HIGH INHIBITORY ACTIVITY OF R 5020, A PURE PROGESTIN, AT THE HYPOTHALAMIC-ADENOHYPOPHYSEAL LEVEL ON GONADOTROPIN SECRETION

Transcription

1 FERTILITY AND STERILITY Copyright c 1977 The American Fertility Society Vol. 28, No. 10, October 1977 Printed in U.s.A. HIGH INHIBITORY ACTIVITY OF R 5020, A PURE PROGESTIN, AT THE HYPOTHALAMIC-ADENOHYPOPHYSEAL LEVEL ON GONADOTROPIN SECRETION FERNAND LABRIE, M,D., PH.D.* LOUISE FERLAND, PH.D.t LISETTE LAGACE, PH.D.t JACQUES DROUIN, PH.D.t JACQUES ASSELIN, PH.D. GENEVIEVE AZADIAN-BOULANGER, M.D., PH.D. JEAN-PIERRE RA YNAUD, PH.D. Medical Research Council Group in Molecular Endocrinology, Le Centre Hospitalier de l'universite Laval, Quebec GIV 4G2, Canada, and Centre de Recherches Roussel-UCLAF, Romainville 93230, France Synthetic "progestins" currently used in the contraceptive pill inhibited the luteinizing hormone (LH) responsiveness to LH-releasing hormone in cells in culture in a way undistinguishable from that of androgens. Moreover, they competed for binding of the 3H-labeled androgen R 1881 to the rat prostate androgen receptor and stimulated seminal vesicle and prostate weight in castrated rats. R 5020, a pure progestin, was without effect on the above-mentioned parameters. However, a complete inhibition of the LH surge measured in the afternoon of expected proestrus was obtained at a dose of R 5020 similar to that of D-norgestrel. The synthetic progestin was also found to inhibit ovulation and to delay vaginal cornification. The present data show that the synthetic "progestins" commonly used in the pill possess intrinsic androgenic activity which could well be responsible, to an unknown extent, for their effectiveness as contraceptive agents. R 5020, a synthetic progestin devoid of androgenic activity, is at least as potent as the most potent 19-nortestosterone derivative, D-norgestrel, in inhibiting gonadotropin secretion and other parameters of the estrous cycle in the rat. The availability of a pure progestin devoid of androgenic activity but highly effective as an inhibitor of gonadotropin secretion could be of great interest for the development of an improved contraceptive. It is well known that progesterone (P) can exert inhibitory and stimulatory effects on luteinizing hormone (LH) secretion. In fact, when injected at different times during the rat estrous cycle, P can advance or inhibit ovulation.!' 2 Inhibition or stimulation of plasma LH levels can also be seen after P treatment in estradiol (E2)-primed, ovari- Received March 30, 1977; revised June 13, 1977; accepted June 14, * Associate of the Medical Research Council of Canada. To whom reprint requests should be addressed at Groupe du Conseil de Recherches Medicales en Endocrinologie Moleculaire, Le Centre de l'universite Laval, 2705 Boulevard Laurier, Quebec G1V 4G2, Canada. tfellow of the Medical Research Council of Canada ectomized rats.3, 4 In the monkey, spontaneous and E 2 -induced preovulatory LH peaks can be blocked by P. 5 When progestational activity was investigated as a means of controlling conception, it was found that two classes of steroids, the androstane and the pregnane derivatives, could be successfully used to prevent ovulation in the rabbit,6 rat,6 and human. 7 The ability of these compounds to inhibit ovulation has been ascribed to their high progestational activity,6, 8 and both hypothalamic 9 and pituitary10 sites of action have been suggested. Although well recognized, marked discrepancies have been reported for the androgenic activity of synthetic "progestins."1l,12

2 Vol. 28, No. 10 HIGH POTENCY OF R 5020 TO INHIBIT LH AND FSH SECRETION 1105 Since we had recently found that androgens can exert a marked inhibitory effect on the LH responsiveness to LH-releasing hormone (LHRH) in anterior pituitary cells in culture13 whereas P had almost no activity,14 it was thought that the synthetic "progestins" might act as pituitary blocking agents through their androgen-like activity. The present paper examines the androgen-like activity of synthetic "progestins" at the pituitary level, using cells in culture, and compares the effect of R 5020, a highly potent progestin devoid of androgenic activity,15 with that of D-norgestrel, P, and 5a-dihydrotestosterone (DHT) as inhibitors of gonadotropin secretion and with other parameters of the estrous cycle in the rat. The present data show that R 5020 is at least as potent an inhibitor of gonadotropin secretion as is any "progestin" currently used in the pill. MATERIALS AND METHODS Animals. Adult male and female Sprague-Dawley rats (obtained from Canadian Breeding