The FSH secretion-inhibiting action of inhibin in vitro under basal conditions and also in the presence of LH-RH is suppressed by the addition of MIX, a phosphodiesterase inhibitor. In the presence of LH-RH, inhibin reduces significantly the intracellular level of cAMP in isolated pituitary cells. In contrast, the simultaneous addition of MIX and inhibin raises the cAMP level, and this stimulation is comparable to the increase observed when MIX is added alone. These observations suggest that one mode of action of inhibin could be mediated by a reduction in cAMP within the pituitary gonadotropic cell

Charlet-Renard, C.

Franchimont, P.

Hazee-Hagelstein, M. T.

Lecomte-Yerna, M. J.

English

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NASA TECHNICAL MEMORANDUM 	 NASA TM-76847
D
INTRACELLULAR "' ,CHANISM OF T.[E ACTION OF INHIBIN ON
THE SECRETION OF FOLLICULAR STIMULATING HORMONE AND
OF LUTEINIZING HORMONE INDUCED BY LH-RH IN VITRO
M. J. Lecomte-Yerna, M. T. Hazee-Hagelstein,
Ch. Charlet-Renard, and P. Franchimont
JUASA -Tll-/bb4 /) IbTt( ALJSLLUL Ab MELUANISA UY	 NdZ- 2Zdb`!
T6E ACTION OF tNHIBIN ON IEE SEC8E1'IGb C^ llO
d^
^l`i'^ 
A01FOLLICULAR STIMULATING WEMCNE AND OF 
LUTEINIZIbG dOBECNE INLUCkE BY LH-Rh I	 inclas
VITRO (National Aeronautics and Space 	 G3/51 09031
Translation of "Un mecanisme intracellulaire de f action de
l'inhibine sur la secretion de 1'hormone folliculo-stimulante
et de l'hormone luteinisante induite par le LH-RH in vitro,"
Comptes Rendus Des Seances de la Societe de Biologie et
de Ses Filiales, Vol. 175, No. 3, 1981, pp. 385-388.
ti
NATIONAL AERONAUTIC: AND SPACE ADMINISTRATION
'r; AS HINGTON , D.C. 	 20546	 APRIL 1982
Leo Kanner Associates
Redwood city, CA, 94063
13. Type of Report and Period Coverer+
ORIGINAL PAGE IS
	
OF POOR (QUALITY	 1
47etI,)ARP 'V 17 r 0A"
1. Report No.	 ^2. Govarnment Accession No.	 } t ' k 	 r	 1	 N.,_
NASA TM-76847
title	 5. Roaar, Dore1. Titl• end Sub INTRACELLULAR MECHANISM OF THE 	 I	 April 1982
ACTION OF INHIBIN ON THE SECRE'T'ION OF FOLLICL)TAR--- -
	 -- - -
STIMULATING HORMONE AND OF LUPEDJIZING HORMONE
INDUC T,^ BY T H RH Tip to n	 _	 -	 ^. - -- - -
7. Author(s)
	
3. Par;c..-.:np :;o_Aa^r:n Repo,,
M.J. Lecomte-Yerna, M.T. Hazee-	 I
Hagelstein, Ch. Charlet-Renard and	 ;10. ;,ark Unit No.
P. Franchimont	 M
- 4_ Pertorm : ng Crgan: Aorton Name and Address 	 ^11.—Cantrocr or 
vrcnl No.
NAS11-3541
12. 5ponsoring Agency Name and Address	 Translation
National Aeronautics and Space Administration ^u. Spon s or i ng
 4^,en:y cad,
Washington, D.C., 20546
	 1
15. 5upplementaryNotes Translation of " Un m6canisme intracellulaire de faction
de 1 1 inhibine sur la secretion de 1 1 hormone folliculo-stimulante et de
l t hormone luteinisante induite par le LH-RH in vitro," Comptes Rendus Des
Seances de la Soci6te de Biologie et de ses Filiales, Vol-175, No. 3,
1981, pp .
 
