The antiovulatory effect of the antiprogestin onapristone could be related to down-regulation of intraovarian progesterone (receptors)

.7. Steroid Biochem. Molec. BioL Vol. 62, No. 1, pp. 107-118, 1997

Pergamon

PII: S0960-0760(97)00012-5

© 1997 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0960-0760/97 $17.00 + 0.00

The Antiovulatory Effect of the Antiprogestin Onapristone Could Be Related to D o w n regulation of Intraovarian Progesterone (Receptors) j. D o n a t h , ' H . M i c h n a 1'2 a n d Y. N i s h i n o 1. 1Research Laboratories of Schering AG, Berlin Miillerstrasse 170, 13342 Berlin, Germany and 2Present address: Department of Morphology and Tumor Research, DSHS Cologne, Carl-Diem-Weg 6, 50933 Cologne, Germany

T h e p r e s e n t s t u d y was u n d e r t a k e n to investigate i n t r a o v a r i a n m e c h a n i s m ( s ) for the a n t i o v u l a t o r y effect o f O n a p r i s t o n e (ON), b e c a u s e a n t i p r o g e s t i n s possessing the s a m e a n t l p r o g e s t a t i o n a l activity a n d in h ib itin g the p r e o v u l a t o r y L H s ur ge to t he s a m e ext ent differ in t h e i r a n t i o v u l a t o r y p o t e n c y . O v u l a t i o n was i n d u c e d by t r e a t i n g i m m a t u r e f e m a l e rat s with p r e g n a n t m a r e s e r u m g o n a d o t r o p i n ( P M S G ) f o r folliculogenesis a n d h C G for t he i n d u c t i o n o f ovulation. T h e a n i m a l s w ere t r e a t e d twice with ON (200 mg/kg 42 h a n d 48 h a f t e r P M S G ) a n d killed at d i f f e r e n t t i m es. T h e ovul at i on r a t e was assessed b y c o u n t i n g t he n u m b e r o f ova in t he fallopian tubes a n d uteri. Bl ood a n d ovaries w er e coll e cted f o r r a d i o i m m u n o a s s a y (RIA) o f s t e r o i d h o r m o n e s a n d histological analysis for 3]~-hydroxys t e r o i d d eh y d r o g e na . ; e (3]~-HSDH), 1 7 ~ - h y d r o x y s t e r o i d d e h y d r o g e n a s e (17~-H SD H ), p r o g e s t e r o n e (PR), e s t r o g e n (ER) :and a n d r o g e n (AR) r e c e p t o r s . T r e a t m e n t with ON totally bl ocked ovul at i o n a n d the p r o g e s t e r o n e ( P 4 ) surge was significantly d i m i n i s h e d in c o m p a r i s o n to the c o n t r o l (6-8 h posth C G ) , w h e r e a s a n d r o g e n levels r e m a i n e d u n a f f e c t e d . T h e d e c r e a s e d P 4 c o n c e n t r a t i o n s c o r r e l a t e d well with a r e d u c e d staining i n t e n s i t y o f 3/~-HSDH in g r a n u l o s a ceils o f t e r t i a r y follicles. M o r e o v e r , we o b s e r v e d a d o w n - r e g u l a t i o n o f P R in g r a n u l o s a cells o f t e r t i a r y follicles. Additionally, in s e c o n d a r y a n d t e r t i a r y folliicles t he e x p r e s s i o n o f AR b e t w e e n 0 a n d 6 h a f t e r h C G was r e d u c e d . T h e s e results suggest t h a t the a n t i o v u l a t o r y effect o f the a n t i p r o g e s t i n ON is r e l a t e d to d o w n - r e g u l a t i o n o f i n t r a o v a r i a n p r o g e s t e r o n e , c a u s e d by a t t e n u a t e d 3 ~ - H S D H activity a n d P R expression. O ne c a n thus a s s u m e t h a t i n t r a o v a r i a n P 4 is an i m p o r t a n t f a c t o r for the i n d u c t i o n o f ovulation. An effect o f ON on th e staining i n t e n s i t y o f 1 7 ~ - H S D H in t h e c a a n d g r a n u l o s a cells could n o t be o b s e r v e d at a n y t i m e. In co n clu s i on, t he i n h i b i t i o n o f ovul at i on i n d u c e d by the a n t i p r o g e s t i n , ON, could be r e l a t e d to d e c r e a s e d i n t r a o v a r i a n p r o g e s t e r o n e p r o d u c t i o n t h r o u g h r e d u c e d 3/~-HSDH activity a n d th e d o w n - r e g u l a t i o n o f PR. © 1997 E l s evi er Sci ence L t d

J. SteroidBiochem. Molec. Biol., Vol. 62, No. 1, pp. 107-118, 1997

INTRODUCTION

ovulatory L H surge to the same extent, but they differ in inhibiting ovulation in rats [1, 2]. Elger et al. [3] reported comparable results for RU 486 and ON and some other progesterone antagonists. They found that both antiprogestins possess the same antiprogestational activity in abortion tests but ON seems to be three times more potent in inhibiting ovulation than RU 486. These findings suggest that the antiovulatory potency of ON may be induced by intraovarian mechanism(s) rather than by its ability to reduce the preovulatory L H surge.