Farms, St. Constant, Quebec), weighing 200 to 225 gm upon arrival, were kept in individual cages and exposed to 14-hour light/lo-hour dark cycles (lights on at 5:00 A.M., off at 7 :00 P.M.) in a soundattenuated and temperature-controlled (24 0 ± 2 C) room. Purina rat chow and water were available ad libitum. Animals were used after a mini 0 mum of 1 week of acclimatization to the conditions of the animal room. In the appropriate experiments, daily examination of vaginal smears was performed between 8:00 and 10:00 A.M. Only animals having completed two or three 4-day cycles were used in the present studies. Materials. LHRH was kindly provided by Drs. Gotz and Degenghi, Ayerst Research Laboratories, Montreal. 6,7-3H-17,B-Hydroxy-17a-methyl-estra- 4,9,11-trien-3-one, 3H-R 1881 (58.2 Ci/mmole), the unlabeled steroid, and 17,21-dimethyl-19-norpregna-4,9-diene-3,20-dione (R 5020) were synthesized at the Roussel Research Center, Romainville, France. The labeled compound was stored at -20 C in toluene-methanol (9:1) and 0 its purity was checked by thin-layer chromatography before use. After evaporation of the solvent under a stream of nitrogen, the labeled steroid was dissolved in buffer B (10 mm Tris-HCl, 1.5 mm ethylenediaminetetraacetic acid, and 10 mm a-monothioglycerol, ph 7.4). The steroid solutions were prepared by appropriate dilution with buffer B for binding studies. The steroids were then added to the incubation medium in a 10-M-l volume; the final ethanol concentration never exceeded 0.2%. In experiments with anterior pituitary cells in culture, stock solutions of steroids were prepared in 0.9% NaCl-l% ethanol and were used at a 100- fold dilution in the incubation medium. Such a concentration of ethanol (0.01%) in the incubation medium did not affect basal or LHRH-stimulated release. Media and sera for tissue culture were obtained from Grand Island Biological Co., Grand Island, N. Y. Dextran-coated charcoal (DCC) adsorbed sera were prepared by overnight incubation of the sera at 4 C under constant agitation 0 with 1% charcoal (Norit-A) and 0.1% Dextran T70, obtained from Fisher Scientific Co., Pittsburgh, Pa., and Pharmacia Fine Chemicals, Piscataway, N. J., respectively. This treatment was found to remove more than 99% of added 3H-E 2 and to lower the E2 concentration to undetectable levels as measured by radioimmunoassay. For in vivo experiments, steroids were dissolved in a minimal volume of ethanol before dilution with appropriate volumes of 1% gelatin-o.9% NaCl. The indicated doses of steroids were injected subcutaneously in a total volume of 0.4 ml. Controls animals received injections of an identical volume of the vehicle d-norgestrel d-norgestrel + E MPA M PA + E2 ::> 0:> "- "- 0:>...,......,...:..., -' 1000, "-... t-..., z: -'...: ::> "" c STEROIO (log M) FIG. 1. Effect of increasing concentrations of D-norgestrel and medroxyprogesterone acetate (MP A) on the LH response to 1 x M LHRH in rat anterior pituitary cells in primary culture incubated in the presence or absence of 1 x 10-9 M 17 f:l-estradiol. The results are presented as means ± standard error of the mean of assays of triplicate culture dishes.

3 1106 LABRIE ET AL. October e -! c "'-0 Control... PROGESTERONE (0.3mg) o-- (1 mg) (3mg) &---0 (9mg) MIN. AFTER lh-rh INJECTION FIG. 2. Effect of increasing doses of progesterone on the plasma LH response to 200 ng of LHRH. Adult rats ovariectomized 1 month previously received daily subcutaneous injections of the indicated doses of progesterone for 1 week. Animals were anesthetized with Surital (50 mg/kg, intraperitoneally), and blood samples were obtained at the indicated time intervals after intravenous LHRH injection. Preparation of Dispersed Anterior Pituitary Cells. Adult female Sprague-Dawley rats at random stages of the estrous cycle were used for preparation of the primary culture of anterior pituitary cells. The cells were prepared as described previously,i6 except that the cell suspension was washed by centrifugation through a layer of 4% bovine serum albumin after the Viokase treatment. Cells (5 to 10 x 10 5 ) suspended in 1.5 ml of Dulbecco's modified Eagle's medium (DMEM) containing 10% DCC-adsorbed horse serum and 2.5% DCC-adsorbed fetal calf serum were plated in 35 x 10 mm Falcon Petri dishes. Cells were usually used 3 days after plating. Incubation Procedure. Cells were washed four times with sterile DMEM without sera before further incubation for about 48 hours in 1.5 ml of DMEM containing DCC-adsorbed sera in the presence or absence of the indicated concentrations of steroids to be tested. These