385-388
16. Aestr,ict The FSH secretion-inhibiting; action of inhibin in vitro under
basal conditions and also in the presence of III-RH is suppressed by the
addition of MIX, a phosphodiesterase inhibitor. In the presence of LH-RH,
inhibin reduces siMificantly the intracellular level of cAM1P in isolated
pituitary cells. In contrast, the simultaneous addition of MIX and inhibin
raises the cAMP level, and this stimulation is comparable to the increase
observed when MIX is added alone. These observations suF,gest that one mode
of action of inhibin could be mediated by a reduction in cAPT within the
pituitary gonadotropic cell.
17, Key Words ( Selected by AuthOr(s)) 	 ; t8, Dufn:. l,on Statement	 ^f
Unclassified Unlimited	 I
19. Sesvr!ry Closni ioi r ,e rep rr)	 econry CI r;: s. ;o.	 " s ; ;e	 tea. u! i'a,e;	 [2.
L =Unclassified	 Unclassified 	 6	 i
rt ACA sin
INTRACELLULAR MECHANISM OF THE ACTICY OF INHIBIN ON
THE SECRETION OF FOLLICULAR STIMULAT,:NG HORMONE AND
OF LUTEINIZING HORMONE INDUCED BY LH-RH IN VI'T'RO
M. J. Lecomte-Yerna, M. T. Hazee-Hagelstein,
Ch. Charlet-Renard, and P. Franchimont
Radioimmunology Laboratory, Faculty of Medicine, University of Liege
C.H.U. 823, Sart Tilman, 4000 Liege 1, Belgium
Summary
The FSH secretion-inhibiting action of inhibin in vitro under
basal conditions and also in the presence of LH-RH is suppressed by
the addition of MIX, a phosphodiesterase inhibitor. In the presence
of LH-RH, inhibin reduces significantly the intracellular level of
cAT4P in isolated pituitary cells. In contrast, the simultaneous ad-
dition of MIX and inhibin raises the cAMP level, and this stimulation
is comparable to the increase observed when MIX is added alone.
These observations suggest that one mode of action of inhibin could
be mediated by a reduction in cANP within the pituitary gonadotropic
cell.
Introduction
Inhibin is a proteinaceous hormone produced by the Sertoli cells
of the testicle and by the cells of the granulosa of the ovary. It
acts on the pituitary level where it specifically reduces synthesis
of follicular stimulating hormone (FSH) under basal conditions, and
preferentially reduces liberation of r H by the hypothalamic releas-
ing factor (LH-RH) [1). We have undertaken a study of the intracel-
lular mechanisms through which inhibin exercises its action. In this
note we will describe the effect of inhibin on the secretion of Bona- /186
dotropins by isolated pituitary cells in culture and on their cAMP
level in the presence and absence of a phosphodiesterase inhibitor.
Material and Methods
Culture of Pituitary Cells
umbers in the margin indicate pagination in the foreign text.
1
This method has been described i-.i detail in a preceding pub-
-	 lication [1]. We succintly restate the stages of it and the par-
ticular methods of this study.
The pituitaries are removed from adult Wistar rats and the
cells are broken up by the method of Hopkins and Farquhar using
trypsin [2]. The broken cells are placed in suspension in "Dul-
becco Modified Eagle's Medium" (DMEM) containing 5% horse serum,
2.5/ fetal calf serum, 1% glutamine, and to nonessential amino
acids. The cells are distributed in 60 mm diameter culture boxes
at the rate of 0.5 x 106 cells per box and incubated at 37°C for
3 days in a water-saturated atmosphere constituted of 95% air and
5% CO2 . After this period the substances under investigation are
added to the culture. The total incubation volume is 1.6 ml.
Incubation is continued for 24 hours. At the end of this period
the culture media are collected for measuring the secretion of
FSH and LH. The cultures are rinsed with fresh medium and set to
incubate for another 6 hours in DMEM with the same substances de-
scribed above, but with the presence of LH-RH at a concentratio.i
of 10- 8 M. At the end of these 6 hours of incubation the culture
media are gathered. Water (1.6 ml) is added to the cells that
are still congealed. After decongelation the soluble cellular
contents are collected by centrifugation and used (after dilution
k
`	 by half) for measuring cyclic AIvT. Each substance or group of
I
	