In a recent study in this laboratory it was demonstrated that antiprogestins such as Onapristone (ON), ZK 136798 and ZK 112993, although possessing the same antiprogestational potencies in antagonizing a progesterone-induced development of mammary glands in rats and transformation of the endometrium in rabbits, differ in their antiovulatory activities in rats [1,2]. These antigestagens are able to reduce the pre*Correspondence to Y. Nishino. Received 10 Jul. 1996; accepted 23 Jan. 1997. 107

J. Donath et al.

108

T h e importance of progesterone for follicular development and the induction of ovulation has been suggested in previous studies [4-8]. In fact, the findings that the antiovulatory effect of O N or mifepristone can be partly reversed by a concomitant administration of progesterone indicate the involvement of progesterone in the process of follicular development and ovulation [2, 9]. It is reasonable to think that the antiovulatory effect of antiprogestins is mediated by the interaction with intraovarian progesterone receptors (PR), because PR has been localized in the ovaries of several species [10-12]. Experiments in PR gene " k n o c k - o u t " mice [13] primed with pregnant mare serum gonadotropin ( P M S G ) / h C G revealed a total blockade of ovulation in these animals. This indicates the importance of PR in the induction of ovulation. Therefore, the present study was undertaken to elucidate intraovarian mechanism(s) contributing to the antiovulatory effect of O N by examining the influence of O N on steroid concentrations, the detection of 3flhydroxysteroid dehydrogenase (3fl-HSDH) and 17flhydroxysteroid dehydrogenase (17fl-HSDH), and the expression of progesterone, estrogen and androgen receptors in the ovary in P M S G / h C G - t r e a t e d immature rats.

T h e y received water and commercially available food (Altromin, Altrogge/Lippe, F.R.G.) ad libitum.

Compounds and formulation P M S G and h C G were dissolved in 0.1 ml 0.9% NaCI solution. T h e antiprogestin Onapristone ( l l f l [4-dimethyl-aminopropyl]- 17~-hydroxy- 17/?- [3-hydroxypropyl]- 13~-methyl-4,9-gonadien-3-one) synthesized in the laboratory of Schering A G [14] was dissolved in 0.2 ml castor oil containing 20% benzyl benzoate for s.c. administration.

Experimental procedure At time 0 (Oh) all animals received 12.5I.U. P M S G (s.c.) to stimulate follicular development, and 5 4 h later 10 I.U. h C G (i.p.) to start the process of ovulation. T h e y were given O N (200 mg/kg) twice, 42 and 48 h after P M S G treatment, and control animals received the vehicle only. This dose was chosen because in dose-response curves it was seen that at this dose of O N totally blocked ovulation in immature rats. T o assess the ovulation rate one group of animals was killed with CO2 13 h after hCG (67 h postP M S G ) to permit counting of the n u m b e r of ova in the fallopian tubes and uteri. Another group of animals was killed by decapitation at varying intervals of time (0, 4, 6, 8 and 1 0 h after hCG). Each group consisted of 10 animals. An overview of the experimental design is given in Fig. 1. T h e blood was collected, centrifuged and the serum used for radioimmunoassay (RIA), and the ovaries were excised, weighed and either frozen immediately in a dry ice-pentane bath for histological examination or stored in liquid N2 for intraovarian hormone evaluation. T h e intraovarian steroids were extracted by homogenizing the ovaries in 1.5 ml absolute ethanol

EXPERIMENTAL

Animals In this study immature female rats (SPF, Wistarstrain) weighing 50-70 g were used. T h e y were kept in a room maintained at constant temperature ( 2 1 + 1 ° C ) and humidity ( 5 0 + 5 % ) on a 1 4 h light:10 h darkness schedule (lights on at 6.00 a.m.).

E x p e r i m e n t a l d e s i g n f o r t h e i n h i b i t i o n o f o v u l a t i o n in i m m a t u r e rats by O n a p r i s t o n e ( O N )

PMSG

(0h)

ON or veh.

ON or veh.

(48h)

(54h)

6

4

4

(42h)

hCG

treatment regime

t=0 0 A: serum and ovaries for RIA and histochemistry

4

6 A

8 10

13

(h after hCG)

B

B: counting the number of ova

Fig. 1. S c h e m e o f t h e e x p e r i m e n t a l d e s i g n . T h e b l a c k a r r o w s a b o v e t h e l i n e i n d i c a t e t h e t r e a t m e n t r e g i m e a n d t h e o p e n a r r o w s u n d e r t h e l i n e t h e t i m e o f killing.

