Binding of progesterone and R 5020, a highly potent progestin, to human endometrium and myometrium

BINDING OF PROGESTERONE PROGESTIN,

D.

AND R 50'20, A HIGHLY

TO HUMAN ENDOMETRIUM

and J.P.

PHILIBERT

POTENT

AND MYOMETRIUM

RAYNAUD

Centre de Recherches, Roussel-Uclaf 93230 RomainvilZe, France

ABSTRACT

The in vitro binding of labelled progesterone and R 5020 to receptor protein from human endometrium and myometrium cytoplasmic fractions has been studied. Both compounds give rise to two separate peaks in density gradient analysis experiments : a "7-8s" progestin-specific peak and a "4-5s" peak accounted for by CBG-like and non-specific binding in the case of progesterone, by non-specific binding only in the case of R 5020. The affinity and specificity of labelled R 5020 binding to the "7-8s" component have been determined by equilibrium dialysis on total cytosol. Identical results to those already obtained for the mouse, rat, rabbit and guinea pig have been recorded. The binding components were further characterized by crossed competition studies following ammonium sulfate precipitation.

Accepted

for

NOVEMBER

publication

September

6,

1974 VOL. 10 NO. 5

1974

457

CONTRACEPTION

INTRODUCTION

Since the pilot studies by O'Malley, Sherman and Toft (1) on the chick oviduct in 1970, a progesteronespecific receptor has been identified in the uterus of various mammals including the guinea pig (2), rabbit (3), mouse and rat (4,s). By using a highly potent synthetic progestin to tag this elusive receptor, it has been possible to determine its binding characteristics and establish a similarity in binding affinity and specificity in all these four species (6,7,8). In the present studies it is proposed to demonstrate that a comparable progesti; receptor is to be found in the human uterus.

MATERIALS

AND

METHODS

Steroids [1,2 3H1 Cortisol (36 Ci/mmole) was supplied by the C.E.A., Saclay, France. [l 3Hl Progesterone (27 Ci/mmole), [6,7 3H] 17,21-dimethyl-19-nor-pregna-4,9-diene-3,2O-dione (R 5020) (51 Ci/mmole) as well as the following radioinert steroids : cortisol, progesterone, R 5020, Sa-pregnane20a-hydroxy-progesterone, 3,20-dione (allopregnanedione), 208-hydroxy-progesterone and 13-ethyl-17-hydroxy-18,19dinor-17a-pregna-4,9,ll-trien-2O-yn-3-one (R 2323), were synthesized by Roussel-Hclaf. 13-Ethyl-17-hydroxy-18,19dinor-17a-pregn-4-en-2O-yn-3-one (norgestrel) was also used.

Radioactivity

measurements

Radioactivity was counted on samples dissolved in 15 ml methoxy ethanol : toluene (2:3, v/v) containing in an naphthalene (8%, w/v) and butyl PBD (0.4%, w/v) Intertechnique SL30 liquid scintillation spectrometer with a standard error of counting of less than 1%. All counts were corrected to 100% efficiency by channel ratio.

Preparation

of endometria2

and

myometriai!

cytoso2

Uteri from three women treated with estrogen prior to hysterectomy were used. The endometrium was carefully separated from the myometrium ; the tissues were then washed in a buffer, p1-r7.4, composed of 10 mV Tris-HCl, 1.5 m.I EDTA, 12 mM thioglycerol and 10% (v/v) glycerol, at O'C for 1 hr, homogenized by three 5 set bursts of an Ultra-Turrax (Janke and Kunkel KG, Germany) followed by ten strokes in an ice-cold Teflon-glass homogenizer and centrifuged at 105,000 g for 1 hr. The resulting supernatant is referred to as "cytosol".

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Sucrose

gradient

anaZysis

The cytosol (l/3, w/v) was incubated with labelled steroid in the absence or presence of 100 nM radioinert competitor for 1 hr at 4”C, then layered (0.3 ml) on a S-20% sucrose gradient prepared in homogenization buffer and centrifuged at 45,000 rpm for 16 hr at 4’C in a Beckmann L350 centrifuge using an SW 50.1 rotor. The radioactivity of 2-drop fractions collected from the bottom of the tubes was counted by liquid scintillation. Protein concentration was measured by the method of Lowry et al. (9).

