Grant. HD12629-06 and. Veteran's. Administration. Medical. Research. Funds and in partial fulfillment of the ... and Sawyer. (1980) reported that chronic removal.
BIOLOGY
OF
REPRODUCTION
37,
33-38
(1987)
Role of the Central Ascending Noradrenergic System in the Regulation of Luteinizing Hormone Responsiveness to Testosterone Negative Feedback in the Adult Male Rat’ DONNA
L. MACCALLA,4
DONALD K. CLIFTON,3 ALVIN and ROBERT A. STEINER2’#{176}
M. MATSUMOTO,6’7
Departments of Obstetrics & Gynecology,3 Physiology & Biophysics,4 Zoology,5 and Medicine,6 University
of Washington
Seattle, Geriatric
Washington
Research,
Veteran
98195
and Education
and
‘s Administration Seattle, Washington
Clinical
Medical 98108
Center7
Center
ABSTRACT is well otropin
It gonad
poorly
established
that testosterone However, the
secretion.
understood.
negative feedback. izing hormone
We To (LH)
depleted Depletion ascending
of hypothalamic of hypothalamic noradrenergic
the
toxin,
were period
neuro
hypothesized test and
that
After
activity, demonstrate
the
dose-response that normal
negative pothalamic
feedback effect NE does not
on alter
of
allowing
weeks
2-3
established
dose-dependent
that
negative
tropin secretion al., 1976) that hypothalamus
(Moore is affected, (Simpkins
1982). However, neither nor the mechanisms by inhibitory
action
to
testosterone
feedback
effect
secretion. sensitivity
(T)
exerts
gonadotropin
hypothalamic
relationships (FSH) in adult
recovery
on
gonado-
from
T-containing reduction
to inhibit action is
sensitivity
to T
between T and luteinmale rats chronically
this
initial
surgery,
or empty (sham) in hypothalamic
all animals implants for noradrenergic
gonad otropins remained unaltered. activity are not essential for they suggest feedback.
of action its central secretion
stimulate
luteinizing
et al., 1979;
1970; Quijada Kaufman et
mechanism to affect Accepted January 13, 1987. Received September 2, 1986. ‘This work was supported by NIH Grant Administration Medical Research Funds
pituitary inhibitory
that
chronic
a
These data T to exert its removal
of
hy-
are known. T may inhibit the gonadotropin-releasing hormone (GnRH) neuron directly, or alternatively might interact with other neuronal networks within the hypothalamus to inhibit the release of GnRH. Strong evidence indicates that the classical neurotransmitter, norepinephrine (NE), acts centrally to
a
Price, 1932; Damassa et in part, at the level of the et al., 1980; Steiner et al.,
regulate
for
Furthermore, to T negative
and
the specific sites which T exerts
the anterior its central
decreases
dose-response hormone
T and the noradrenergic
INTRODUCTION
It is well
activity
the
with subcutaneous a profound chronic
between plasma hypothalamic
gonadotropin gonadotropin
the brain and which T exerts
(NE) to the dose-response curves exhibited by control animals. by two independent methods: 1) by bilateral transection of the of the mesencephalon, and 2) by intracerebroventricular infusion of
were outfitted that despite
relationship amounts
on by
noradrenergic
we compared follicle-stimulating
norepinephrine NE was achieved system at the level
and 3 weeks later hours. We observed
feeds back mechanism
central
this hypothesis, between T and
6-hydroxydopamine.
castrated, of 48
(T) precise
hormone
(LH)
by which NE influences LH secretion remains
Barraclough and Wise, 1982). In addition to affecting GnRH tropin release, T has been shown noradrenergic activity (Chiocchio
HD12629-06 and Veteran’s and in partial fulfillment
of the Master of Science degree requirements for Donna L. MacCalla in the Department of Physiology and Biophysics at the University of Washington. 2Reprint requests: Robert A. Steiner, Department of Obstetrics and Gynecology, RH-20, University of Washington, Seattle, WA 98195.
