BIOCHEMICAL
Vol. 76, No. 3, 1977
INHIBITION
WITH
A POTENT
Auclair,
Paul
A.
LUTEINIZING
Kelly,
Medical
Research
Centre
Council
Hospitalier
and
*Department
and
VA Hospital,
Received
de of
HORMONE-RELEASING
V.
Group
H.
Coy*,
Endocrinology,
Laval, Tulane
Orleans,
David
Molecular
l'universite
New
Labrie,
and
Schally*
in
Medicine,
HORMONE
GONADOTROPIN
Fernand
Andrew
RESEARCH COMMUNICATIONS
HORMONERECEPTOR LEVEL BY
OR HUMAN CHORIONIC
AGONIST Claude
LUTEINIZING
OF TESTICULAR
TREATMENT
AND BIOPHYSICAL
Quebec
University
La
Le
GlV
462,
School
of
Canada, Medicine,
70112
21,1977
April
SUMMARY: Treatment of adult male rats with a potent luteinizing hormone-releasing hormone agonist, CD-LeuG, Des-Gly-NH2101 LHRH ethylamide or human chorionic gonadotropin for one week caused a marked decline in luteinizing hormone receptors in testicular tisTesticular weight was also significantly decreased by these sue. treatments while plasma testosterone levels were decreased by treatment with the LHRH agonist and increased after hCG. A single injection of the LHRH agonist or hCG also induced a long-lasting loss of testis LH binding sites with receptor levels returning to control values at 8 days. These findings indicate than an elevation of endogenous LH, whether induced by single or repeated injection of a potent LHRH agonist, is capable of down regulating testicular LH receptors with a resultant long-term effect of reduced testicular size and decreased plasma testosterone concentration. A marked se
to
loss
administration to
induce
loss
insulin,
LH receptors (2)
has
sues
on
the
of
ovine
LH or
of
their
own
that
treatment
of
of
hCG.
receptors
oligospermia
Abbreviations mone-releasing
in
own
and the
effect
101 rat
used: LH, hormone;
the and in
used
In and or
the
repeated hCG on
of
catechotarget
important currently
single
effect
respective
infertility. of
systemic
gonadotropins to
hormone
in
LHRH ethylamide
respon-
after
of
growth
are
male
rat
analogous
extremely
analogues
steroidogenic the
ability
is receptor
becomes its
and in
This
receptors
their
concept
LHRH and
we have studied port, of CD-LeuG, Des-Gly-NH2 LH/hCG
2)
observed
hormone,
level This
(3-8). fact
(1,
been
thyrotropin-releasing
lamines the
of
gonadotropins
view in
tisof
the
present
re-
injections the
level
of
testis. luteinizing hCG, human
Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
hormone; chorionic
LHRH, luteinizing gonadotropin.
hor-
855
ISSN
0006-291X
Vol. 76, No. 3, 1977
BIOCHEMICAL
AND BIOPHYSICAL
MATERIALS
AND METHODS
RESEARCH COMMUNICATIONS
Animals: Adult male Sprague-Dawley rats weighing 185-205 g upon arrival were obtained from Canadian Breeding Farms, St. Constant, Quebec. Animals were housed 2 per cage in a temperature (20-22'C)and light (14-h light, -10-h darkness)-controlled room and given food and water ad libitum. Hormones: Purified hCG (CR 119, 11,600O IU/mg) was generously supplied by the Center for Population Research of the NICHHD, NIH. For in vivo treatment, a preparation of hCG (APL, approximately 2600IU/m9J obtained from Ayerst was used. Ovine LH (NIH-LH-S19 1.01 x NIH-LH-Sl) was a gift of the National Pituitary Agency, NIH. CD-Leu6, Des-Gly-NH2101 LHRH ethylamide was synthesized in a solidphase system and purified as described (9). Treatment with the LHRH agonist or hCG: In the first experiment, the animals (8 per group) were injected with either 0.6, 3, 15 or 75 pg of the LHRH agonist, hCG (50 IU) or the vehicle alone (0.15M 1600 and 2400 h) for 7 days. NaCl) three times a day (at 0800, Animals were sacrificed 16-18 h after the last injection of the In a second experiment, the effect of sinLHRH analogue or hCG. gle injection of the LHRH analogue (125 Pg) or hCG (200 IU) was studied at the indicated time intervals after injection. In the two studies, animals were killed by decapitation between 0800 and 1000 h, testis removed and plasma collected for testosterone RIA. LH/hCG receptor assay: Decapsulated testes were homogenized in 40 ml of O.lM Tris-HCl, pH 7.4, 0.25 