Antiovulatory Effect of Antiprogestin Onapristone and leaving them at 4°(2 overnight in the tubes. After centrifugation next morning, the ethanol fraction was transferred to clean tubes, evaporated to dryness with nitrogen gas at 45°C and the extracts redissolved in 0.5 ml 0.05 M gelatine phosphate buffer. In previous experiments, the efficiency of the extraction of the various steroids from the homogenate was estimated by the addition of iodinated (125I) steroids to a series of ovaries before homogenization. T h e efficiency of extraction was 85% or greater for all steroids.

Histology For the (immuno-)histological examination the granulosa and theca ce]lls of primary, secondary, early tertiary (<500 pm), and late tertiary (>500 #m) follicles were observed. A classification of the staining patterns was used as follows. 0 denotes no staining and 3 indicates maximal staining intensity.

Histochemical study of 3fl-HSDH activities Cryostat sections of 5 # m were m o u n t e d on glass slides, air-dried, and washed (15 min) in modified Krebs solution (0.21 M N a C I - 1 0 0 0 ml, 0.21 M KC14 0 m l ; 0.21 M M g S O 4 . T H 2 0 - 1 0 m l ; 0 . 1 2 M phosphate buffer 360 ml). T h e y were then incubated at 37°C for 50 min with the reaction mixture containing 10 m M dehydroepiandrosterone (dissolved in 0.5 ml acetone), 1.3 m M N A D in 7.5 ml modified Krebs solution, 40 m M nicotinamide in 1 ml distilled water, and 0.5 mg/ml nitro blue tetrazolium chloride in distilled water. T h e reaction was stopped by washing with modified Krebs solution, and the slides were fixed with 10% formalin (pH 7.4). Control sections were incubated in the absence of the substrate.

Hiswchemical study of 17fl-HSDH activities T h i n frozen section,; (5 pm) were prepared with a cryostat, air-dried, and incubated at 37°C for 50 min in darkness with the incubation medium ( 1 0 m M androstenediol in 2.5 ml dimethyl formamide, 5 mg NAD, 0.1 M K C N , 0.05 M MgC12, 20% (w/v) PVA, 1 mg/ml nitro blue tetrazolium chloride, 4 ml 0.1 M phosphate-buffered saline (PBS) (pH 7.4) and 10 ml aq. dest.). Before starting the reaction the incubation medium was filtrated and adjusted to p H 7.5. After termination of the reaction with PBS, post-fixation in 10% formalin followed. Slides incubated without substrate served as negative controls.

Immunohistochemical staining of PR, ER and AR Cryostat sections of 5 #m were m o u n t e d on glass slides coated with 2% silane in acetone. For PR and E R staining the sections were immersed in 3.7% formaldehyde in PBS fc,r 10 min and washed in PBS twice for 5 min. NexL they were immersed in 100% methanol at - 2 0 ° C for 4 m i n , then in acetone a t - 2 0 ° C for 2 min, and subsequently washed twice in

109

PBS. T h e fixation of AR was performed as follows: the sections were immersed in acetone + CaC12 (anhydrous) at a concentration of 100 mg/l at - 8 0 ° C for 2 days. T h e y were then post-fixed in a solution containing 0.2% picric acid, 2% paraformaldehyde and 1.5% polyvinylpyrrolidone (PVP) in 0.4 M PBS at 4°C for 10 min. In the next step the sections were immersed in 1.5% PVP in 85% ethanol for 4 min, twice in 0.05% NaBH3 in PBS +1.5% PVP for 2 min, and at least three times in PBS +1.5% PVP +0.1% gelatine. Each incubation was followed by washing three times with 1.5% PVP in PBS. After fixation, the sections were incubated with rat serum (PR and ER) or normal goat serum (AR) for 30 min at room temperature in a humidified air chamber, and then incubated at 4°C overnight with either anti-PR (MA1140, Dianova, Hamburg, Germany), anti-ER (H222, Abbot Labs, biotinylated from D r Bunte, Schering AG, Berlin, Germany) or anti-AR (PG-21; obtained from Kathy Flanders, Chicago, U.S.A.) monoclonal antibody and, as control, with mouse IgG (Dianova, Hamburg, Germany), fl-galactosidase (biotinylated, Sigma, St Louis, U.S.A.), or rabbit IgG (Dianova, Hamburg, Germany), respectively. For positive controls sections of prostate (AR) or uterus (PR and ER) were used. T h e slides were treated with 0.6% H202 in methanol for 20 min to block the endogenous peroxidase reaction. For PR and AR they were incubated for 3 0 m i n with anti-mouse F(ab)2 biotinylated (Dianova, Hamburg, Germany) or anti-rabbit Ig F(ab)2 biotinylated (Amersham, Bucks, U.K.), respectively. Subsequently, the sections (for PR, E R and AR) were incubated for at least 30 min with the components avidin and biotin of the ABC staining kit (Vector, Burlingname, U.S.A.). Each incubation was followed by washing in PBS. Finally, the immunoreaction was visualized by incubating the sections in a solution consisting of diaminobenzidine (DAB) in Tris-buffer solution containing 0.1% H202 for 12 min (PR and AR) or in a solution containing Ni-DAB in acetate-buffer to which 100 units glucose oxidase had been added (ER).