kins
et al.,
castration tivity as 33
1980). increases assessed
release
(Kamberi
et al., 1973; Negro-Vilar al., 1985). However, the
Removal
the unclear
system review,
activity and gonadoto influence central et al., 1976; Simp-
of T negative
hypothalamic by changes
GnRH (see
et al., precise
in
feedback
noradrenergic NE content
by acand
MACCALLA
34 turnover
in
while
specific
regions
T replacement
dosages secretion
that of
et
acutely
activity al.,
1976;
These
rise data
regulating
the
rats,
that
feedback
NE depletion in LH levels
greater
LH
and
suppression
by
to
(Clifton The
the
estradiol of the priming
the
sensitivity effects
the
changes
in
central
feedback regulation adult male rat. To
the and
the
rate of and a
below the dura sham-cut animals.
in
laterally
in either
direction
After 30,000
surgery, U procaine
steroids
to
determine
system
sensitivity
to
of gonadotropin test this hypothesis,
negative
secretion in the we examined
cement. sham distilled gelatin
one
of
tion. Two implant,
NE
between allowed
experiment,
chronic
bilaterally
transecting
system. In another adult male rats were (6-OHDA)
to
NE
depletion
the
ascending
experiment, treated with
chronically
deplete
was
achieved
noradrenergic
a second group 6-hydroxydopamine hypothalamic
content to control for nonspecific effects of the surgical lesions. These animals were also castrated, and the LH and FSH response to a single replacement dose of T was examined and compared with vehicleinfused
animals. MATERIALS
Animals Adult tories,
and
AND
METHODS
Accommodations
male Bellevue,
Sprague-Dawley WA), weighing
receiving were
Animals according
were to the
Pellegrmno et al. (1967), steel blade was placed ± 1.5 and lowered 8 mm animals was then to cut
animals penicillin
the
and 6 mm in moved 0.1 mm fibers.
received an injection of i.m. to prevent infection
Laborag, were
10,
or
20,
25,
an and
empty 30
(sham)
mm
Empty 20-mm implants implants. All implants were water/phosphate-buffered (20:1) for at least 48 h prior
separated assayed
in
before After under with implant.
length,
were
days after implantation blood samples were 1300 to clot
and 1400 at 4#{176}C for
by centrifugation for
LH,
FSH,
of either a T or sham obtained by decapitation h. The trunk blood 24 h, the serum was and
and
were used for equilibrated in saline with 1% to s.c. implanta-
stored
T by
was then
at -20#{176} C until
radioimmunoassay
(RIA, see below). Immediately after decapitation, brains were rapidly removed and frozen on dry ice and stored at -40#{176} C for subsequent hypothalamic dissection and catecholamine determination. Hypothalamic sections from all ANS-cut animals, representative sham-cut animals (from each size of T implant)
rats (Tyler 250-350
anesthesia. instrument
ANS-cut The knife
all
of hypothalamic
made using Silastic tubing (i.d. 0.062 in [1.57 mm], o.d. 0.125 in [3.18 mm] ; Dow Corning, Midland, MI) filled with crystalline T (Sigma Chemical Co., St Louis, MO) and sealed at both ends with Silastic
the T-LH and T-follicle-stimulating hormone (FSH) dose-response relationships in animals chronically depleted of hypothalamic NE, and compared this response with that exhibited by control animals. In by
and
lesioning the ascending at the level of the
group of animals served as controls. sham-cut operations
used by stainless 0.0, ML
a T-containing
T-implants,
mediates T
food
and were allowed a 2- to 3-wk recovery period undergoing any further surgical manipulations. this recovery period, all animals were castrated ether anesthesia and 3 wk later were outfitted
brain-pituitary
was
and
depletion
under ketamine in a stereotaxic
either study
of animals,
release.
findings secretion
gonadal
group
coordinate system and a 2-mm-wide bilaterally to AP
replacement.
the
noradrenergic
hypothalamic
to
14L: IOD schedule, ad libitum.
mesencephalon. Another sham lesions (sham-cut) Both ANS-cut and performed oriented
LH surge resulting was dramatically
of
and Sawyer, 1980). objective of this
whether
LH
animals. These gonadotropin of
the
reported
input negative on
in
T on
in a slower ovariectomy
upon
feedback
of
on a available 1
In one
post-
involved
maintained water were
NE was achieved by bilaterally noradrenergic system (ANS-cut)
1973).
(1980)
estrogen
in these NE-depleted that NE may regulate
decreasing
axis
be
action
resulted following
In addition, the magnitude from estrogen/progesterone increased suggest
may
Sawyer
of
the
AL.
