sign in sign up
<<
Home , Desogestrel, Gestrinone

Endocrinol. Japon. 1989, 36 (3), 387-394

Inhibition of Rat Ovarian 3ƒÀ-Hydroxysteroid Dehydrogenase (3ƒÀ-HSD), 17ƒ¿-Hydroxylase and 17,20 Lyase by Progestins and

SATOKO ARAKAWA, MIZUE MITSUMA, MASATO IYO, RYOICHI OHKAWA, AKIRA KAMBEGAWA, SHoicm OKINAGA AND KIYOSHI ARAI

Department of Obstetrics and Gynecology, Teikyo University School of Medicine 2-11-1 Kaga Itabashi-ku, Tokyo, Japan

Abstract

The site of action of synthetic progestins or danazol in the treatment of

is considered to be mainly the hypothalamo-pituitary level, but

the direct action to the uterine and the is also suggested. We investigated the effect of these synthetic to rat ovarian steroido-

genic . The effect of , , danazol, gestri- none, and 3-keto-desogestrel was studied . The sources of the enzymes were prepared from of immature rats treated either with

pregnant mare serum (PMS) and human chorionic gonadotropin

(hCG) for 3ƒÀ-hydroxy dehydrogenase (3ƒÀ-HSD), or with PMS for 17ƒ¿- hydroxylase and 17,20 lyase. The substrates used were (P5) for

3ƒÀ-HSD, (P4) for 17ƒ¿-hydroxylase, and 17ƒ¿-hydroxy-progesterone

(17ƒ¿-OH-P4) for 17,20 lyase. The substrates were incubated with the sources and coenzymes, and the products formed were measured. All the

steroids inhibited 3ƒÀ-HSD, and the inhibition by (Ki= 3.0ƒÊM) and 3-keto-desogestrel (17.5ƒÊM) was particularly marked. Only desogestrel (Ki= 30.3ƒÊM) and danazol (168ƒÊM) inhibited 17ƒ¿-hydroxylase. All the steroids

inhibited 17,20 lyase, and the inhibition by desogestrel (Ki= 0.70ƒÊM), danazol

(0.80ƒÊM), and gestrinone (30ƒÊM) was particularly marked.

The site of action of synthetic progestins and danazol (Barbieri et al.; 1977, Barbieri and danazol in the treatment of endo- et al.; 1981) inhibit ovarian enzymes di- metriosis is considered to be mainly the rectly. In the following study, we investi- hypothalamo-pituitary level (Franchimont gated the effect of synthetic steroids on rat and Cramilion, 1977; Franchimont et al., ovarian 3ƒÀ-hydroxy steroid dehydrogenase 1970; McDonald and Gilmore, 1971; (3ƒÀ-HSD), 17ƒ¿-hydroxylase, and 17,20 Cullberg et al., 1982; Garmendia et al., lyase. 1976). But the direct effect of synthetic steroids on ovarian enzymes was also re- ported. Norethisterone (Shinada et al., Materials and Methods 1978;Johansson, 1971), levonorgestrel (Mukherjee et al.; 1972, Johansson; 1971) Chemical Received January 20, 1989 [4-14C]-pregnenolone (P5) (57.2 mCi/mmol), Endocrinol. Japon. 388 ARAKAWA et al. June 1989

[4-14C]-progesterone (P4) (57.2 mCi/mmol),[4-14C]- hibitor. For assays of 17ƒ¿-hydroxylase and 17ƒ¿-hydroxy progesterone (17ƒ¿-OH-P4) (50 mCi/ 17,20 lyase, each incubation mixture (1ml) con-

mmol), [1, 2, 6, 7-3H(N)]-P4 (106.8 Ci/mmol) and tained a preparation of from PMS treated 17ƒ¿-[1, 2-3H(N)] hydroxy P4 (57.4 Ci/mmol) were rats (12-15ƒÊg protein), 0.5ƒÊmol of NADPH, 14C- or 3H-steroid (about 0 purchased from New England Nuclear Corp. .03ƒÊCi, 0.1-4 mol) (Boston, MA, USA). NAD and NADPH were and an inhibitor. Labelled P4 was employed the products of Boehringer (Mannheim, Ger- for a 17ƒ¿-hydroxylase assay, and labelled 17ƒ¿- many). Norethisterone, levonorgestrel, pregnant OH-P4 for a 17, 20 lyase assay. The mixture