incubations were followed by a 5-hour incubation with 1 x LHRH. These short-term incubations were performed in DMEM without sera in the presence or absence of the indicated concentrations of steroids. At the end of the incubations, media were centrifuged at 100 x g for 5 minutes at 4 0 C and the supernatants frozen at _20 0 C until assayed. LH Response to LHRH in Vivo. For measurement of pituitary sensitivity to LHRH, rats were anesthetized with sodium thiamylal (Surital, 50 mg/kg, intraperitoneally) 1 hour before injection of the decapeptide and were kept deeply anesthetized by the administration of small quantities of the anesthetic. LHRH (200 ng), in 0.4 ml of 0.01 M acetic acid-0.9% NaCI, was injected subcutaneously between noon and 1:00 P.M. At the time intervals indicated, blood samples (0.6 md were withdrawn into heparinized syringes via a catheter inserted into the right superior vena cava a few minutes after the first injection of Sur ita 1. A volume of 0.6 ml of 0.9% NaCI solution containing heparin (200 U/mI) was injected into the catheter after each blood sampling in order to maintain the blood volume. Hormone Assays. LH, follicle-stimulating hormone (FSH), and prolactin (PRL) were measured by double-antibody radioimmunoassay using rat hormones (rat LH-I-3, rat FSH-I-1, rat PRL-I-1 and LH-RP-1, FSH-RP-1, and PRL-RP-1) and rabbit antisera (anti-rat LH serum 1, anti-rat FSH serum 6, and anti-rat PRL serum 2) kindly supplied by Dr. A. F. Parlow of the Rat Pituitary E... ;[ 300 <>:, :5 I- Z co: ::: > '" DHT.--.. NORGESTREl NORETHINDRONE NORETHINDRONE ACET. ".,, ETHYNODIOl DlACET. NORETHYNODREl <> MEDROXYPROGESTERONE ACET MEGESTROl ACET CHlORMADINONE ACET. T M--M R \ \ \ -* t \,.... " '1: g STEROID FIG. 3. Effect of increasing doses ofthe synthetic "progestins" commonly used in the pill, DHT, and R 5020 on plasma LH levels in orchidectomized rats. Animals castrated 2 weeks previously received injections daily for 7 days of the indicated doses of steroids before measurement of plasma LH.

4 Vol. 28, No. 10 HIGH POTENCY OF R 5020 TO INHIBIT LH AND FSH SECRETION 1107 TABLE 1. Inhibition of 3H_R 1881 Binding to Rat Prostate Cytosol by "Progestins" and Other Steroids" Steroid R N ortestosterone 5a-Dihydrotestosterone Testosterone D-N orgestrel Medroxyprogesterone acetate Norethindrone Chlormadinone acetate Cyproterone acetate Megestrol acetate 3a-Androstanediol Progesterone R 5020 Norethynodrel 17 f3-estradiol Ethynodiol diacetate Displacing ability (%) "Rat ventral prostate cytosol was incubated with 6 x 10-9 M 3H-R 1881 (6,7-3 H-17f3-hydroxy-17a-methyl-estra-4,9,1l-trien- 3-one, methyltrienolone; 58.2 Cilmmole) in the presence or absence of increasing concentrations of the indicated unlabeled steroids for 15 to 17 hours at 0 C as described?5 Bound radioactivity was separated by dextran-coated charcoal (0.5%). The displacing ability was calculated from the concentrations of unlabeled steroids giving a 500/0 inhibition of 3H-R 1881 binding. Hormone Program, National Institute of Arthritis, Metabolism and Digestive Diseases, Bethesda, Md. Calculations. Radioimmunoassay data were analyzed with a Hewlett-Packard desk-top calculator using a program based on model II of Rodbard and Lewald.19 Dose-response curves and 50% effective doses (ED50) were calculated by using an iterative and nonlinear least-squares regression according to the method described.20 Statistical significance was analyzed according to the multiple-range test of Duncan-KramerY All data are expressed as means ± standard error of the mean. RESULTS Androgenic Activity of Synthetic "Progestins" Effect of "Progestins" on Gonadotropin Secretion in Anterior Pituitary Cells in Culture. A representative of each class of synthetic "progestins" was first studied for its effect on the LH and FSH responsiveness to LHRH in anterior pituitary cells in culture. In agreement with our previous data,22 E2 led to an increase of the LH response to LHRH (Fig. 1). Preincubation for 48 hours in the presence of increasing concentrations of norgestrel ormedroxyprogesterone acetate (Provera) led to a progressive inhibition of the LH response to 1 x M LHRH. The norgestrel ED50 value was 8.2 ± 1.1 versus 9.9 ± 0.9 x M in control and E2-treated cells, respectively, whereas values of 4.6 ± 0.7 and 4.7 ± 1.6 x 10-9 M were found for medroxyprogesterone acetate. In fact, the two "progestins" led to same maximal inhibition of LH release at about 20% of control. This effect is similar to that obtained with testosterone (T) or DHT,13 thus indicating that the two "progestins" exert full androgenic activity of LH release. Under the same conditions, P had no effect on basal or LHRH-induced LH release. 