	
substances is studied in five different cultures of isolated pi-
tuitary cells.
Tested Substances
The preparation of inhibin used was extracted from the li-
quid of the rete testis by filtration on Sephadex G100 (Pharmacy)
(Pic 1A). Its molecular weight is greater than 10,000 daltons
[3]. This preparation was investigated at the rate of 10 µg/ml
of culture medium.
3- isobutyl-l-methyl-xanthine (VIX). MIX possesses the prop-
erty of inhibiting phosphodiesterase activity and therefore per-
2
- .. . ..
	 - . -
mits an accumulation of cAMP within the cell. The concentration
investigated was 0.2 mM.
Measurements
FSH and LH were measured in the culture media by a radioim-
munological method using the material graciously supplied by
NIADIDD, whom we thank. The results were expressed in ng of hor-
mone per ml of medium.
cAMP way also measured b-.\ a radioimmunological method [41.
Separation of the antigen 'r._d in antibodies from free antigen is
carried out by the technique of double precipitation. The re-
sults were expressed in pmol/ml.
Results
Under basal conditions, inhibin significantly reduces secretion
of FSH. MIX has no effect when it is used alone, but it hinders cne /18Z
inhibitory action of inhibin on FSH (Table 1).
At the time of stimulation by LH-RH, liberation of FSH and LH is
more significant in 6 hours than the basal secretion in 24 hours.
Liberation of FSH is significantly reduced in the presence of inhibin,
but this effect disappeared when inhibin was incubated with MIX (Tabl--
2). Inhibin does not change the secretion of LH under base conditions
nor under the influence of LH-RH.
The intracellular level of cA^Tl (Table 2) was significantly re-
duced in the presence of inhibin. In contrast, in the presence of MIX
the intracellular level of CAMP was significantly increased.
The simultaneous addition of MIX and inhibin to the culture me-
dium brings about an increase of the intracellular level of CAMP in
comparison to standard cultures and to those treated with inhibin.
3
Discussion	 /^88
Inhibin would seem to operate on the pituitary gonadotropic cell
through specific intermediate receptors, different from those of LH-RH
C5,6]. This activation leads to a reduction in production of intra-
cellular cAPP, usually induced by LH-RH C73, at the same time as a re-
duction in liberation of FSH.
MIX, which is an inhibitor of phosphodiesterase, hinders the deg-
radation of cAMP C83. It accompanies an accumulation of cAMP within
the pituitary cells, which persists in the presence of inhibin. One
can therefore think that inhibin operates in reducing the amount of
cAMP formed under the influence of LH-RH. The concentration of the
intracellular level of cAMP under base conditions has not yet been de-
termined. Nevertheless, observe-'ion of an inhibitory effect of inhi-
bin on the basal secretion of Y H, and of its disappearance in the
presence of MIX, brings to mind the possibility that inhibin operated
in reducing the production of cAMP.
With the objective of defining the intracellular mechanism of
action of inhibin, studies are underway to demonstrate a possible ac-
tion of inhibin on the different enzymes implicated in the metabolism
of cAlel.
lw ork subsidized by grant number 3.4501.80 of F.N.R.-S.
4
ORIGINAL PACE 19
OF POOR QUALITY
TABLE 1. SPONTANEOUS SECRETION
OF FSH AND LH IN CULTURE MEDIUM
Treatment FS111'0Ki1111 LII ('1nKlml
^VIA11hU-F^Illtl'^1 ^V1.1^i1^h
	 1.11	 111'^i
S tandnrd 524 ± 41 574 7t- 172
Inhibin	 10 µg 403 ± 25('') 551 ± 125
At IX 0.2 mM 493 -±- 35 572 ± 105
MIX 0.2 mM
+ Inhibin	 10 µg 470 ± 37 672	 114
(#} Mean + 1 standard deviation
() p < 0.05
TABLE 2. SECRETION OF FSH ANR LH IN CULTURE MEDIUM
IN THE PRESENCE OF LH-RH (10- M) AND CORRESPONDING
INTRACELLULAR CONCENTRATIONS OF CYCLIC AMP.
FS11 ('1
	
1,11('	 ^r ^p
n K/ml	 ngjml	 Mdi'tnl I1Treatment iN'IAMD-FSII-IIP,)
	
IXIaMD - LH-HPI)
Standard 1033 ± 65 3334 :t 299 0.129 ? U,026 (a)
Inhibin	 10;tg 832	 71(") 3055 ± 606 0.096 i- 0.024(b)
MIX 0 92 mM 1070 ± 54 3559 ± 283 0.214 ± 0.042 (c)
Inhibin	 + MIX 1073 ± 78 3720 t 194 0.173 --- 0,028 (d!
(#) Mean + 1 standard deviation
(#) p < 0.05
a versus b and a versus d: p< 0.025s a versus c:
P < 0.005; c versus d : not significant; b versus c
P < 0.0005: b versus d : p < 0.0005.
w
5
^j
REFERENCES
1. Franchimont, P., J. Verstraelen-Proyard, M.T. Hazee-Hagelstein,
Ch. Renard, A. Demoulin, J.P. Bourguignon, and J. Hustin, Vit.
orm. .2Z, 243 (1979) •
2. Hop':ins, C.R. and M.G. Farquhar, J. Cell. Biol. 2, 276 (1973)•
3. Franchimont, P., A. Demou1in, J. Verstraelen-Proyard, M.T. Hazee-
Hagelstein, J.S. Walton, and G.M.H. Waites, Int. J. Androl.
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4. Steiner, A.L., C.W. Parker, and D.M. Kipnis, J. Biol. Chem. ,g4,
1106 (1972)•
5. Sairam, M.R., M.R. Ranganathan, and P. Lamothe, Biol. Re -prod. 18
Suppl. 1. 58 (1978)•
6. Labrie, F., J. Massicotte, M. Godbout, L. Lagace, V. Giguere, and
J.H. Dorrington, Proceedings of the II Pan American Con gress of
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7. Labrie, F., P. Borgeat, N. Barden, M. Godbout, M. Beaulieu, L.
Ferland, and M. Lavoie, "Polypeptide Hormones", Beers, R.F., Jr.
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6


Intracellular mechanism of the action of inhibin on the secretion of follicular stimulating hormone and of luteinizing hormone induced by LH-RH in vitro

Intracellular mechanism of the action of inhibin on the secretion of follicular stimulating hormone and of luteinizing hormone induced by LH-RH in vitro

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