mare serum gonadotropin (PMS) and human was incubated in a Dubnoff-type incubator at chorionic gonadotropin (hCG) were provided 37•Ž for various time intervals. The incubation

by Teikoku Co.(Japan, Tokyo). was terminated by chilling in an ice bath. Desogestrel and 3-keto-desogestrel were a gift Steroids were extracted with ether (5ml). A from Organon Co.(Holland). Danazol was mixture of nonradioactive carrier steroids was provided by Tokyo Tanabe (Japan), and gestri- added to each extract, which was then applied none by Roussel Uclaf (Paris, France). All to a silica gel thin layer plate and developed

chemicals and reagents used were of analytical in a solvent system of benzene-acetone (4:1, grade. v/v). Steroids which were not separable in this system were sub jected to further thin layer Preparation of enzymes chromatography in the following solvent system: Immature female rats of the Wistar-Imamichi cyclohexane-ethyl acetate (1:1, v/v) was employ-

strain were used. Ten IU of PMS was injected ed for the separations of from subcutaneously (s. c.) at 9:00 h, on the 23rd day 3ƒ¿-hydroxy-5ƒ¿-pregnan-20-one and of 17ƒ¿-OH- of life. PMS treated rats were decapitated at P4 from 3ƒÀ-hydroxy-5ƒ¿-pregnan-20-one. Andro- 17:00 h two days later. Another group of rats stenedione and 17ƒ¿-hydroxy-5ƒ¿-pregnane-3,20- were given an injection of 30 IU of hCG at dione were completely separated by developing

14:00 h two days after PMS injection, and killed the plate three times in ethyl acetate-n-heptane three hours after the last treatment. Ovaries (2:5, v/v). Spots of the carrier on thin layer were homogenized in a teflon glass homogenizer chromatograms were located under an UV light with 0.25 M sucrose/0.05 M Tris HC1 buffer and (254 nm) or by being exposed to iodine vapor. then centrifuged at •~ 800 g for 20 min. The The 14C-spots were detected by autoradiography. supernatant was used as enzyme sources. Pro- Each spot was scraped off the plate, added with tein in the supernatant was determined by the 0.1ml methanol and 3ml toluene scintillator, Bradford's method (Bradford, 1976). and the radio-activity was measured in a liquid

scintillation spectrometer (Aloka LSC 703). The Enzyme assay recovery of steroids was 55-70%. For an assay of 3ƒÀ-HSD, each incubation The enzyme activity was expressed as the mixture (1ml) contained a preparation of tissue sum of metabolites formed from radioactive from PMS-hCG treated rats (2.5-100ƒÊl of the substrates. Some of the results were analyzed enzyme; 1.5-61.1ƒÊg protein), 0.5ƒÊmol of NAD, by the method of Lineweaver-Burk (Lineweaver 14C-P5 (about 0 .03ƒÊCi, 1-100 nmol), and an in- and Burk, 1934).

Table 1. Title;Identification of radioactive metabolites formed by repeated recrystallizations to constant specific radioactivities. Regend; Methanol-chloroform or methanol-water were used as the recrystallization solvent. Vol. 36, No. 3 INHIBITION OF OVARIAN ENZYME 389

Fig. 1a Statistical Analysis Statistical analysis of the results was done by Student's t-test, and a P value of less than 0.05 was considered significant.

Results

Autoradiography 3ƒÀ-HSD: A metabolite formed from P5 was only P4 under the experimental condi- tions used and the 3ƒÀ-HSD activity was expressed by the amount of P4 17ƒ¿-hy- droxylase: Metabolites formed from P4 were Fig. 1b 17ƒ¿-OH-P4, androstenedione and a small amount of other steroids. The 17ƒ¿-hy- droxylase activity was expressed as the sum of 17ƒ¿-OH-P4 and androstenedione formed.

17, 20 lyase: Metabolites from 17ƒ¿-OH-P4 were androstenedione and a small amount of other steroids. The 17, 20 lyase activity was expressed by the amount of andros- tenedione. These metabolites were crystallized re-

peatedly with the corresponding standard steroids. Constant specific radioactivity of the crystals is shown in Table 1.