14 That norgestrel and medroxyprogesterone acetate exert androgenic activity at the pituitary level was further indicated by the observation of an absence of a potentiating effect of E2 on the stimulatory effect of the two steroids on FSH cell content (data not shown). These findings are in marked contrast with the effect of P, which is potentiated by E2, but are compatible with that of androgens. 13 While norgestrel and medroxyprogesterone acetate inhibited LHRH-induced LH release at ED50 values of approximately 9 x M and 5 x 10-9 M, respectively, the following ED50 values were found for norethindrone, norethindrone acetate, norethynodrel, and ethynodiol diacetate: 6 x 10-9 M, 2 X 10-8 M, 2 X 10-8 M, and 8 x 10-8 M. Megestrol acetate and chlormadinone acetate had higher ED50 values at 2 and 1 x 10-7 M, respectively. It should be remembered that > "" > : '00! DHT NORGESTREl o----a NORETHINORONE NORETHINORONE ACET. - ETHYNODIOl OIACET.... NORETHYNODREl <>- - -<> MEDROXYPROGESTERONE Am ---. MEGESTROl ACET CHlORMADINONE ACET. R 5020 :-:--:.:.: V9 STEROID FIG_ 4_ Effect of synthetic "progestins" commonly used in the pill, DHT, and R 5020 on seminal vesicle weight_ Adult rats orchidectomized 2 weeks previously received injections daily for 7 days of the indicated doses of steroids.

5 1108 LABRIE ET AL. October 'E1200 "- co.900 x "'0 PROGESTEROfH PR.OGESTERONE +RUI (] R R RU 1&117 STEROID (pg 1100g. OW.) FIG. 5. Effect of increasing doses of progesterone or R 5020 alone or in combination with the fixed dose ofru (0.25 JLg/100 gm body weight), injected at 9:00 A.M. and 4:00 P.M. on the day of estrus, on the plasma LH concentration measured in the afternoon (4:00 P.M:) of expected proestrus in 4-day cycling rats. T and DHT had respective ED50 values of 5 and 1.5 x M.13 Action of"progestins" on Plasma Gonadotropin Levels in Gonadectomized Rats. Although P was known to have no effect on plasma LH levels in castrated rats,3 it remained possible that a change in the LH response to LHRH induced by P could be compensated by an inverse change in LHRH secretion. As is illustrated in Figure 2, the injection of P for 1 week in ovariectomized rats at daily doses ranging from 0.3 to 9 mg had no significant effect on either the basal plasma LH levels (time zero) or the plasma LH response to 200 ng of LHRH. There was only a small but insignificant tendency to an inhibitory effect of the steroid at the relatively high doses of 3 and 9 mg. LHRH, at the dose used, had no effect on FSH release in any of the groups (data not shown). The absence of an effect of P on the LH responsiveness to LHRH supports our in vitro data13,22 which show that androgens (but not P alone) inhibit LH release at physiologic concentrations. Moreover, the observation of unaltered plasma LH levels under P treatment in castrated animals indicates that P treatment alone does not affect LHRH secretion. Since androgens are well known to be potent inhibitors of LH secretion in the castrated ani- mal,2:j the present in vivo system provides a good model for measuring the androgenicity of synthetic "progestins." As observed ih pituitary cells in culture, pure progestins should have no effect on LH secretion whereas those sharing androgenic activity should inhibit plasma LH concentration. As is illustrated in Figure 3, all of the 19-nortestosterone derivatives used were relatively potent inhibitors of LH secretion in rats orchidectomized 2 weeks previously. As estimated from their inhibitory effect on the plasma LH concentration, norgestrel, norethindrone, norethindrone acetate, ethynodiol diacetate, and norethynodrel have 10% to 30% the potency ofdht itself. The 17-acetoxyprogesterone derivatives medroxyprogesterone acetate, chloramidone acetate, and megestrol acetate had no significant effect at daily doses of up to 150 J1-g. At the 1500-J1-g dose, medroxyprogesterone acetate led to a 50% inhibition of plasma LH levels (208 ± 28 versus 446 ± 47 ng/mi). As was observed previously,23 DHT exerted an almost maximal inhibition of plasma LH concentration at the daily dose of 25 J1-g. R 5020, as expected from its lack of binding to the androgen receptor,24,25 had no significant effect on plasma LH levels. These findings support the in vitro data (Fig. 1) indicating that co..., a:: :z: FIG. 6. Effect of increasing doses of R 5020, injected at 9:00 A.M. and 4:00 P.M. on the day of estrus, on the plasma LH concentration measured in the afternoon (4:00 P.M.) of expected proestrus in 4-day cycling rats.