Equilibrium

dialysis

A Nojax dialysis bag containing 1 ml of cytosol was introduced into 15 ml of 0.5 r&l radioactive steroid, 0.5 to 250 nM radioinert steroid in homogenization buffer. After magnetic stirring at 4’C for 24 hr, the radioactivity of two 0.2 samples from inside and outside the bag was determined. The intrinsic dissociation constant and number of binding sites were evaluated as described previously (10). Dialysis was performed on total cytosol (l/SO, w/v) and on precipitates obtained by treating cytosol with ammonium sulfate. Cytosol (l/S, w/v) was precipitated with 40% ammonium sulfate, the supernatant was precipitated by adjusting to 80% ammonium sulfate. Precipitates were taken up in 10 ml homogenization buffer. In competition studies, various concentrations of radioinert steroid were added to a tracer concentration were expressed by (0.5 nM) of labelled R 5020. Results plotting the log of the expression b/(bmax-b) (where bmax = fraction bound in absence of competitor), denoted against competitor concentration. The logit fraction bound, concentration of test steroid required to reduce bound radioactive R 5020 to the value b,ax/2 (i.e. logit fraction bound = 0) was determined. RESULTS

Binding

to human

endometrial

and

myometria2

cytosol

As shown in Figure la, a steroid-receptor complex zone is formed on incubation of sedimenting in the “7-8s” labelled R 5020, a highly potent progestin, with human endometrial cytosol. Addition of either radioinert R 5020 or progesterone results in the suppression of this peak, the radioactivity being recovered in the “4-5s” zone. Labelled progesterone gives rise to two distinct peaks : peak. Labelled cortisol gives a “4-5.5” peak and a “7-8s” a sharp peak in the “4-5s” zone.

NOVEMBER

1974 VOL. 10 NO. 5

CONTRACEPTION

The distribution of radioactivity on incubation of labelled steroid with endometrial cytosol obtained from another uterus is shown in Figure lb. Labelled R 5020 exhibits the same pattern as in Figure la except for slight binding in the "4-5s" zone as witnessed by the small hump at fraction 19. Labelled progesterone gives rise to a "4-5s" peak and a "7-8s" peak. Addition of radioinert progesterone virtually suppresses both these peaks ; radioinert R 5020 and I? 2323 (an anti-progesterone agent exerting moderate competition for the progesterone receptor in the rabbit (11)) displace the radioactivity towards the "4-5s" region, whereas radioinert cortisol, on the contrary, enhances binding in the "7-8s" zone and suppresses the "4-5s" peak. The gradient patterns for myometrial cytosol (Figure 2) show that, as for endometrial cytosol, labelled R 5020 and, to a lesser extent, progesterone are bound in the "7-8s" region. Addition of radioinert R 5020, R 2323, norgestrel or progesterone results in the total suppression of this peak, the radioactivity being recovered in the "4-5s" zone. The displacement of radioactivity away from the "7-8s" peak on addition of radioinert progestins reflects the progestin-specificity of the "7-8s" binding component in endometrial and myometrial cytosol (Figures la,b and 2). Binding in the "4-5s" region is non-specific in the case of R 5020 (Figure 2), but specific for progesterone as demonstrated by the suppression of both peaks on addition of radioinert progesterone and of the "4-5s" peak on addition of radioinert cortisol (Figure lb).

Physicochemical

binding

parameters

The absence in human endometrial and myometrial cytosol of any specific R 5020 binding other than binding receptor has enabled the to the "7-8s" progestin-specific determination of. binding parameters on total cytosol by equilibrium dialysis (7). Results have been recorded in Table I. The value obtained for the dissociation constant (0.5x10-9M) concords well with that already found for the mouse, rabbit and guinea-pig (7,8).

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Table

I. Intrinsic

dissociation

constant II/KS) and number binding sites (iv,) in nM of R 5020 binding to human endometrial and myometrial cytosol (l/SO) as determined by equilibrium dialysis

of

specific

1 /KS

NS

KnsNns::

0.19 + 0.03

Endometrium (lmg protein/ml)

0.45 + 0.04

1.4 k 0.1

Myometrium (lmg protein/ml)

0.60 f 0.15

1.1 f 0.2

:: K,,N,, = non-specific binding from binding curves (10).

deduced

Unmeasurable

mathematically

FRRCTIONS

FRACTIONS

Figure

1. In vitro binding

to human endometria2

cytosol

Following incubation of cytosol with 6 nM labelled R 5020 (a) or progesterone (b), alone _ . or in . the presence ., of 100 nM radioinert competitor, the bound sterotd corny plexes were separated from free steroid by centrlfugatlon of a 0.3 ml cytosol sample (S!" 50.1 rotor) at 45,000 rpm for 16 hr at 4'C in a 5-20% sucrose gradient. The radioactivity of 2-drop fractions was counted and expressed as percent of the total layered on the gradient.

