Aug 20, 1982 - GnRH agonists share a common mechanism of action on follicle-enclosed oocytes. Alternatively, experiments were conducted to study whether.
BIOLOGY
OF REPRODUCTION
A Comparative
28,
Study
161-166
of the Mechanism
and a Gonadotropin N.
(1983)
Releasing I. SHERIZLY,
DEKEL,’
of Action Hormone
of Luteinizing
Analog
A. TSAFRIRI
Department The Weizmann
of Hormone Institute
Rehovot
Hormone
on the Ovary
and
Z. NAOR
hormone
(GnRH)
Research of Science
76100,
Israel
ABSTRACT The
mechanism
of
action
of a gonadotropin ovary has been studied
releasing
agonistic
analog
(ED-Ala’] GnRH) on the rat in comparison to similar effects of luteinizing hormone (LH). Stimulation of meiosis resumption in vitro in follicle-enclosed oocytes by both LH and ED-Ala’ ] GnRH, was blocked by elevated levels of cAMP as demonstrated when either dibutyryl cAMP or the phosphodiesterase inhibitor methylisobutylxanthine was present in the culture medium. In vivo, the prostaglandin synthase inhibitor indomethacin, which blocks LH-induced ovulation, also inhibited ovulation induced by the GnRH analog in hypophysectomized rats. On the other hand, the potent GnRH-antagonist ED-pGIu’, pClPhe2, D-Trp3’#{176}IGnRH which blocked the stimulatory effect of the agonist on oocyte maturation and ovulation had no effect on LH action. It is concluded that while a GnRH-like peptide does not seem to mediate LH action on the
ovarian follicles, both post-receptor locus.
LH
and
GnRH
agonist
share
INTRODUCTION
Direct hormone both
effects (GnRH)
of and
stimulatory
ovary
have
vitro
in
inhibitory,
and been
review see stimulatory
gonadotropin-releasing. its agonistic analogs,
recently
on
the
demonstrated
Hseuh and Jones, effects, resumption
follicle-enclosed
1981).
oocytes
(for
lating
hormone
mone
(LH)
and
on
and
and
LH-induced
Jones,
sites
are
al.,
1979)
tracted
prolactin
and thus follicular
present and
from
not yet implications
ovarian
1981).
rat
clear for
Since GnRH the effect of prostaglandin
weight
Although in
ovarian
tissue
a GnRH-like ovaries whether normal
(Ying these gonadal
et al.,
(Hseuh receptor
(Clayton
peptide
was
1981),
findings physiology.
was
under-
and
GnRH
GnRH-like action
in the coordination and function.
AND
et ex-
Organon,
it is
Holland)
(D-Ala’
have
CA)
I GnRH
was
or
of
METHODS
a dose
(GnR}la,
given
24
of
Peninsula
h later.
hypophysectomized
rats
500 ng per rat of Lab., San Carlos,
A second was
injected
group
of
with
one
the of
these hormones in combination with either 5 ig per rat of a GnRH-antagonist [D-pGlu1, pCIPhe2, D-Trp34)GnRH (kindly provided by Dr. D. Coy, Tulane University, New Orleans, LA) or 2 mg per rat of indomethacin (1-(p-chlorobenzoyll -5-methoxy-
and its analogs seem to mimic LH on the ovary in terms of formation, oocyte maturation
2-methylindol-3-acetic
Israel). Accepted Received
LH
at a
Hypophysectomy was performed on mature Wistar proestrous female rats and followed by a subcutaneous injection of 15 IU of pregnant mare’s serum gonadotropin (PMSG, Gestyl, Organon, Holland) in OJ ml of 0.9% NaCI. PMSG treatment was previously shown to preserve the majority of the preovulatory follicles in hypophysectomized rats (Braw et a!., 1981). An intraperitoneal injection of either the indicated dose of human chorionic gonadotropin (hCG, Pregnyl,
hor-
gain
study
substance with the gonadotropin
MATERIALS
by
formation,
GnRH
present
participate development
Corbin
luteinizing receptor
pathways
whether
and
1981,
on
the
investigate
(Hillensjt$
Inhibition follicle-stimu-
(FSI-I)-induced
to
the locally produced activity may mediate
a!.,
et included.