6 Vol. 28, No. 10 HIGH POTENCY OF R 5020 TO INHffiIT LH AND FSH SECRETION ' " C :L 900 v.o 0- ; 600 <C :> ::::> c:::i 300 0,,-0 NORGESTREl...- NORGESTREl + RU a - OHT...- 5a-DHT+RU STEROID (pg /100g, B.W) FIG. 7, Effect of increasing doses of 5a-DHT or norgestrel alone or in combination with a fixed dose of RU (0,25 p,g/loo gm body weight), injected at 9:00 A.M. and 4:00 P:M, on the day of estrus, on the plasma LH concentration measured in the afternoon (4:00 P.M.) of expected proestrus in 4-day cycling rats, all of the 19-nortestosterone derivatives tested were relatively potent stimulators of seminal vesicle weight, while chlormadinone acetate and megestrol acetate showed lower but significant potency. Only R 5020 had no significant effect on seminal vesicle weight. Similar results were obtained with ventral prostate weight (data not shown). The present data indicate a close correlation between the effect of synthetic "progestins" and' DHT on prostate and seminal vesicle weight and their potency as inhibitors of plasma LH levels in castrated rats. Although the 17 a-acetoxy derivatives of progesterone had less intrinsic androgenic activity, they did exert stimulatory effects at the daily dose of 150 p,g on prostate and seminal vesicle weight. R 5020, on the other hand, was completely devoid of androgenic activity. Actions of R 5020 on Gonadotropin Secretion Inhibitory Effect of R 5020 on the Proestrous Surge of Gonadotropin Secretion. We next examined the inhibitory activity ofr 5020 on gonadotropin secretion and compared its action with that of the "progestins" commonly used in the pill. The the inhibitory action of "progestins" on LH secretion is at least partially secondary to their intrinsic androgenic activity. Affinity of "Progestins" for the Rat Prostate Androgen Receptor. It was then felt to be of interest to compare the androgenic activity of synthetic "progestins" measured in vitro and in vivo on LH release with the affinity of these steroids for the androgen receptor and their potency in a classic test of androgenic activity-rat ventral prostate weight. The affinity of the different "progestins" for the rat ventral prostate cytosol androgen receptor was measured by competition of the unlabeled steroids for binding of3h-r 1881 (Table 1). In agreement with previous reports,24.25 it was found that DHT, 19-nortestosterone, and R 1881 had a somewhat higher affinity than T for the prostate receptor. N orgestrel, medroxyprogesterone acetate, and norethindrone had 43%, 23%, and 23% the activity of T, respectively, roughly paralleling their inhibitory effect on LH release. Chlormadinone acetate and megestrol acetate had higher binding than LH-release-inhibiting activities, whereas norethynodrel and ethinodiol diacetate had a greater potency to inhibit LH release than for competition of 3H-R 1881 binding. Effect of "Progestins" on Seminal Vesicle and Prostate Weight. As is illustrated in Figure 4, 500 4oo E E " ḡ'300 " ::0: Control RU16117 R &020 RU16117+R5020 :/ i /'... it"".... " " "... " '" " '" ol- -L L- -L o m lw MIN. AFTER lh-rh INJECTION FIG, 8. Effect of R 5020 (25 p,g/loo gm body weight) or RU (0.25 p,g/loo gm body weight), injected alone or in combination at 9:00 A.M. and 4:00 P,M. on the day of estrus, on the plasma LH response to 200 ng of LHRH injected in the afternoon (2:00 P.M.) of expected proestrus. The LHRH test was performed under Surital (50 mglkg, intraperitoneally) anesthesia.