Effect of incubation time and volume of the tissue preparation Fig. 1c Fig. la shows the relation between the

product (P4) formed and the amount of enzyme in the 3ƒÀ-HSD assay. The con- centration of the substrate was 10ƒÊM, and the incubation time was 20 min. Fig. 1b and c show the relation between the amount of products formed and the incubation time in 17ƒ¿-hydroxylase and 17, 20 lyase. The concentration of the substrate was 1ƒÊM and the amount of the enzyme was 25ƒÊl

(16.2ƒÊg protein). A subsequent study was performed in which the enzymatic reaction was not saturated. For the experiment on 3ƒÀ-HSD, 25ƒÊl of the enzyme (15.3ƒÊg Fig. 1. Title; Effects of the amount of the tissue preparation used for a) 3ƒÀ-HSD protein) was incubated for 20 min. For assay and of incubation time for b) 17ƒ¿- 17ƒ¿-hydroxylase, 25ƒÊl of the enzyme was hydroxylase and c) 17, 20 lyase. incubated for 10 min. For 17, 20 lyase, the Endocrinol. Japon. 390 ARAKAWA et al. June 1989

*P<0 .05

Fig.2 c **P<0. 01 ***P<0 .001

*P<0 .05 Fig.2 b **P<0 .01

*P<0 .05

Fig .2 a

Fig. 2. Title; Inhibition of a) 3ƒÀ-HSD, b) 17 ƒ¿-hydroxylase and c) 17,20 lyase by various steroids. Regend; Vertical bars indicate mean•}standard error. 391 Vol.36, No.3 INHIBITION OF OVARIAN ENZYME

Fig. 3a Fig. 3c

LEVONORGESTREL PROGESIERONE NORETHISTERONE NOFIETHISTERONE 100μM 20μM 20μM 100μM

LEVONORGESTREL DESOGESTREL 3KETO-DESOGESTREL 20μM 20μM DESOGESTREL 100μM 2μM

GESTRINONE 3KETO-DESOGESTREL 2μM 20μM DANAZOL GESTPINONE 2μM 100μM

DANAZOL 20μM

Fig. 3. Title; Lineweaver Burk's plot in the inhibition study of a) 3ƒÀ-HSD,

b) 17ƒ¿-hydroxylase and c) 17,20 lyase by various steroids.

3b

DESOGESTREL DANAZOL

ficantly when the concentration was 20μM enzyme volume was 20 or 25ƒÊl and the incubation time was 16 or 20min. (Fig. 2b). Most of the steroids inhibited 17,20 lyase significantly (Fig. 2c).

Inhibition of enzymes by steroids Fig. 3 shows Lineweaver Burk's plot Fig. 2a shows the effect of various indicating the inhibitory effects of steroids steroids on 3ƒÀ-HSD activity. All steroids on enzymes. Table 2 summarizes the inhibited 3ƒÀ-HSD significantly when 20ƒÊM results of the kinetic studies on steroida- was used. However, no steroid, except genic enzymes. The inhibition of 3ƒÀ-HSD desogestrel, inhibited 17ƒ¿-hydroxylase signi- by gestrinone (Ki = 3.0ƒÊM) and 3-keto- Endocrinol. Japon. 392 ARAKAWA et al. June 1989

desogestrel (17.5ƒÊM) was particularly mark- serum level of P4, and ed. When the steroid concentration was in women (Cullberg et al., 1982). This is 100ƒÊM, only desogestrel (Ki = 30.0ƒÊM) and mainly due to the inhibitory effect of its danazol (168ƒÊM) inhibited 17ƒ¿-hydroxy- active form, 3-keto-desogestrel on gonadot- lase, whereas in the assay of 17,20 lyase, ropin release from the pituitary, but the

the inhibition by desogestrel (Ki =0.70ƒÊM), possibility now arises that both desogestrel danazol (0.80ƒÊM) and gestrinone (30ƒÊM) and 3-keto-desogestrel directly inhibit ovarian was particularly marked. steroidogenesis. It is reported that levonor-

The Km values for the enzymes were gestrel (Mukherjee et al., 1972; Nygren and 2.73•}1.34 (3ƒÀ-HSD, P5 as a substrate), Johansson, 1975; Garmendia et al ., 1976) 2.9 (17ƒ¿-hydroxylase, P4 as a substrate) and and danazol (Menon et al., 1980) suppress 0.635•}0.0365ƒÊM (17,20 lyase 17ƒ¿-OH-P4 the serum steroid levels, and their direct as a substrate). The Ki values are listed action on ovarian enzymes may also be in Table 2. considered now.