ovulation,
taken
the in
sectomized
(Ekholm
and
mechanistic
Among of meiosis
formation (Clark of both oocyte vivo in hypophy-
rats
common
agonists share a common mechanism of action on follicle-enclosed oocytes. Alternatively, experiments were conducted to study whether
rat
LeMaire, 1980), prostaglandin et a!., 1981) and stimulation maturation and ovulation in and Bex, 1981), are GnRH is demonstrated
some
August 20, 1982. May 12, 1982.
h
after
examined completeness
Correspondence.
161
The
acid,
rats
were
injection.
for
killed
The
Assia
oviducts
the
presence
of
hypophysectomy
Chemical disolaction were removed
by cervical
of ovulated was
oocytes. checked
Labs.,
20 and
The by
162
DEKEL
ET AL.
ca
RESULTS
>
In
(9
effect
the
first
part
of
LH
and
meiosis
5
in
of
this
> 0 0
GnRHa
follicle-enclosed
induced
0
manner
4-
with
2x109
a)
by
a maximal
M)
was
0
M.
an The
0)
by
0
IO” [GnRHa]
108 (M)
LH
is
oocytes
1. Dose
dependency
of
GnRHa
effect
on GV
to
breakdown in follicle-enclosed oocytes. Isolated follicles were incubated in the indicated concentration of GnRHa for 17 h. Cumulus-oocyte complexes were recovered and the oocytes were examined for the presence of GV’s as described in Materials and Methods. The numbers associated with the graph represent the amount of oocytes examined at each experimental
GnRH,
1).
GV of
in
while than for
meiotic
addition
of
50%
of
division
the
(Fig. GnRH
D-Trp3’6]
i0
effect this
8 h
3 h, a 5-h 95% of the
a potent
of
stimulatory
by
responded breakdown
DpClPhe2,
a concentration
(i0’
oocytes
oocytes of GV
faster;
of
breakdown
GnRHa
of the
their
meiosis
a concentration
dose
However,
com-
dose-dependent
after less was enough
resume
the
a
of
[D-pGlu’,
at
blocked (Table
at
in 93%
Concomitant
antagonist, FIG.
ED50
relatively
responded period
of
in
50% of the 2). Induction
oocytes incubation 2).
As
resumption
effective
of incubation; after 4 h (Fig.
of
was
GnRH-antagof both these shown in Fig. 1,
stimulation
completed
vitro
a potent action
oocytes
0
in
resumption
oocytes
the
follicle-enclosed
the
on
pared and the effect of onist on the stirnulatory hormones was examined.
C’,
study
GnRHa
antagonist
M, of
totally GnRHa
failed
to
point.
>
I00 -c 4-
S C,,
examination of the sells turcica and the peripheral serum was assayed for the presence of LH. Follicles were dissected from 26-day-old female rats injected subcutaneoulsy with 15 IU of PMSG, and killed by cervical dislocation 48 h later. Groups of 50 individual follicles were incubated in 2.5 ml of Leibovitz’s L-15 tissue culture medium (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (Sera-Lab, England), penicillin (100 units/rn!) and streptomycin (100 Mg/ml) (Gibco), in the presence or absence of the indicated concentrations of the following agents: GnRHa, GnRH antagonist, ovine LH (oLH, NIH LH S-20), dibutyryl 3’:5’-cyclic monophosphate (dbcAMP, Sigma Chemical Co., St. Louis, MO) or methylisobutyixanthine (MIX) (Sigma Chemical Co.). Incubations were carried out in a N2 :03 (1:1) atmosphere in an oscillating bath at 37#{176}C. At the indicated time the follicles were incised and the cumulus-oocyte complexes were recovered. The oocytes were examined using an inverted microscope equipped with Nomarski interference contrast optics. Resumption of meiosis was indicated by the absence of the germinal vesicle (GV) in the oocyte. For each study, the data of several individual experiments were combined and the results are reported as the fraction of oocytes with GV breakdown (GVB).