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CONTRACEPTION

+ NORGESTRE

FRRCTlONS ::

Figure

:

labelled

compound,

P = progesterone.

2. In vitro b?:nding to human myometrial Legend as in Figure

cytosoZ

1.

Separation of binding entities by ammonium sulfate precipitation and their characterization The histograms in Figure 3 show that following precipitation of human myometrial cytosol with 40% ammonium sulfate, a high affinity progestin-specific binding component is isolated. Both labelled progesterone and R 5020 bind to this component ; in each case, the radioactivity is displaced by addition of radioinert compound establishing the specificity of the binding. Radioinert cortisol has virtually no effect on bound labelled progesterone thus distinguishing this binding from CBG-like binding. Labelled cortisol binds very slightly, in all likelihood to a contaminating fraction present in the precipitate. The supernatant recovered on precipitation with 40% ammonium sulfate was precipitated by raising the ammonium sulfate concentration to 80%. Labelled progesterone? R 5020 and cortisol bind to this fraction, but R 5020 binding is entirely non-specific, as radioinert R 5020 does not compete either with labelled progesterone nor with labelled R 5020. Radioinert progesterone, on the other hand, competes with labelled progesterone, thus indicating the presence of specific binding. This binding is comparable to cortisol binding since radioinert cortisol competes in a similar manner. These results were confirmed by competition for labelled cortisol binding ; both radioinert cortisol and progesterone compete unlike radioinert ?? 5020.

NOVEMBER

1974 VOL. 10 NO. 5

CONTRACEPTION

!

.

5

! iil ;’ :

i-

Progertaron.*

R

5020*

C0rtk.l’

100 nM radioinert steroid were added to 0.5 nM labelled steroid (indicated by asterisk). Figure

3. Competition for human myometrial cytosol receptors following precipitation with ammonium sulfate.

Competition

studies

Endometrial cytosol precipitated by 40% ammonium sulfate in order to isolate the progestin binding component was used for the determination of competitive effect as illustrated in Figure 4. Steroid concentrations giving rise to a 50% competitive effect have been entered in Table II and the activity of each steroid has been compared to that of progesterone. Only radioinert R 5020 is a more effective competitor than progesterone. R 2323 and allopregnanedione have approximately a third of the activity of progesterone, whereas the progesterone metabolites, ZOa- and ZOB-hydroxyprogesterone, virtually do not compete.

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1974 VOL. 10 NO. 5

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CONTRACEPTION

Figure 4 and Table II. Competition for the human endometria2 cytosol progestin receptor tagged with radioactive R 5020 as measured by equilibrium diaZysis. Results have been represented according to the graphical representation detailed in Materials and Methods and are expressed as concentrations of radioinert steroid giving rise to a 50% competitive effect on the binding of labelled R 5020 (Iso) and as a ratio of concentration of progesterone (P) required to achieve 50% inhibition to concentration of test compound (C) required to achieve the same inhibition.

ISOP IsoC

I50 nM P. 5020 Progesterone Allopregnanedione R 2323 20B-OH-progesterone 20a-OH-progesterone