7 1110 LABRIE ET AL. TABLE 2. Effect of Treatment with R 5020 (25 pgl100 gm) and Norgestrel (25 pgl100 gm) Alone or in Combination with RU (0.5 pglloo gm) on Vaginal Cornification in the Rat" Treatment Control R5020 Norgestrel RU R RU Norgestrel + RU Delay of vaginal cornification (mean ± SEM) days o 0.8 ± ± ± ± ± 0.3 "The indicated doses of steroids were injected at 9:00 A.M. and 4:00 P.M. on the day of estrus, and vaginal smears were examined daily for 2 weeks. finding of a potent inhibitory effect of R 5020 on gonadotropin secretion could then make possible the use of a contraceptive steroid devoid of secondary androgenic effects. Since we previously found that RU 16117, a new steroidal antiestrogen, was a potent inhibitor of the proestrous LH surge when administered 3 days previously (during estrus) to 4-day cycling rats,26 we first examined the effect of increasing doses of R 5020 administered alone or in association with a small dose (0.25 ILg/100 gm) of RU on the plasma LH levels measured in the afternoon of expected proestrus. In the same experiment, the effect of R 5020 was compared with that of progesterone, norgestrel, and DHT. When injected at 9:00 A.M. and 4:00 P.M. on the day of estrus, P, at the dose of 200 ILg/100 gm, led to approximately a 50% inhibition of plasma LH concentration; complete inhibition was observed at the 1000-lLg dose (Fig. 5). R 5020 led to a complete inhibition of plasma LH concentration at the lowest dose used (50 ILg), thus indicating a potency of R 5020 at least 20 times that of P itself. RU alone led to a 70% inhibition of plasma LH, and the addition of the dose of 200 ILg of P led to a further inhibition (to approximately 20% of control values). In order to obtain a more precise assessment of the potency of R 5020 on the spontaneous preovulatory LH surge, we next studied the effect of doses of 1, 5, 20, and 50 llg of R 5020 injected TABLE 3. Effect of Treatment with R 5020 (25 pglloo gm) and RU (025 pgl100 gm), Injected Alone or in Combination at 9:00 A.M. and 4:00 P.M. on the Day of Estrus, on Ovulation in 4-Day Cycling Rats Treatment Control R5020 RU R RU No. of rats ovulating 8/8 3/8 1/8 0/8 No. of eggs October 1977 twice on the day of estrus. While the 5-lLg dose led to approximately a 30% reduction of the plasma LH concentration, an 85% inhibition was observed at the 20-lLg dose of R 5020 (Fig. 6). Norgestrel was about 3 times more potent than DHT in inhibiting the proestrous LH surge (Fig. 7). Approximately a 90% inhibition of plasma LH was in fact obtained with 30 ILg of norgestrel/100 gm body weight, while a similar effect was obtained with the 90-lLg dose ofdht. These data indicate clearly that, despite its lack of androgenic activity, R 5020 has a potency to inhibit the spontaneous proestrous LH surge which is equal or slightly superior to that of norgestrel, a compound having both progestin and androgenic activities. Inhibitory Effect of R 5020 on the Pituitary Sensitivity to LHRH. Since the preovulatory LH surge is accompanied by an increased sensitivity of the LH pituitary response to LHRH,27 we next examined the effect of treatment with R 5020 on this sensitive parameter. Although the 0.25-lLg dose of RU alone had no significant effect on the LH response to LHRH, it did potentiate the inhibitory action ofr 5020 (Fig. 8). It should be mentioned that higher doses of RU would also, by themselves, inhibit the LH responsiveness to the neurohormone.27 Effect of Treatment with R 5020 on Vaginal Cornification. As is shown in Table 2, the administration of norgestrel (25 ILg/100 gm), R 5020 (25 ILg/100 gm), or RU (0.5 ILg/lOO gm) alone led to a similar delay of vaginal cornification (0.8 to 1.1 days). When administered in combination withru 16117, both R 5020 and norgestrel TABLE 4. Effect of Treatment with Increasing Doses of RU and R 5020, Injected Alone or in Combination with a Fixed Dose of the Other Steroid, on Ovulation" Control R 5020 (25 /kg) R 5020 (50 /kg) RU (0.25 /kg) RU (0.50 /kg) RU (2.5 /kg) Treatment R 5020 (25 /kg) + RU (0.25 /kg) R 5020 (25 /kg) + RU (0.5 /kg) R 5020 (25 /kg) +.RU (1 /kg) R 5020 (50 J.Lg) + RU (0.25 /kg) R 5020 (50 /kg) + RU (0.5 /kg) R 5020 (50. /kg) + RU (1 /kg) No. of animals ovulating 8/8 5/8 1/8 7/8 2/ /8 2/8 0/8 3/8 0/8 0/8 "The indicated amounts of steroids were injected at 9:00 A.M. and 4:00 P.M. on the day of estrus, and ovulation was assessed at 10:00 A.M. on the morning of the next expected estrus in 4-day cycling rats.