Radioirnmunoassay Serum and ovarian levels of estradiol and androstenedione were measured by the double antibody methods using kits from Diagnostic Products Co. (I_A, U.S.A.) and Diagnostic Systems Labs Inc. (Webster, U.S.A.). Serum and ovarian progesterone and testosterone were determined by the solid phase methods using kits from Diagnostic Products Co. (LA, U.S.A.). O N showed no crossreaction to these antibodies in the RIA kits used.

Statistics All data are presented as means ±SD. Differences between control and treated groups were compared

110

J. Donath et al. 17.1 + 12.9 (mean + SD) ova (Table 1). These results could be confirmed by histological examination of ovaries 10 h p o s t - h C G (data not shown). In the control group, the start of luteinization of the ovulated follicles was observed, whereas ON-treated animals showed no signs of luteinization. Additionally, in the O N group we found preovulatory follicles with an ovum. The serum and ovarian levels of estradiol (E2), androstenedione (AD), and testosterone (T) in control animals decreased after h C G treatment, a slight but not significant increase in the serum level just after h C G was observed. This may have been caused by the h C G treatment. In animals which received O N there was a significant difference in the peripheral levels of E2, whereas the levels of A D and T were unchanged in both serum and ovary (Figs 2-4). At the time when E2, AD, and T were elevated, the control level of progesterone (Pa) started to increase and reached serum and intraovarian peak values at around 6 and 8 h after hCG-treatment, respectively,

Table 1. Effect of Onapristone on PMSG/hCG-induced ovulation in immature rats No. of ova

Proportion of rats showing ovulation

% Inhibition of ovulation

17.1 + 12.9 0

8/10 0/10

20 100

n = 10 Control Onapristone

by Student's t-test. The results were considered to be significant if P < 0.05.

RESULTS Effects o f O N on ovulation a n d on serum a n d intraovarian steroid concentrations

Treatment with the antiprogestin O N (200 mg/kg) at 42 and 48 h after P M S G priming resulted in a total blockade of ovulation and no oocytes were found. In the control group eight out of 10 animals ovulated with

I

I control Onapristone (200mg/kg), s.c.

n=10

160O

~ 1200.c_ 800 -

T

0

4

a)

6

8

10

8

10

h after hCG

2O

o

.=_ -5

0

b)

0

4

6 h after hCG

Fig. 2. I n f l u e n c e o f O N o n p e r i p h e r a l (a) a n d o v a r i a n Co) levels o f e s t r a d i o l a t d i f f e r e n t t i m e s in i m m a t u r e r a t s t r e a t e d w i t h P M S G / h C G (*significant d i f f e r e n c e in c o m p a r i s o n w i t h c o n t r o l . S t u d e n t ' s t - t e s t , P < 0.05). T h e a n i m a l s r e c e i v e d P M S G a t t i m e 0 a n d h C G 54 h l a t e r . O N w a s a d m i n i s t e r e d t w i c e a t 42 a n d 48 h a f t e r P M S G treatment.

Antiovulatory Effect of Antiprogesdn Onapristone I

111

I control Onapdatone (200mg/kg),s.c.

n=10

40 30 t-

O

20

i

lO

a)

6

8

10

8

10

h after hCG

40

t~

E

30

O

E

'-® 20 e..

® 10

0

0

4

b)

6 h after hCG

F i g . 3. I n f l u e n c e of' O N o n p e r i p h e r a l (a) a n d o v a r i a n (b) l e v e l s o f a n d r o s t e n e d i o n e at d i f f e r e n t t i m e s i n i m m a t u r e r a t s t r e a t e d w i t h P M S G / h C G . T h e a n i m a l s r e c e i v e d P M S G at t i m e 0 a n d h C G 54 h l a t e r . O N w a s a d m i n i s t e r e d t w i c e at 42 a n d 48 h a f t e r P M S G t r e a t m e n t .

after which they dropped again. This preovulatory P4 surge was significantly ,,mppressed by O N (Fig. 5).

Effects of O N on histochemically detected ovarian 3flH S D H and 17~-HSDH activities In control animals treated with P M S G and h C G the histochemical stair.Ling of 3 f l - H S D H was present in theca cells of secondary and tertiary follicles between 0 and 10 h after h C G . In contrast, only granulosa cells of tertiary follicles were stained positively. T h e staining of granulosa cells at primary and secondary follicular stages was negative. It was obvious that the granulosa cells of ].ate tertiary follicles were more strongly stained than the others. In theca cells staining was relatively intensive throughout the given period. As can be seen in Fig. 6, treatment with O N caused a significant reduction in the staining intensity in granulosa cells of tertiary follicles 6 h after h C G . This suppression of histochemical 3 f l - H S D H activity is

observed 0, 4 and 6 h post h C G - t r e a t m e n t , and before the reduction in P4 levels. T h e histochemical study of 1 7 f l - H S D H activity (data not shown) revealed that in granulosa as well as in theca cells of secondary and tertiary follicles, staining was present but not in the primary follicular stage. T h e histochemical activity of granulosa cells in late tertiary follicles was stronger than in secondary and early tertiary follicles. In theca cells the staining was always relatively strong in secondary and tertiary follicles. O N had no appreciable effect on the 17flH S D H activity of the different stages of follicular development at any time examined in this study.