M sucrose, 5 mM MgCl with a Sorval tissue homogenizer at a speed setting of 10 for P 5 sec. After centrifugation of the homogenate at 20,000 xg for 15 min, the pellet was resuspended in 40 ml of buffer and recentrifuged. The pellet was then suspended in 3 ml of buffer (without sucrose but with 0.1% bovine serum albumin) and appropriate dilutions made so that each assay tube received 5 mg equivalents of tissue. For 2.5 ug of hCG and 1000 ng of chloramine iodination, 500 PCi ~12511, The hormones were then repuT were incubated on ice for 2-3 min. As calculated rified on a column (0.9 x 100 cm) of Sephadex G-100. the specific activity was 41.6 uCi/p'g. by isotope recovery, LH/hCG binding was measured by duplicate incubation for 1416 h at room temperature of 100 ~1 of the testis suspension with 200 ,.,l buffer, 100 ~1 of Cl2511 hCG (approximately 100,000 cpm or 40 fmol) in the presence or absence of an excess of unlabeled hormone (20 pg/ml ovine LH) in 12 x 75 mm glass tubes. The reaction was stopped by dilution with 4 ml ice-cold buffer and the mixture centrifuged at 2000 rpm for 15 min. The supernatant was decanted and the pellets counted in a LKB autogamma spectrometer with Difference of the cpm bound in the a counting efficiency of 74%. absence and presence of excess unlabeled hormone (specific binding) was expressed as fmoles of bound hormone. Testosterone assay: Plasma concentrations measured by a specific double-antibody with benzene and the extract assayed developed against testosterone-7a-undecanoate ne-7a-butyric tyrosine methyl ester. 5 and 5000 pg testosterone per tube. as described (10).
856
of
testosterone were Plasma was extracted using an antibody and C125IltestosteroThe limits of the assay were Calculations were performed
RIA. directly
BIOCHEMICAL
Vol. 76, No. 3, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
a3
Oobh 0.6
3
[D-LEU*,OES
-L :u”
75
15 GkMz’“]
41
I_
0.6
3 [II-LEU*,DES
ETHYLAMIDE Fig.
1.
75
50 IU
cg
hCG
LHRH
15
Gu-NH~lo] LHRH ETHYtAMlOE
hCG
Effect of treatment with increasing doses of CD-Leu6, Des-Gly-NH2101 LHRH ethylamide (0) or hCG (e) on binding of C125I1 hCG to testicular LH/hCG receptors (A) and Animals were intestis weight (B) in adult male rats. jected three times a day with the indicated doses of the LHRH agonist or hCG for one week.
RESULTS Long-term lamide cular
a marked
tissue
greater
from
decrease
Injection
of
sites.
(Fig. From
LH/hCG
CD-Leu6, of
to
140
seen
at
an
also
Fig.
1,
binding
higher
doses
complete
of
loss
significantly
LHRH ethy-
sites
in
(Fig.
1A).
of: 16 fmol/testis
almost
was
Des-Gly-NH2101
LH/hCG
the
of
A
LHRH analog.
LH/hCG
reduced
testi-
by
binding these
treat-
1B). the
data
receptor on
testosterone
LHRH analogue
?1 53 even
weight
of after
507 was
expressed plasma
of reduction
hCG caused
Testis
ments in
administration
caused
a basis while
hCG administration
in
levels of
was testis levels
increased (Table
it
can
also
be
calculated
observed
weight. was
Also
observed
plasma I).
857
androgen
that
when
hCG binding
a 55 after
a decline
to
75% reduction
treatment levels
was
were
with found
the
Vol.76,
No. 3, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE Testicular tration CD-Leu6,
in
LH/hCG receptor levels and plasma testosterone male rats injected with increasing concentrations Des-Gly-NH2 loI LHRH-ethylamide or hCGa* b.
Treatment
Dose
Control
Bound hCG fmol/g test‘i s
0
CD-Leu6, Gly-NH2101 LHRH
Des-
hCG
*,
I concenof
Plasma testosterone rig/ml
438
k 55
6.5
+ 1.0
0.6
ug
137
k 13**
2.9
k 0.4
3
ug
118
f
18**
2.5
f
0.3*
15
u9
132
k 34**
2.2
f
0.3"
75
P9
108
?r ll**
1.6
+ 0.2*
IU
50
5 f
2""
26.6
e 3.0**
p < 0.05;
** , p < 0.01
(exp
vs
control)
a Animals were the indicated
injected doses
bCalculations
based
A single ethylamide
thereafter.
weight
declined
8 days
(Fig.
It a small
can
subcutaneously 3 times a day for 7 days the LHRH agonist or 50 IU of hCG a molecular
of
IU
125
weight
ug
hCG induced
(Fig.
until
values
levels
200
levels
maximal
on
injection or
receptor ed
of
2A).