0
0 0 9-
0 0)
0 4-
C a) 0
a) 0
Hours FIG.
2. Time
course
in of
GV
Culture breakdown
in follicle-
enclosed oocytes. Isolated follicles were incubated in the presence of either GnRHa (10’ M) or oL}1 (5 ng/ml) for the indicated times. Cumulus-oocyte complexes were recovered and the oocytes were exainined for the presence of GV’s. The numbers associated with the graph represent the amount of oocytes examined
at each
experimental
point.
GnRH
ACTION
ON
I
‘
108
-
I +GnRPI
-
in follicle-enclosed
GnRHa
8060
/
in
7
113
FIG.
I 0.1
soluble
dibutyryl
derivative
or the present
phosphodiesterase in the culture
(pg/ml)
experimental
inhibit
cAMP
breakdown
stimulated effectively
TABLE 1. follicle-enclosed
antagonist
Effect
in
by
LH
of GnR}l oocytes.
antagonist,
Additi
GnRHa injection
on of
GnRHa
(500
of in
MIX
as of
low the
vivo
(Fig.
rats
were to of
the
induced
effect
by
of
the by
demonstrated dose of hCG lation. On the
revealed
zM
totally effective in
the
examine both LH
used
and and
As seen in (either 2 or ovulation
hypophysectomized
blocked inhibition
4)
as 50 maximal
study effects
ovulation. either hCG ng)
levels totally stimulatory
A dose-dependent
of
this
(Tsafrir,
Fig. 3). matu-
in the
and
MIX
on
et al.,
LH
PMSG-
completely
antagonist antagonist
(5 j.tg), could
1973)
could
or
also rats
syntheovulation
block
(Table
GnRHa-induced
GV
to Mediuma
ovulation 3).
breakdown
in
GVB
GNRHa (10 M)
antagonist
dbcAMP
MIX
Percentage
Total no. of
(10
(5 mM)
(0.2 mM)
of oocytes
oocytes
-
-
-
-
-
+
-
-
-
-
-
+
-
-
-
-
-
+
-
-
-
-
-
-
+
-
+
+
-
-
+
-
+
-
-
-
i.
-
-
+
-
-
+
(0.1
tzg/ml)
1-
M)
-
+
-
+
-
+
-
-
+
-
alsolated with or without for the presence
follicles
were
incubated
either GnRH antagonist, or absence of GVs.
in the
no be
even when the minimal effective (2 EU) was used to induce ovuother hand, indomethacin, which
GNRH
0LH
2, or
However,
was
GnRHa
GnRH GnRH
Table 4 IU) in
rats.
GnRHa-stimulated
either
the
of cAMP
is an effective inhibitor of prostag!andin sis and is known to block LH-induced
dbcAMP
ons
part the
while
follicle-enclosed
(Table 1 and LH-induced
blocks
effect
GV
either
of cAMP as GnRHa
maturation.
the
second compare
treate4
point.
GV
oocyte
of GnR}l
oocyte of
of
can of
inhibitor MIX was As seen in Table
medium.
modulators LH as well on
et al.,
incidence
membrane
doses of both hormones. Hypophysectomized
1.0
on LH-induced maturation in follicle-enclosed oocytes. Isolated follicles were incubated in the presence of the indicated concentrations of 0LH with or without GnRH antagonist (10 M) for 17 h. Cumulus oocyte complexes. were recovered and the oocytes were examined for the presence of GV’s. The numbers associated with the graph represent the amount of oocytes examined at each
3. Effect
the
when
that a concentration prevented the action
001I
[oLH]
system, monitored
analysis
/
this
(Dekel
was
action
Antagonist 127,
oocytes
whether this nucleotide the stimulatory action
breakdown
1, both blocked
40200d
163
1981). To examine also interfere with
72
//
OVARY
ration if
too
THE
presence
dbcAMP
of the or MIX.