Effect of O N on immunohistochemically detected PR, ER and A R in the ovary Detection of P R in the ovary was first observed 4 h after h C G in theca and granulosa cells of tertiary follicles. In contrast, at 0 h no staining was visible. Primary and secondary follicles were negatively

112

J. Donath et al. [~

control Onapristone (200mg/kg), s.c.

n=10

t

030

_

50

!,o )

2o lO o

o

a)

4

6

8

lO

8

10

h after hCG

60 5O O o)

~ 4o 0 E ~_ 30 r-

0

4

b)

6 h after h C G

Fig. 4. I n f l u e n c e o f O N o n p e r i p h e r a l (a) a n d o v a r i a n (b) l e v e l s o f t e s t o s t e r o n e at d i f f e r e n t t i m e s i n i m m a t u r e r a t s t r e a t e d w i t h P M S G / h C G . T h e a n i m a l s r e c e i v e d P M S G at t i m e 0 a n d h C G 54 h l a t e r . O N w a s a d m i n i s t e r e d t w i c e at 42 a n d 48 h a f t e r P M S G t r e a t m e n t .

stained. In the granulosa cells of animals which received O N we observed a down-regulation of PR starting 8 h after h C G , and the theca cells were unaffected (Fig. 7). In control animals E R expression was apparent in granulosa as well as in theca cells of the preantral and antral follicles, except those of primary follicles. Rats treated with O N presented a significant reduction in the staining intensity in granulosa cells of secondary and tertiary follicles 10 h after h C G (Fig. 8). Like E R expression the staining of AR was seen in secondary and tertiary follicular stages but the staining intensity was weaker. Nevertheless, O N significantly reduced the staining intensity in the granulosa and theca cell layers of tertiary follicles at 0, 4 and 6 h after h C G (Fig. 9).

in immature rats may suggest intraovarian mechanism(s) for the antiovulatory effect of this antiprogestin, because this blockade of ovulation occurred in spite of hCG-treatment. Recent studies from these laboratories demonstrated that, compared to ON, some antiprogestins such as Z K 112993 and Z K 136798, which suppress the preovulatory L H surge to the same extent as does ON, are distinctly less effective in inhibiting ovulation in rats [1, 2]. In contrast to ON, these antiprogestins are not able to suppress the preovulatory surge of progesterone. Therefore, it seems that the antiovulatory effect of O N in rats is related to an interference with the intraovarian activity of progesterone during the process of ovulation. Effect o f O N on progesterone (receptors)

DISCUSSION

T h e finding of Tsafriri et al. [15] that mifepristone (RU 486) partly inhibits ovulation induced by h C G

In the present study treatment with O N blocked PMSG/hCG-induced ovulation completely, and decreased the induced secretion of progesterone in

Antiovulatory Effect of Antiprogestin Onapristone I

113

I control Onapristone (200mg/kg), s.c.

n=lO

1200 1000 0

E ¢o

800

600

p

400

e~

200 0

a)

4

6

8

lO

8

10

h after hCG

160 ~" 14o 0

o~ 120 "6 100 .c_

8o

i ~

60 40-

£ Q.

20-

,

b)

7- , ~ 0

I[///1

4

6 h after hCG

Fig. 5. I n f l u e n c e o f O N o n p e r i p h e r a l (a) a n d o v a r i a n (b) levels o f p r o g e s t e r o n e at different t i m e i n t e r v a l s in i m m a t u r e rats t r e a t e d w i t h P M S G / h C G (*significant d i f f e r e n c e in c o m p a r i s o n w i t h c o n t r o l , S t u d e n t ' s t-test, P < 0.05). T h e a n i n u d s r e c e i v e d P M S G at t i m e 0 a n d h C G 54 h later. O N w a s a d m i n i s t e r e d t w i c e at 42 a n d 48 h after P M S G t r e a t m e n t .

the serum and ovary. Additionally, our histochemical observation indicates that O N causes a decrease in the 3 f l - H S D H activity in granulosa cells of tertiary follicles at the time before the elevation of progesterone levels, as could also be demonstrated for R U 486 [16]. T h e reduction of intraovarian progesterone synthesis caused by R U 486 has been related to the decrease in the ovarian sensitivity to L H stimulation [ 17], and/or to the inhibition of the autocrine effect of progesterone on its own production [16]. T h e results that serum progesterone levels show a reduction before ovarian levels after O N - t r e a t m e n t is unexpected, but m a y be explained by the findings of Brann and Mahesh [18![ who demonstrated that progesterone levels increased in the adrenal 2 h before the ovary. According to Park arm M a y o [19] little or no P R m R N A is found in the ovaries of P M S G - t r e a t e d rats, but a high expression of the m R N A is recognized in

the granulosa cells of large follicles when P M S G primed animals are treated with h C G . Until now, there have been no reports concerning the influence of antiprogestins on P R expression in the ovary. T h e present results revealed the detection of P R in the theca and granulosa cells of tertiary follicles after h C G stimulation, but not during elevated estradiol levels after P M S G - p r i m i n g . It seems that P R expression in the ovary is not estrogen- but L H - d e p e n dent [20], so it is possible that O N down-regulates P R by decreasing the ovarian sensitivity to h C G .