464

*-a o-o *-* :z; t-t

5 26 63 72 'L 600 %lOOO

NOVEMBER

5.2 1 0.4 0.35 QO.04 $0.025

1974

VOL. 10 NO. 5

CONTRACEPTION DISCUSSION

The above results have confirmed the presence of a “7-8s” progestin-specific binding component in human endometrial and myometrial cytosol (12,13). As in several other mammals, the determination of the binding affinity of progesterone for this component is complicated by the presence of marked specific progesterone binding to a “4-5s” CBG-like rec,eptor (10,14). Several techniques have already been developed to dissociate these two binding systems in order to measure the true affinity of progesterone for each receptor ; numerous others can yet be envisaged. For instance, affinity may be measured on the pooled fractions corresponding to a single peak of a sucrose gradient (7), on the precipitate obtained following treatment of total cytosol with 40% ammonium sulfate, on cytosol in which the CBG-like receptor has been saturated with cortisol or retained on a cortisol affinity column. In the present studies on human uterus, the use of a highly potent progestin R 5020 which is specifically bound by the “7-85” and not “4-5s” progesterone receptor (6,7) was favored for the detection of the “7-8s” receptor. Ammonium sulfate precipitation was then used to isolate this “7-8s” binding and distinguish it conclusively from the specific CBG-like progesterone binding. Results for binding affinity and specificity obtained by these last mentioned techniques (use of R 5020 as a tag and ammonium sulfate precipitation) concord well with those already published for the mouse, rat, rabbit and guinea pig (6,7,8). The same intrinsic dissociation constant is recorded for R 5020, namely, O.SX~O-~M ; the same specificity is observed with regard to R 5020, progesterone and R 2323 and also allopregnanedione (2,s). The similarity in results for different species using different techniques points to the existence of a single progestin-binding entity present in the uterine cytosol of a wide variety of species. The cytosol receptor-steroid the first molecular event in the induction of interaction is therefore the same whatever a progestomjmetic response, the species and consequently the basic structural requirements for a compound with high affinity in man can be deduced with confidence from animal binding studies. However, owing to inter-species differences in pharmacokinetics and the ideal clinically active form of this compound metabolism, can only be determined by clinical pharmacology studies. since there is no species discrimination at the Furthermore, the only fundamental criterion in progestin receptor level, the choice of species for the long-term toxicity of these compounds is that their metabolism in the species selected be close to that in man. ACKNOWLEDGMENTS

We are deeply indebted kindly supplying the uteri. S. Viet for their competent NOVEMBER

1974

to Professor J.P. Gautray for We thank A.M. Tremblet and technical assistance.

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CONTRACEPTION

REFERENCES

1. O'MALLEY, B.W., SHERMAN, M.R. and TOFT, D.O. (1970). Progesterone "receptors" in the cytoplasm and nucleus of chick oviduct target tissue. Proc. Natn. Acad. Sci. USA, 67, 501-508. 2. MILGROM, E., ATGER, M. and BAULIEU, E.E. (1970). Progesterone in uterus and plasma. IV. Progesterone receptor(s) in guinea-pig uterus cytosol. Steroids, 16, 741-754. 3. WIEST, W.G. and RAO, B.R. (1971). Progesterone binding proteins in rabbit uterus and human endometrium. In : Advances in the Biosciences 7, Schering Workshop on Steroid Hormone IIReceptorsII, Berlin 1970, p.251, Pergamon Press, Vieweg, New-York. 4. McGUIRE, J.L. and DeDELLA, C. (1971). In vitro evidence for a progestogen receptor in the rat and rabbit uterus. Endocrinology, 88, 1099-1103. 5. FEIL, P.D., GLASSER, S.R., TOFT, D.O. and O'MALLEY, B.W. (1972). Progesterone binding in the mouse and rat uterus. Endocrinology, 91, 738-746. 6. PHILIBERT, D. and RAYNAUD, J.P. (1973). Progesterone bindSteroids, ing in the immature mouse and rat uterus. 22, 89-98. 7. PHILIBERT, D. and RAYNAUD, J.P. (1974). Progesterone binding in the immature rabbit and guinea-pig uterus. Endocrinology, 94, 627-632. G. 8. RAYNAUD, J.P., PHILIBERT, D. and AZADIAN-BOULANGER, Progesterone-progestin receptors. XIIIth (in press). International Symposium on Physiologic and Genetic Aspects of Reproduction, Bahia 1973. 9. LOWRY, O.H., ROSEBROUGH, N.J., FARR, A.L. and RANDALL,R.J. (1951). Protein measurement with the folin phenol reagent. J. BioZ. Chem., 193, 265-275. 10. BAULIEU, E.E. and RAYNAUD, J.P. (1970). A "proportion graph" method for measuring binding systems. Eur. J. 13, 293-304. Biochem., G. and RAYNAUD, J.P. (1974). 11. SAKIZ, E., AZADIAN-BOULANGER, Ivth International Congress of Endocrinology, Washington Excerpta Medica, Amsterdam. 1972. ICS 273, 988-994. K., JANNE, O., LUUKKAINEN, T. and VIHKO, R. 12. KONTULA, protein in human myometrium. (1973). Progesterone-binding Bfochim. Biophys. Acta., 328, 145-153. 13. MURUGESAN, K. and LAUMAS, K.R. (1973). Binding of 6,7 3H norethynodrel to the rat uterine cytosol and to human endometrium and myometrium. Contraception, 8, 451-470. on Endocrinology, 14. WESTPHAL, U. (1971). In : Monographs Interactions, p.213, Springervoz.4, Steroid-Protein Verlag, Berlin.

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