A Comparative Study of the Mechanism of Action of ... - CiteSeerX

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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