Effect of O N on estrogens (receptors) Although estrogens are not apparently involved in the m e c h a n i s m of intraovarian regulation of PR, they clearly play an active role in follicular development [21]. T h e presence of intraovarian estrogen receptor (ER) has been found in different species [22-24]. Changes in intraovarian levels of estrogens m a y thus

114

J. Donath et al.

Fig. 6. Histochemical demonstration of ovarian 3 ~ - H S D H in granulosa (GLC) and theca (TC) cells o f a tertiary follicle 6 h post hCG-treatment. (A, control; B, ON; ×290). The positive reaction is indicated by arrows.

be involved in the mechanism of anovulation induced by ON. In adult rats showing spontaneous cycles, O N and R U 486 has been shown to increase serum estradiol as the result of aromatization of excessively produced androgens [25, 26]. These authors suggested that the high levels of serum estrogens and/or androgens in animals treated with O N or R U 486 contribute to the disturbance of physiological follicular development and to the block of ovulation. In the present study, however, we found a decrease in the serum estradiol and no significant change in the ovarian level of estradiol in P M S G / h C G - t r e a t e d immature rats after ON-treatment. This contrasting effect of O N is not surprising because O N cannot

cause counterregulation in PMSG/hCG-treated immature rats as opposed to adult rats showing regular estrus cycles. Although the mechanism by w h i c h O N decreases peripheral estradiol without changing the ovarian estradiol level in immature rats treated with exogenous gonadotrophins is still obscure, the present results at least suggest that O N has no influence on the sensitivity of the ovaries to exogenous gonadotrophins in producing estrogens. The inhibition of the peripheral estrogen levels in ON-treated rats might be caused by delayed follicular maturation. These follicles may have a low capacity to secrete E2. In fact, in a recent study Schubert et al., [25] detected a significant increase in "late" tertiary

Antiovulatory Effect of Antiprogestin Onapristone

115

Fig. 7. I m m u n o h i s t o c h e m i c a l detection of p r o g e s t e r o n e receptors in granulosa (GLC) a n d t h e c a (TC) cells of a tertiary follicle 8 tt post h C G - p r i m i n g in a control (A) and O N - t r e a t e d (B) a n i m a l (x290). T h e positive reaction is indicated by arrows.

follicles in ovaries from ON-treated rats. In comparison, R U 486 had no effect on serum E2 levels in immature rats [9], but the doses used in these studies were not able to block ovulation totally. Another possibility for the decreased serum E2 may be the insufficient aromatizatic,n of androgens by an extraovarian aromatase complex, and should be ascertained in a further study. The expression of E R in the ovary has been d e m o n strated by Richards [27], S t u m p f [28], and Suzuki et al. [24] and our immunohistochemical study shows the presence of E R in granulosa as well as in theca cells. Treatment with O N resulted in a low intensity of staining of estrogen receptors in theca and granulosa cells of secondary and tertiary follicles, demon-

strated for the first time of antiprogestins. This decrease in E R may, however, hardly be regarded as a significant aspect related to O N - i n d u c e d anovulation, because it was observed at a very late stage (64 h post P M S G and 10 h post h C G ) after O N treatment.

Effect of O N on androgens (receptors) As mentioned above, Sfinchez-Criado et al. [26] reported that androgens, which increase in rats treated with R U 486, are involved in atresia and anovulation induced by this antiprogestin. In contrast, a recent study from this laboratory has clearly demonstrated that the antiovulatory effect of O N - - R U 486 and O N are known to have antiandrogenic properties [3] - - can be partly restored by a concomitant admin-

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et aL

Fig. 8. l m m u n o h i s t o c h e m i c a l detection of estrogen receptors in g r a n u l o s a (GLC) and theca (TC) cells o f a tertiary follicle 10 h p o s t h C G - p r i m i n g in a control (A) and O N - t r e a t e d (B) a n i m a l (×290). T h e positive reaction is indicated b y arrows.

istration of D H T and can be potentiated by the antiandrogen, flutamide, in rats. This evidence indicates that androgens seem to exert not only inhibitory but also stimulatory effects in the process of follicular development and ovulation. According to previous studies, intraovarian androgens are able to induce follicular atresia [29-31] and to stimulate follicular growth in rats [32]. T h e finding that the antiandrogen, flutamide, inhibits the growth and maturation of rat follicles [33], and that the anovulation induced by antiserum to progesterone can be reversed by testosterone or D H T in P M S G / h C G - t r e a t e d rats [34],

suggests the active participation of androgens in the induction of ovulation. So far, no data have been presented concerning the influence of antiprogestins on androgens (receptors) in immature rats. T h e present results show no change in the peripheral and ovarian levels of testosterone and androstenedione, but a lowered intensity of staining for androgen receptors (AR) in granulosa and in theca cells. Just parallel to the decrease in AR we detected histochemically a reduction in 3 f l - H S D H in granulosa cells. On the basis of these findings it can be concluded that treatment with O N leads to the

