model, negative
that
gonadectomy increases and E2 suppresses pituitary GnRH receptor numbers [48]. In male catfish, castration increases and androstenedione suppresses pituitary GnRH receptor numbers
GnRH
domperidone suggesting
secretion.
in GnRH
E2 on
may
activity
different.
enhancement of GTh in the goldfish, since GnRH receptor binding
responsiveness gonadectomy tuitary GnRH
are quite known.
of I and goldfish
it is clear
were observed at any time of the steroid response in a gonadectomized
paradigm is not
intact
secretion physiological
of aromatase and reductase activity in varies amongst the vertebrates. results in elevated blood GTh, and stetherapy in gonadectomized teleosts re-
the
action
in the
the experimenbetween studies,
these elevations in GTh [11, 12]. In the case of intact and female goldfish, however, no negative actions of
gonads! suggesting
positive
teleost species, DA has a strong inhibitory GnRH-induced GTh secretion [16,51], and of various DA antagonists potentiates the acagonists on GTH secretion. The increase in
GTh
alterations seasonality influence
case had
the
secretion
subsequently,
fish and other influence on administration tions of GnRH
of
[45]. Esin frogs
to GnRH. not
and,
[52] and goldfish,
E2 inhibits
that
Gill
It is possible that gonadal steroids act to modulate dopamine (DA) inhibition of GTH secretion. In rats, gonadal steroids act to affect hypothalamic catecholamine turnover
serum
in a turtle [47], the same research gonadectomy elevated and I and E2 (but
suggest
levels inin this
be related to changes in pituitary GnRI-I receptor This hypothesis is currently under investigation.
of
In go-
responsiveness responsiveness
LHRH-A-induced in steroid action
species employed
and I and
response
feedback
responsiveness
I and
effect on differences
making
duces male
study
both
represent true tal protocols
in vivo
in vivo in vitro
pituitary
of goldfish,
the
cold-blooded
frogs,
and DHT augments tradiol also inhibits
increase
[11], and serum gonada! steroid seasonal reproductive development
GnRH-induced
gonadotrophs
a direct secretion
parison, in the immature trout, both I rectly on the pituitary in vitro to enhance
ET AL.
increase in the
drogen and estrogen secretion; however, failed to demonstrate
with
cause
resolved. by using
paradigm,
have
adult teleost sonally. We levels
during
a gonad-intact,
demonstrated
enhance species; suggest gonadal
LHRH-A-induced
result
an-
in successful
for
of estrogen and goldfish steroid-treated
the GTh
first
recrudescence
spawning
may
time
secretion
in the case of E2, the effect that elevations in endogenous
tary and ensure that changes in release, in response to environmental [59],
Gill
that
modulate the effect of GnRH on GTH our limited experiments thus far have similar effects for CE. Whether there
remains to be In conclusion, E2 can
cat-
on
indicate
difference in the mechanism secretion in the Mrican catfish
and
for
DA deg-
DA effects clearly
is a fundamental action on GTh
we
DA
decreased
inhibitory goldfish
in-
the brain and/or to CE. These in-
compete
and
the
varies seasteroid the
pituiGill cues
species.
I
in an
GnRH [57, 58] and and pheromonal in this
prime
that
SEX
STEROIDS
POTENTIATE
GONADOTROPIN
ACKNOWLEDGMENTS
RELEASE
inverse
on pituitary
effects
in the immature The
authors
acknowledge
and B. Wakeford
Smith,
ful comments and
Dr.
and
discussions
G. Somoza
forming
the
are
hCG
great
with with
also
appreciation
of this study. Furthermore,
Dr. J. Chang,
Dr. M. Kobayashi,
The authors
appreciated.
cross-reactivity
the help of C. Nahomiak,
the duration
throughout
T.
the help-
Dr. P. Rosenblum, C.K. Murthy for per-
thank
Kagawa
gonadal
Sci Fish. 25. 26.
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