indicated After
2
65
93 97
96 149
3
33
9 11
47 56 79
99 13 1
concentrations
20 h the
oocytes
5
42
4
45
of either were
70 70
recovered
0LH
or GnRHa
and examined
DEKEL
164
ET AL.
oocyte maturation hypophysectomized have -C
shown
and ovulation rats. In
that
a
GnRH
in vivo, addition,
antagonist
in we
which
a)
effectively blocked these stimulatory actions of GnRHa on the ovary failed to interfere with LH action. However, both hormones were inhibited
> 0
in vitro
0
when
(‘3
0
seems
It
‘4-
0 0)
of
by elevated prostaglandin
the
g!andin
0
as
0.I
[MIx]
as
completely in vivo
(mM)
cultured
The
rats,
a!.
(5 Mg/mI) or GnRHa (10’ M) for 17 h. Cumulusoocyte complexes were recovered and the oocytes were examined for the presence of GV’s. The numbers associated with the graph represent the amount of oocytes examined at each experimental point.
influence
and Bex, a GnRH
1981) agonistic
we have analog
the resumption oocytes in vitro,
TABLE
of as
2. Effect
Hormone
of GnRH
of
GnRH hCG hCG GnRIl hCG bCG GnRH
(4 (4 (2 (2
in follicle-enclosed as a trigger of
antagonist
on
either
study
that of
antagonist
implies
et
under
the
as yet.
the
were
1981).
Ekholm
occurs direct
action
followed
of
by
the
tissue-specific
sites
that
operate mechanism
in the
induced
maturation
both
is blocked
GnRHa
or hCG-induced
ovulating (500 ng) (500 ng) antagonist lU) IU) + antagonist IU) IU) +
a!.,
by
stereospecific,
gonadal
et
which
in PGE synthesis in the GnRH-
clear of
cells
1982),
(Clark
Our
for
the
and
other
hormone reports
(Behrman et a!., 1980) suggest that GnRH does not share its receptors with LH, since a potent GnRH antagonist blocked GnRH but not LH actions on the ovary. Nevertheless, our present
Fraction
administered
(dose/rat) GnRHa GnRHa
meiosis well
meiosis
demonstrations
et
the
in our study, clearly the mechanism by
is not
gonada!
et a!., cells
originally
of
of GnRH
characterization
reports (Hillensj#{246} and et al., 1981; Corbin also demonstrated is a potent inducer
the rise ovulation
this
on
of
indomethacin,
reinitiation
The
DISCUSSION earlier Ekholm
that
which
GnRH
response
a result
(Ekholm
supports
(Clayton
to 1980;
is
confirmed idea. However,
and
high-affinity
Similar LeMaire,
fact
as shown
(1982)
ovulatory
granulosa
abolished blocked
also
treated
FIG. 4. Effect of MIX on LH- or GnRH-induced maturation in follicle-enclosed oocytes. Isolated follices were incubated in the presence of the indicated concentrations of MIX with or without either oLH
in vivo
in
1981).
the
GnRHa
in vivo
of this hormone on prostawhich has been demonstrated
ovaries
well
a!.,
that to
effect synthesis,
in whole
a)
likely
follicles
stimulatory
0 C
levels of cAMP and synthesis was blocked.
these
two
hormones
follicle via a related at a post-receptor
by
in follicle-enclosed
cAMP
(Dekel
may
or common locus. LH-
et a!.,
oocytes 1981),
as well
in hypophysectornized
ovulation
of
rats.a
Oocytes/ovulating
rat
rats
6/6
8.3
(5 zg)
1/7 4/5
9.0 11.0
(5 Mg)
5/6 5/8
12.0 8.8
(5 tag)
10/14
8.4
+
5GnRH antagonist (5 zg) was injected concomitantly with the indicated hypophysectomized mature rats. The oviducts were isolated and examined 20 h after the injection.