Experiment
Moreover,
McCann,
noradrenergic both the
effects
at
(Chioc-
inhibits
feedback
removal of increased
positive
1980).
and
NE
Clifton
Chronic increase
al.,
(Ojeda
negative
that chronic hypothalamus
et
levels
synthesis
LH
animals
precastration
NE in
hypothalamus,
postcastration hyperrestores hypothalamic
Simpkins
suggest
hypothalamus. In female
the to
suppressing
castration
the
orchidectomized
suppress gonadotropins
noradrenergic chio
to
of
ET
isolated NE and
and
intact
adult
males
of
similar
for comparison of hypothalamic dopamine (DA). Brains from
age
were
content of intact adult
NE
male
rats
operations
were
used
had
disrupted
catecholamine
to
assess the
REGULATION
whether
OF
RESPONSE
was
hypothalamic
content.
To
control
cuts
and
ment
1,
for
to
nonspecific
corroborate
results
HCL
was
depleting
hypothalamic
6-OHDA Co.) was
HC1, freshly
ascorbic
acid,
experiment
used
as an
free base in saline
(vehicle)
5.5,
kept
lowered
into
the
ventricle
sinus, DV: -7.0 from presence of cerebrospinal
stylet attached 5 .zl of 6-OHDA a Hamilton
were
animals an s.c.
confirmation A 28-gauge
PE2O tubing was (3 mg/ml) or vehicle
syringe.
Solutions
allowed
a 2-wk
then and
were
recovery
were castrated and 25-mm T implant.
produced dose-response
T levels curve
probability 6-OHDA
of and
centrifuged, assayed removed
close and
3 wk This
animals were serum
dissection
i.c.v.
at
Next,
all
T-LH the
between 48
the h after
were killed by decapitation. collected, allowed to clot, was
stored
for LH, FSH, and and frozen on dry
pothalamic
filled with connected
outfitted with of T implant
At
at
-20#{176} C until
T. Brains were rapidly ice for subsequent hy-
and
catecholamine
Luteinizing
odology
LH,
hormone
used
previously
for
in
(Steiner
FSH
Details
RIA.
the etal.,
LH
RIA
1982).
rat LH RIA kit (RP-2 standard; sure serum LH concentrations. was 0.5 ng/ml. The interassay (CV)
was
14%
and
the
and
intraassay
the
by
Matsumoto
1.5
ng/ml,
and
4%,
Health
Inter-
with
reagents and
described
intraassay
5.1%,
al.
inter-
Organization,
previously
and
and
et and
respectively.
measured
been
RIA
CVs
by for
respectively.
the
T assay
ng/ml.
Determination
Sample
preparation.
rapidly
dissected
Whole
from
stainless cut at
frozen
hypothalami
brain
tissue
steel blade was the level of the
second
transverse
border traversed
of the mammillary the anterior
cut
was
made
were
and
used optic
to make chiasm.
through
bodies, commissure,
weighed.
the
while
the
caudal
dorsal lateral
and
a A cut cuts
bordered the lateral ventricles. These hypothalamic sections, weighing 44.1 ± 0.8 mg (mean ± SEM, n 48), were immediately placed in a tissue homogenizer with 400 jtl 0.1 N perchioric acid containing 50 ng/ml of 3 ,4-dihydroxybenzylamine ical Co.) as an internal centrifuged supernatants
for 20 were
modification
of the
Anton (300
1
ml
for
2600 rpm at 4#{176}C,and and extracted by a
alumina
extraction
tris
M
Buffer 2
mm
aspirated twice
The tubes
aluminum alumina, grade
0.5
(Tris)
and and
with
tube The
and distilled,
chromaCo.) and
aminomethane
Each
centrifuged. of
of
acid extract 10 mg of
oxide (Type WN-2 1, Sigma Chemical
8.6).
discarded 1 ml
procedure
perchloric containing
(hydroxymethyl)
(pH
Chemwere
mm at aspirated
and Sayre (1962). jil) was added to
acid-washed tographic
(DHBA, Sigma Homogenates
standard.
=
the
was
vortexed
supernatant
alumina
deionized
was
was
washed
water.