8 Vol. 28, No. 10 HIGH POTENCY OF R 5020 TO INHIBIT LH AND FSH SECRETION 1111 TABLE 5. Effect of R 5020 (50 JlIIIRat) + RU JlIIIRat) on Plasma LH and PRL Concentrations" Treatment Stage of cycle Plasma LH PlasmaPRL at sacrifice nglml Control Proestrus 798 ± ± 129 R RU Proestrus 61.7 ± 8.6b 22.6 ± 5.7b R RU Diestrus II 83.3 ± 12.3b 7.6 ± 1.5b R RU Diestrus I 46.2 ± 7.3b 15.1 ± 3.2b R RU Estrus 44.7 ± 6.9b 19.2 ± 7.7b "Injections of R RU were given at 9:00 A.M. and 4:00 P.M. on the day of estrus, and plasma LH and PRL concentrations were measured at 5:00 P.M. on the afternoon of expected proestrus. IIp < showed the same effect, the vaginal cornification being delayed to 1. 7 days. Inhibitory Effect of R 5020 on Ovulation. While individual treatment with R 5020 or RU led to incomplete inhibition of ovulation as determined by examination on the morning of the next expected estrus, combined treatment led to complete inhibition of ovulation (Table 3). A more detailed study of the effect ofru and R 5020 on ovulation is described in Table 4. It can be seen that the inhibitory effect of both steroids on ovulation was additive. Early Effects of R R U on Afternoon Plasma LH Levels. Since P is known to be able to advance ovulationl. 2 and can have early stimulatory effects on plasma LH in E2-primed, ovariectomized rats,3. 4 we studied the effect of combined treatment with R 5020 and RU 16117, injected during estrus, on plasma LH levels measured at 5:00 P.M. on the 4 days of the cycle. As is shown in Table 5, no peak of plasma LH or PRL could be detected on any day of the cycle, thus indicating that such treatment leads exclusively to an inhibitory effect on gonadotropin secretion. DISCUSSION The present data show clearly that the synthetic "progestins" currently used as contraceptives in the pill (especially the 19-nortestosterone derivatives) have an important androgenic activity at the anterior pituitary level on LH secretion both in vitro and in vivo. The important affinity of these steroids for the rat prostate androgen receptor and their ability to stimulate seminal vesicle and prostate weights in gonadectomized rats are in complete agreement with these findings. We have previously shown that androgens lead to a marked inhibition of the LH responsiveness to LHRH in anterior pituitary cells in culture while P alone has no effect Moreover, while E2 potentiates the stimulatory effect of P on FSH release, the effect of androgens is not affected by E2.22 The present findings of a marked inhibitory effect of "progestins" on the LH responsiveness to LHRH and the lack of a potentiating effect of E2 on the stimulatory effect of "progestins" on the FSH responsiveness to LHRH are in every respect characteristic of androgenic activity. That these "progestins" have intrinsic androgenic activity is clearly illustrated by their affinity for the androgen receptor (Table 1) and their stimulatory effect on seminal vesicle and prostate weights (Fig. 4). The synthetic "progestins" were thought to inhibit ovulation by an action at the hypothalamicpituitary level.s. 1o While the hypothalamus was proposed as a site of blockage of ovulation by norethindrone in the rabbit,9 a pituitary site of action was also suggested.10 The present data obtained with pituitary cells in culture demonstrate that both 19-nortestosterone and 17a-acetoxyprogesterone derivatives can inhibit the LH response to LHRH by a direct action at the pituitary level and that, contrary to the current dogma, the androgenic activity of these compounds may well be largely responsible for their inhibitory activity. The marked inhibition of the proestrous LH responsiveness to LHRH after treatment with R 5020 on the day of estrus suggests that the decreased sensitivity of the LH responsiveness could explain the inhibitory effect ofr 5020 on the spontaneous LH surge. However, since injections of low doses of LHRH have been shown to enhance markedly the responses to later injections of the neurohormone,23 it is possible that a decreased secretion of LHRH resulting from an inhibitory action of R 5020 at the hypothalamic level could lead to a secondary suppression of the pituitary responsiveness to the neurohormone. We have recently found that, although progesterone alone has no effect on LH release in anterior pituitary cells in culture, it can markedly inhibit the estradiol-induced increased LH responsiveness to LHRH.14 It is thus likely that at least part ofthe potent inhibitory action ofr 5020