doses for
the
of either presence
GnRHa of ovulated
or hCG oocytes
to
GnRH
TABLE
3. Effect
Agent
of indomethacin
on
ACTION
ON
GnRHa-induced
GnRHa
(500 (500
by
(2 mature
mg)
rats.
was
injected
The
caused
oviducts
were
with
isolated
and
by
the
1976). elevated
presence
inhibitor GnRHa
a complete inhibitory a phosphodiesterase GnRH action, agree
which demonstrates phosphodiesterase is
that GnRH hibition of decreasing cAMP
direct
MIX, action.
or
not only It seems
GnRH in cultured
activity Catt,
1981).
blocked may
by
examined
dose for
the
we
cAMP
somatic
of the hormone as an outcome of
cells
of
the
Whether
GnRH
follicle.
for
(500
ng)
presence
of GnRHa
of
to hypophy-
ovulated
oocytes
has
dogenous
GnRH
However,
since
possible
the
on
is open role
20
h
possible regulation
of
still
that the is much
be time more
origin.
studies suggest the of a GnRH-like peptide(s) cells (Ying et al., 1981), a
ovarian role
hypothalamic
for oocyte considered.
the
GnRH-lIke maturation Our
factor and
present
can
serve
However,
release findings,
course of GnRH action on meiosis retarded than that of LH1 suggest
the
to inhibit maturation as
a
mediator
fact
that
the
the and
LH ovu-
for
this
that
the
support
it is still
possible
gonadal peptide is similar but not identical to the pituitary GnRH. Thus, the antagonist to the pituitary hormone may not interfere with the of
action
the
ovarian
to
the
of
or not,
obscure
Until
a specific
GnRH-like
substance
question remains open. a GnRH-like substance
regulator
to
ligand.
ovarian
get
it
an of
the
ovarian still
insight
acts
function
can
provide
into
the
mammalian
and a very
as useful
mechanism oocyte
a
develof
which
is
as yet.
ACKNOWLEDGMENTS
This
was made possible in part by funds granted by the Charles H. Revson Foundation to N. D. and Z. N. and by The Israel Academy of Science and Humanities to N. D. This work was supported in part by grants from the Ford Foundation and the Rockefeller Foundation to H. R. Lindner. study
in
recent
production
the
of
lation,
maturation
effect
a physiological
failed oocyte
also
as a local
addition,
antagonist on both
opment
into
act
cannot
In
GnRH effects
suggest
with
action
action.
means
Determination
GnRH
hormone
LH
local
by leading
its
this
for
oocyte
Since
cAMP,
that
is used, this Whether
the control of ovarian function is still not clear. Although the gonadal binding sites to GnRH fulfill all the requirements of classical hormone receptor interaction, the ovary is normally not exposed to effective concentrations of en-
may
ovulatory
antagonist
increases granulosa
induce maturation accumulation
of the cellular target for further investigation.
by
an
speculation.
cAMP levels in the oocyte. A drop in levels in the oocyte can be a result of a interaction as well
gamete,
dbcAMP
effect. Our findings, inhibitor interferes with a recent study
that
and
maturation
either
study cAMP
effect of MIX in our study is to its inhibitory influence on activity, since relatively low (IC50 =30 I.LM) of this agent ex-
concentrations
(Knecht
et
The present levels of of
also
likely that the attributed solely phosphodiesterase
(Tsafriri
inhibitors
Hi!lensj6, that
the phosphodiesterase inhibit LH, but
the
concomitantly
injection.
phosphodiesterase
cells
7.0
0/8
(2 mg)
a!., 1972; demonstrates
with
rat
+
alndomethacin sectomized after the
Oocytes/ovulating
rats
3/3
ng)
ng)
indomethacin
rats.a
of
ovulating
GnRha
165
in hypophysectomized
Fraction
administered
hibit that
OVARY
ovulation
(dose/rat)
as
THE
in
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