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Fig. 9. I m m u n o h i s t o c h e m i c a l detection of androgen receptors in granulosa (GLC) and theca (TC) cells of an "early" tertiary follicle 4 h post h C G - p r i m i n g in a control (A) and ON-treated (B) a n i m a l (x290). The positive reaction is indicated by arrows.

down-regulation of AR followed by the suppression of progesterone synthesis, because androgens and FSH have been demonstrated to stimulate 3/3-HSDH activity synergistically [35]. In conclusion, the inhibition of ovulation induced by the antiprogestin, ON, could be related to decreased intraovarian progesterone production through reduced 3/3-HSDH activity and down-regulation of PR. These results confirm the importance of progesterone in the induction of ovulation in rats by ON. Acknowledgements--We

wish to thank Mrs Bahr, Mrs Meindlschmidt, Mrs Wagener, M r Gr6ning and M r Leidecker for

their skillful assistance. W e would also like to express our appreciation to M r Johnston (Schering AG) for the kind revision of the English text.

REFERENCES 1. Schubert C., D o n a t h J., M i c h n a H. and Nishino Y.: T h e antiovulatory activity of progesterone antagonists is not correlated to their antiprogestational potency in the rat. ft. Steroid Biochem. Molec. BioL 59 (1996) 75-82. 2. Nishino Y., Schubert C. and H a s a n S.: Increase in the antiovulatory potency of the antiprogestin Z K 112993 by combination with the antiandrogen flutamide in the rat. Exp. Clin. Endocr. 102 (1994) 111. 3. Elger W., Beier S., Puri C. P., Pollow K., Chwalisz K., F/ihnrich M., H a s a n S. H. a n d N e e f G.: Comparative p h a r m a cological studies on new progesterone-antagonists in cyclic and

118

J. D o n a t h et al. pregnant animals and their therapeutic perspectives. Nichidoku-

Iho 33 (1989) 171-186. 4. Rondell P.: Role of steroid synthesis in the process of ovulation. Biol. Reprod. 10 (1974) 199-215. 5. Takahashi M., Ford J., Yoshinaga K. and Greep R.: Induction of ovulation in hypophysectomized rats by progesterone. Endocrinology 95 (1974) 1322-1326. 6. Rao I. and M a h e s h V.: Role of progesterone in the modulation of the preovulatory surge of gonadotropins and ovulation in the pregnant mare's s e r u m gonadotropin-primed i m m a t u r e rat and the adult rat. Biol. Reprod. 35 (1986) 1154-1161. 7. Mori T., Suzyju A., N i s h i m u r a T. and K a m b e g a w a A.: Inhibition of ovulation in i m m a t u r e rats by antiprogesterone antiserum. J. Endocr. 73 (1977) 185-186. 8. Snyder B., B e e c h a m G. and Schane H.: Inhibition of ovulation in rats with epostane, an inhibitor of 3fl-hydroxysteroid dehydrogenase (41865). Proc. Soc. Exp. Biol. Med. 176 (1984) 2 3 8 242. 9. Iwamasa J., Shibata S., T a n a k a N., M a t s u u r a K. and O k a m u r a H.: T h e relationship between ovarian progesterone and proteolytic enzyme activity during ovulation in the gonadotropin-treated i m m a t u r e rat. Biol. Reprod. 46 (1992) 3 0 9 - 3 1 3 . 10. Iwal T., Fuji S., N a n b u Y., Nonogaki H., Konishi I., Mori T. O k a m u r a H.: Effect of h u m a n chorionic gonadotropin on the expression of progesterone receptors and estrogen receptors in rabbit ovarian granulosa cells and the uterus. Endocrinology 129 (1991) 1840-1848. 11. Hild-Petito S., Stouffer R. and Brenner R.: Immunocytochemical localization of estradiol and progesterone receptors in the monkey ovary throughout the menstrual cycle. Endocrinology 123 (1988) 2896-2905. 12. Iwal M., Yasuda K., Fukuoka M., Iwai T., Takakura K., Tall S., Nakanishi S. and Mori T.: Luteinizing h o r m o n e induces progesterone receptor gene expression in cultured porcine granulosa cells. Endocrinology 129 (1991) 1621-1627. 13. L y d o n J. P., De Mayo F. J., Conneely O. M. and O'Malley B. W.: Reproductive phenotypes of the progesterone receptor null m u t a n t mouse..7. Steroid Biochem. Molec. Biol. 56 (1996) 6 7 77. 14. N e e f G., Beier S., Elger W., H e n d e r s o n D. and Wiechert R.: N e w steroids with antiprogestational and antiglucocorticoid activities. Steroids 44 (1984) 349-372. 15. Tsafriri A., Abisogun A. and Reich R.: Steroids and follicular rupture at ovulation. J. Steroid Biochem. 27 (1987) 359-361. 16. T a n a k a N., Iwamasa J., M a t s u u r a K. and O k a m u r a H.: Effects of progesterone and anti-progesterone R U 486 on ovarian 3flhydroxysteroid dehydrogenase activity during ovulation in the gonadotrophin-primed i m m a t u r e rat. J. Reprod. Fertil. 97 (1993) 167-172. 17. Uilenbroek J., Sfinchez-Criado J. and Karels B.: Decreased luteinizing hormone-stimulated progesterone secretion by preovulatory follicles isolated from cyclic rats treated with the progesterone antagonist R U 486. Biol. Reprod. 47 (1992) 368-373. 18. Brann D. W. and M a h e s h V. B.: Regulation of gonadotropin secretion by steroid hormones. Front. Neuroendocr. 12 (1991) 165-207.