9 1112 LABRIE ET AL. October 1977 observed in vivo is exerted at the pituitary level. An inhibitory action of R 5020 at the pituitary level is also supported by the findings that progesterone given 16 to 19 hours before LHRH was able to block the ovulatory response to an intrapituitary infusion of the neurohormone in the rabbit. 28 The present data show that R 5020, a progestin devoid of androgenic activity, is at least as potent as the most potent "progestin" used as a contraceptive, D-norgestrel, in inhibiting gonadotropin secretion. This is clearly demonstrated by its effect on the spontaneous preovulatory LH surge, on ovulation, and in delay of vaginal cornification. It is also of interest to mention that the inhibitory activities of R 5020 and RU are additive to all ofthe parameters studied. Since masculinizing effects are not infrequently associated with the current use of "progesterone" as contraceptives, the availability of an equally potent but "pure" progestin devoid of androgenic activity could be of great value. REFERENCES 1. Everett JW: Progesterone and estrogen in the experimental control of ovulation time and other features of the estrous cycle in the rat. Endocrinology 43:389, Zeilmaker GH: The biphasic effect of progesterone on ovulation in the rat. Acta Endocrinol (Kbh) 51:461, Caligaris L, Astrada JJ, Taleisnik T: Biphasic effect of progesterone on the release of gonadotropin in rats. Endocrinology 89:331, Kalra PS, Kalra SP, Krulich L, Fawcett CP, McCann SM: Involvement of norephinephrine in transmission of the stimulatory influence of progesterone on gonadotropin release. Endocrinology 90:1168, Spies HG, Niswender GD: Effect of progesterone and estradiol on LH release and ovulation in rhesus monkeys. Endocrinology 90:257, Pincus G, Chang MC, Zarrow MX, Hafez ES, Merrill A: Studies of the biological activity of certain 19-nor steroids in female animals. Endocrinology 59:695, Garcia CR, Pincus G, Roch J: Effects of three 19-nor steroids on human ovulation and menstruation. Am J Obstet Gynecol 75:82, Pincus G: Control of conception by normal steroids. Science 153:495, Kawakami M, Sawyer CH: Effects of sex hormones and antifertility steroids in brain thresholds in the rabbit. Endocrinology 80:857, Hilliard J, Croxatto HB, Hayward IN, Sawyer CH: Norethindrone blockage of LH release to intra pituitary infusion of hypothalamic extract. Endocrinology 79:411, Perrine JW: The anabolic, androgenic and antigonadotrophic properties of five synthetic 19-nortestosterone analogs. Acta Endocrinol (Kbh) 37:376, Desaulles P A, Krakenbuhl C: Comparison of the antifertility and sex hormonal activities of sex hormones and their derivatives. Acta Endocrinol (Kbh) 47:444, Drouin J, Labrie F: Selective effect of androgens on LH and FSH release in anterior pituitary cells in culture. Endocrinology 98:1528, Drouin J, Lagace L, Labrie F: Interactions of 17f3-estradiol on progesterone in the control of LH and FSH release in anterior pituitary cells in culture. Endocrinology. In press 15. Raynaud JP, Bonne C, Bouton MM, Moguilewsoky M, Philibert D, Azadian-Boulanger G: Screening for antihormone by receptor studies. J Steroid Biochem 6:615, Labrie F, Pelletier G, Lemay A, Borgeat P, Barden N, Dupont A, Savary M, Cote J, Boucher R: Control of protein synthesis in anterior pituitary gland. In 6th Karolinska Symposium on Research Methods in Reproductive Endocrinology, Edited by E Diczfalusy. Acta Endocrinol (Suppl 180), Geneva, 1973, p Midgley AR: Radioimmunoassay for human follicle-stimulating hormone. J Clin Endocrinol Metab 27:295, Odell WD, Rayford PL, Ross GT: Simple partially automated method for radioimmunoassay of human thyroid stimulating, growth, luteinizing and follicle-stimulating hormones. J Lab Clin Med 70:973, Rodbard D, Lewald JE: Computer analysis of radioligand assay and radioimmunoassay data. In 2nd Karolinska Symposium on Research Methods in Reproductive Endocrinology, Edited by E Diczfalusy. Acta Endocrinol (Suppl 147), Geneva, 1970, p Rodbard D: Apparent positive cooperative effects in cyclic AMP and corticosterone production by isolated adrenal cells in response to ACTH analogues. Endocrinology 94: 1427, Kramer CY: Extension of multiple range tests to group means with inequal numbers of replications. Biometrics 12:307, Drouin J, Lagace L, Labrie F: Estradiol-induced increase of the LH responsiveness to LH-releasing hormone (LHRH) in anterior pituitary cells in culture. Endocrinology 99:1477, Ferland L, Drouin J, Labrie F: Role of sex steroids on LH and FSH secretion in the rat. In Hypothalamus and Endocrine Functions, Edited by F Labrie, J Meites, G Pelletier. New York, Plenum Press, 1976, p Bonne C, Raynaud JP: Assay of androgen binding sites by exchange with methyltrienolone (R 1881). Steroids 27:497, Asselin J, Labrie F, Gourdeau Y, Bonne C, Raynaud JP: Binding of ("Hlmethyltrienolone (R 1881) in rat prostate and human benign prostatic hypertrophy (BPH). Steroids 28:449, Ferland L, Labrie F, Kelly PA, Raynaud JP: Inhibitory effects of RU 16117, a potent estrogen antagonist, on the estrous cycle in the rat. BioI Reprod. In press 27. Ferland L, Borgeat P, Labrie F, Bernard J, De Lean A, Raynaud JP: Changes of pituitary sensitivity to LHRH during the rat estrous cycle. Mol Cell Endocrinol 2:107, Hilliard J, Schally A V, Sawyer CH: Progesterone blockage of the ovulatory response to intrapituitary infusion of LHRH in rabbits. Endocrinology 88:730, 1971