Cate-
meth-
at -70#{176} C until assayed. Catecholamine assay. NE and DA pothalamic extracts were determined
have
been
detailed
We used
the
NIADDK
S-6 antibody) to meaThe assay sensitivity coefficient of variation CV,
have
FSH
cholamines were then desorbed from the alumina by adding 300 zl 0.1 N HC1O4/0.825 mM cysteine, vortexing for 2 mm, centrifuging, and recovering 250 pl of the supernatant. Alumina extracts were stored
T of
World
The
samples,
deter-
mination. Radioimmunoassays
the assay
0.1
21%
T was
9.8%
was
A sharp transverse
for third infusion
period.
groups.
were
Catecholamine
animals received penicillin i.m.
later size
assay
sensitivity
sagittal
infused
differences
vehicle-infused
and
et al. (1982).
following
to the middle of the was selected to maximize
detecting
T-implantation, Blood samples
Steiner
were
ML:
a rate of 5 jil/min. Following surgery, an injection of 30,000 U of procaine and
of
was
were
RIA.
of the
the top of the dura. The fluid (CSF) flowing through
used as placement.
to
the
bregma,
CVs
details T
RIA.
assay
of the
Animals
at
#{247}0.5 from
was cannula
morning
intraassay by
in the
described
sensitivity
supplied
(Sigma Chemical containing 0.1% used.
previously Assay
of
and placed in a stereotaxic stainless steel cannula was
third
AP:
the cannula ventricular
infusion
variability hormone
Testosterone
Experi-
method
on the
at 4#{176}C until
with ketamine A 22-gauge
coordinates:
in
alternative
and
surgical
NE.
pH
anesthetized instrument.
the
(i.c.v.)
3 mg/ml prepared
and
of
obtained
intracerebroventricular
6-OHDA
to
effects
35
of replicate
was
(1986).
2
T
1.3%. Follicle-stimulating
used Experiment
TO
by analysis
sham-cut
normal
LH
as determined
phase, matograph
ion-pairing, with
tection previously
(HPLC/ECD). by Matsumoto
levels in hyby a reverse-
high-performance liquid amperometric electrochemical The
assay has et al. (1986).
been
chrode-
described
MACCALLA
36 NE,
E, DHBA, and with the HPLC
mm response at 2nA was
range full-scale
2.1%
NE
and
on rat assays,
3.1%
for
Norepinephrine
eluted in less than 13 described. The linear
of the detector with the sensitivity was 60 pg to 8 ng. The intraassay
for
determined consecutive
DA were system
ET AL.
DA;
the
whole brain was 6.0% for
set CV
interassay
2.0
2.0
±
CV, 0
.2
.2 -
0
o, 0.8 Statistical
0,
regression
was
examined
Two-way
using
analysis
test for maintained
an
used
examine
to
All to
used
to
fit
two-tailed
t-test.
was
used
the
content.
effect
of
were
T
on
analyzed of the
the adequacy level below
two
NE for
cuts.
NE The
7
‘1
FIG.
1.
of the ANS-cut was standard deviations
a
Serum mm, and
spectively.
cholamine values pared with those of the sham-cut each from
of the sham-cut
from obtained group,
from
analysis.
Cate-
groups. were also
found in intact adult males levels for ANS-cut, sham-cut, compared by using Student’s
NE and compared
norepinephrine
m&an serum T values 0.1, 0.91 ± 0.1, and
±
Serum
levels pendent
DA levels to those
cut
of similar age. NE and DA and intact animals were two-tailed t-tests.
of
animals
not
T levels
in ANS-cut
T reduced serum LH levels manner in both the ANS-cut (linear
regression
of
similar
pothalamic 0.01 ng/mg to wt., not male
85%
content in ANS-cut wet wt.) control
(Fig.
r
a dose-dethe sham=
0.91
analysis that
of this
and the re-
#{149} ANS-cut o sham-cut Regression-ANS
NE levels in sham-cut from intact male rats
age
(1.84
ng/mg
DA wet
content in ANS-cut animals (0.34 wt.) was also decreased significantly
of sham-cut
±
±
0.10
animals
0.04
levels from ng/mg
(0.40
wet
±
0.02
wt.).
ng/mg
wt.).
LH
ng/mI
1.0
00
#{176}#{176} ,#{149}
o#{149} o#{149} o.
Hy0
±
0.5
.0
1.5
Testosterone
wet
in sham-cut animals did levels found in intact wet
10.0
1).
animals (0.57 was depleted to animals (1.65 ±
wet wt., p