19. Park O.-K. and Mayo K.: Transient expression of progesterone receptor messenger R N A in ovarian granulosa cells after the preovulatory luteinizing h o r m o n e surge. Molec. Endocrinol. 5 (1991) 967-978. 20. Park O.-K. and Mayo K.: Regulation of the progesterone receptor gene by gonadotropins and cyclic adenosine 3', 5'm o n o p h o s p h a t e in rat granulosa cells. Endocrinology 134 (1994) 709-718. 21. H s u e h A., Adashi E., Jones P. and Welsh T. Jr: H o r m o n a l regulation of the differentiation of cultured ovarian granulosa cells. Endocr. Rev. 5 (1984) 76-127. 22. Saiduddin S. and Z a s s e n h a u s H.: Estradiol-17fl receptors in the i m m a t u r e rat ovary. Steroids 29 (1977) 197-213. 23. K i m I. and Greenwald G.: Effect of estrogens on follicular development and ovarian and uterine estrogen receptors in the i m m a t u r e rabbit, guinea pig and mouse. Endocr. ffapon. 34 (1987) 8 7 1 - 8 7 8 . 24. Suzuki T., Sasano H., K i m u r a N., T a m u r a M., Fukaya T., Yajima A. and N a g u r a H.: I m m u n o h i s t o c h e m i c a l distribution of progesterone, androgen and oestrogen receptors in the h u m a n ovary during the menstrual cycle: relationship to expression of steroidogenic enzymes. Human Reprod. 9 (1994) 1589-1595. 25. Schubert C., M i c h n a H. and Nishino Y.: Disturbance of follicular development and endocrine reactions induced by the antiovulatory effective progesterone antagonist Onapristone. Ann. Anat. 177 (1995) 139-146. 26. Sfinchez-Criado J., Galiot F., Bellido C., Gonzalez D. and T r b a r M.: Hypothalamus-pituitary-ovarian axis in cyclic rats lacking progesterone actions. Biol. Reprod. 48 (1993) 9 1 6 - 9 2 5 . 27. Richards J.: Maturation of ovarian follicles: actions and interactions of pituitary and ovarian h o r m o n e s on follicular cell differentiation. Physiol. Rev. 60 (1980) 51-89. 28. S t u m p f W.: Nuclear concentrations of 3H-estradiol in target tissues. Endocrinology 85 (1969) 31-37. 29. Payne R., H e l l b a u m A. and Owens J. Jr: T h e effect of androgens on the ovaries and uterus of the estrogen-treated hypophysectomized i m m a t u r e rat. Endocrinology 59 (1956) 2 7 5 - 2 7 8 . 30. Hillier S., Knazek R. and Ross G.: Androgenic stimulation of progesterone production by granulosa cells from preantral ovarian follicles further in vitro studies using replicate cell cultures. Endocrinology 100 (1977) 1539-1549. 31. Erickson G.: An analysis of follicle development and o v u m maturation. Semin. Reprod. Endocr. 4 (1986) 233-254. 32. Beyer C., Cruz M., Gay V. and Jaffe R.: Effects of testosterone and dihydrotestosterone on F S H s e r u m concentration and follicular growth in female rats. Endocrinology 95 (1974) 722-727. 33. K u m a r i G., Datta J., Das R. and Roy S.: Evidence for a role of androgens in the growth and maturation of ovarian follicles in rats. Hormone Res. 9 (1978) 112-120. 34. Mori T., Suzuki A., N i s h i m u r a T. and K a m b e g a w a A.: Evidence for androgen participation in induced ovulation in i m m a t u r e rats. Endocrinology 101 (1977) 623-626. 35. Welsh T., Jones P., Ruiz de Galarreta C., Fanjul L. and H s u e h A.: Androgen regulation of progestin biosynthetic enzymes in FSH-treated rat granulosa cells in vitro. Steroids 40 (1982) 691-700.