3 days
and
After
CD-Leu6, a rapid
The effect progressively
a slight
steadily
in
for
was
groups
37,900
Des-Gly-NH2101 decline
in
apparent increased
increase
both
hCG of
at and
12
remained
h,
with
(15,
LHRH-
testis
LH/hCG
at 12h, toward
remainnormal
testicular low
up
until
1B). be
followed increase
seen the 12
in
Table
same h after
IIthat
the
pattern
when
injection
858
changes expressed of
CD-Leu6,
of per
LH/hCG g testis. Des-Gly-NH2
receptor After loI
16)
Vol. 76, No. 3, 1977
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
A M o-o
000.
GLY-NH2’0]LHRH ETHYLAMIDE
[O-LEIJ*,DEs
hCG
,
600
2.z s \
2 400 u ,” s z -
2oa
0
L
0
2
4
6
I 2
OO
0
4
4 0
6
DAYS
Fig.
2.
Time-course of the effect of a single Leu6-Des-Gly-NH 101 LHRH ethylamide (200 IU) on bin 5 ing of C12sIl hCG to receptors (A) and testis weight (B)
LHRH ethylamide, and
plasma
returned
injection rone
to of
levels
crease
at
at
hCG led up
to
5 and
testosterone
normal to
levels
later
time
after
6 days
and
data
clearly
were
(Table
elevation
its
of
to
levels
at
3 days
A single testoste-
with
normal
at
2).
plasma
administration
return
of CDhCG LH/hCG male rats. or
decreased
intervals
a sustained
3 days
injection (125 pg) testicular in adult
a small
de-
8 days.
DISCUSSION The
present
injection
of
ethylamide
the
can
lead
LH receptors. depleted while of
course
effect
pg of of
to
the
a marked
similar
peptide
effect
of
125
down
to
the
was a day
LHRH agonist
a59
ug
of
25%
of
measured for
one is
or
repeated
Des-Gly-NH2"l loss of testicular
sustained of
magnitude 3 times
single
CD-Leu6,
and
injection
LH receptors of
the
that
LHRH agonist
A single testicular
an 0.6
potent
demonstrate
very
the
peptide
control
similar
analog levels
after week.
LHRH
injection The
timeto that
BIOCHEMICAL
Vol. 76, No. 3, 1977
AND BIOPHYSICAL
II
TABLE Time-course of 1H2101 Des-Gly-P tor levels and rats
the response to LHRH ethylamide plasma testosterone
Treatment
Days after injection
RESEARCH COMMUNICATIONS
a single injection of CD-Leu6, or hCG on LH testicular recepconcentration in adult male
Bound
hCG fmol/g testis
0
596
+ 50
0.5
409
1
ethylamide
(125 us)
Control CD-Leu6,
Des"ILHRH
Gly-NH2
hCG
IU)
(200
Plasma testosterone nglml
8.2
+ 1.7
+ 42**
13.3
k 2.5"
361
+ 18**
8.2
k 0.9
2
156
it 16**
5.5
+ 0.4
3
168
k 32**
8.2
+ 1.9
4
332
+ 42**
8.5
f
6
488
+ 66*
6.9
+ 1.1
8
525
t
7.0
+ 1.4
0.5
364
+ 47**
26.4
f
55
2.0
2.5**
1
24
+
8**
17.0
+ 2.1**
2
11
+
5**
13.9
f
3
11 +
5**
13.5
L 2.4*
4
55
+ 13**
6.1
2 0.8
6
298
+ 29**
6.2
+ 0.6
8
443
f
8.4
t
37**
1.6"
1.0
* , p < 0.05; ** , p < 0.01
obtained
in
hCG or ment the
ovine
with exogenous
(exp
these
vs
(Figs
LH (1, hCG could hormone,
control)
1 and
2). partly the
2)
Although
and
previous
the
reflect delayed
effects
experiments measured
occupancy of and sustained
860
binding effects
using after
treat-
sites by measured
Vol.76,No.3,1977
after
BIOCHEMICAL
a single
negative
regulation
previously with to
LHRH
48
12
h after
(111,
ovine
LH levels
injection
of
findings up
indicate
slow
data, ed
after
led
to
Such
LH receptors.
injection
of
ovine
only
or
lation
of
lead testis
Since
is
of
These
a 50
of
to
plasma
3 days
after
single
severely
terone
response
levels
Although influence of
with
those
has
been
observ-
of
hCG,
which
LH receptors
(2).
induced
or
exogenous
by
en-
gonado-
of
the
and
and
be
an of
the
hCG in 10
IU
almost
steroidogenic
the mechanisms treatment with
of
however
popu-
in
plasma
men
has hCG
in
complete of the
for
and
possible assessed.
absence
of the
levels the
presen-
ability
of
the
LH receptor and
observed
while
of
hCG-producing
the
rat to
levels of
in
partial been
the
accom-
been
having
LH receptors
also
LH was
testosterone
inhibition
response
role the
suggests of
automa-
testicular
LHRH agonist
compensate loss
be
(12,
secretion.
not for
levels
vitro
of
The
normal
LH receptors to
to
testosterone
has
the
testis
the
LHRH analog
explanation
of
in
androgen
weight.
LH secretion provide
required
expected of
plasma
prostate
between
an
not
of
number
present
with
injection
of to
are
the
a 75% decrease
decrease
finding
to
injection
tors
delayed
testicular
LH levels
of
decrease that
system
A dissociation
led
of
alterations
excess
treatment
depressed
loss.
IU)
note
75%
hypophyso-gonadal
(ZOO
was
LHRH analogue
dose
testis
marked
would
testosterone The
single
in
a parallel
could
(14).
of
and
secretion
testis
tumors ce
obtained
agreement
peptide
sustained
endogenous
findings
elevated
LH-releasing
a 7-day
of
changes
plasma
to
a reduction
levels the
a small
the
by
after by
or
of
LH receptors
interesting
panied
low In
of
LH receptors
followed
receptors
observed
loss
LH receptors
LH (1)
changes
testosterone
a loss
tically
As data
LH receptors.
spare
maximal
a
back to normal within 10 1 LHRH ethylamide
of
that to
be
of
8% occupancy
exogenous
can
to
injection
testicular
suggest
receptor
II),
Table
after
of
about
LH.
present
Des-Gly-NH2
of
tropins
It
recovery
the
likely
loss
dogenous
13),
2,
indicate
endogenous
2),
maximal
are
4 days
clearly
LH levels.
a sustained
data
for
that
CD-Leu6,
(Fig. to
(1,
plasma
plasma
the
by
hCG
reveal
RESEARCH COMMUNICATIONS
LHRH analog
LH receptors
maximal
LH receptors
the
LH and
analog
h after
Since
of of
with
the
24
injection
AND BIOPHYSICAL
the after
a higher both
gonadotropin
testosthe dose
LH recep(2).
testicular receptor loss and the LHRH analogue on endogenous LHRH
861
Vol. 76, No. 3, 1977
secretion,
LH
relevant
hormonal
data
indicate
sing
peptides
timal
dose
and
BIOCHEMICAL
responsiveness
to
secretions
that
the
will
greatly
time
of
AND BIOPHYSICAL
LHRH,
remain
successful
to
steroidogenesis be
benefit
from
the
therapy careful
of
and
elucidated,
long-term
administration
RESEARCH COMMUNiCATlONS
the
with
evaluation
other
present LH-releaof
op-
drug.
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Hsueh, A.J.W., Dufau, M.L. and Catt, K.J. (1976) Biochem. Biophys. Res. Commun. 72, 1145-1152. Hsueh, A.J.W., Dufau, M.L., and Catt, K.J. (1977) Proc.Natl Acad. Sci. 74, 592-595. Kahn, C.R., Neville, D.M., and Roth, J. (1973) J. Biol. Chem. 248, 244-250. Gavin, J.R., Roth, J., Neville, D.M., De Meyts, P. and Buell, D.N. (1974) Proc. Nat1 Acad. Sci. 71, 84-88. Hinkle, P.M. and Tashjian, H.H. (1976) Biochemistry 14, 38453851. Lesniak, M.A., and Roth, J. (1976) J. Biol. Chem. 251, 3730-3739. Lesniak, M.A., Roth, J., Gorden, P., and Gabin, J.R. (1973) Nature New Biol. 241, 20-21. Mukherjee, C., Caron, M.G., and Lefkowitz, R.J. (1975) Proc. Nat1 Acad. Sci. 72, 1945-1949. Coy, D.H., Coy, E.J., Schally, A.V., Vilchez-Martinez, J.A., Debeljuk, L., Carter, W.H., and Arimura, A. (1974) Biochemistry 13, 323-326. F. (1976) Endocrinology 98, 1444Drouin, J., and Labrie, 1451. Vilchez-Martinez, J.A., Coy,D.H., Arimura, A., Coy, E.J., Hirotsu, Y., and Schally, A.V. (1974) Biochem. Biophys. Res. Commun. 59, 1226-1232. Mendelson, C., Dufau, M.L., and Catt, K.J. (1975) J. Biol. Chem. 250, 8818-8823. Catt, K.J., and Dufau, M.L. (1973) Adv. Exptl. Med. Biol. 36, 379-418. Kirschner, M.A., Wider, J.A., and Ross, G.T. (1970) J. Clin. Endocrinol. Metab. 30, 504-511. Bellisario, R., Carlsen, R.B., and Bahl, O.P. (1973) J. Biol. Chem. 248, 6796-6809. Bahl, O.P. and Swminathan, N. (1973) J. Biol. Carlsen, R.B., Chem. 248, 6810-6827.
862