All the steroids inhibited 3ƒÀ-HSD . The synthetic steroids except desogestrel and Table 2. Title; The Ki values for steroidogenic danazol inhibited 3ƒÀ-HSD more effectively enzymes in rat ovary. than they did 17ƒ¿-hydroxylase and 17 Regend; N.D.; inhibition was not ,20 lyase. From these results, it may be pos- detected. tulated that the inhibition by the synthetic steroids of 3ƒÀ-HSD is the most important step among the three ovarian enzymes studied

to bring about a decrease in steroidogenesis .

In the present experiment, 17 ,20 lyase was very readily inhibited by the synthetic

steroids but 17ƒ¿-hydroxylase was not . In natural steroid biosynthesis the inhibition of 17,20 lyase has less significance than 17ƒ¿-hydroxylase, because this enzyme is closely related to 17ƒ¿-hydroxylase, and the Discussion rate limiting step from P4 to androstenedione is 17ƒ¿- (Lieberman et al., 1984; Synthetic progestins and danazol in the Eckstein et al., 1985). Moreover, the enzyme treatment of endometriosis are considered complex is lipophilic, so that P4, which is to act mainly at the hypothalamo-pituitary more lipophilic than 17ƒ¿-OH-P4, can readily level, but the present experimental results attach to the enzyme, and thus P4 becomes suggested that they may also have direct a better substrate to the enzyme complex effects on ovarian steroidogenic enzymes. than 17ƒ¿-OH-P4 (Johnson and Griswold, Desogestrel, which is an inactive form, 1983; Johnson and Griswold, 1984; does not bind to Matsumoto and Samuels, 1969; Eckstein et (Cullberg, 1985), and consequently has no al., 1985). In our experiment, when P4 was progestational effect. But the present study used as a substrate for 17ƒ¿-hydroxylase, the showed that it inhibited 17ƒ¿-hydroxylase androstenedione/17ƒ¿-OH-P4 ratio was stable and 17,20 lyase more markedly than 3-keto- with or without added synthetic steroids desogestrel (the active form) did. Cullberg (data not shown). Thus, androstenedione et al. reported that desogestrel reduced the production from P4 was liable to less in- 393 Vol.36, No.3 INHIBITINN OE OVARIAN ENZYME hibition than progesterone production from quantities of protein utilizing the principle of P5. protein-dye binding. Analytical Biochemistry 72, 248-254. It is reported that 5a-reductase activity Cullberg, G., G. Lindstedt, P. Lundberg and is high in the ovary of the immature rat K. Steffensen (1982). Central and peripheral (Shinada et al., 1978). In the present series effects of desogestrel 15-60ƒÊg daily for 21 of experiments, a few products which were days in healthy female volunteers. Acta Obstet. presumed to be metabolites of 5ƒ¿-reductase Gynecol. Scand. suppl. 111, 21-28. were found in small quantities on a thin Cullberg, G.(1985). Pharmacodynamic studies layer chromatograms. Since these radioac- on desogestrel administered alone and in combination with . Acta Obstet. tive metabolites formed were limited in Gynecol. Scand. suppl. 113, 1-29. amounts, no attempt was made to identify Eckstein, B., O. Greenbaum and S. Cohen (1985). them. Kinetic studies on ovarian C-17,20-lyase ac- The Km value of rat ovarian 17,20 tivity: effect of surge. lyase is reportedly 62.2ƒÊM (Weiss and Endocrinology 117, 2376-2382.

Eckstein, 1984) or 13.6ƒÊM (Eckstein et al., Franchimont, P., G. Cesson, D. Ayalon, A. 1985). The present study showed that it Mutsers and J. J. Legros (1970). Suppressive was considerably lower than the reported action of and acetate on gonadotropin (FSH and LH) levels: Radio- values. Different experimental conditions immunoassay in eugonadal and postmenopausal may be responsible for the discrepancy, and women. Obstet. Gynecol. 36, 93-100. further studies are needed concerning this. Franchimont, P. and C. Cramilion (1977). It is concluded that all the steroids The effect of danazol on anterior pituitary

inhibited 3ƒÀ-HSD, and the inhibition by function. Fertil. Steril. 28, 814-817.

gestrinone and 3-keto-desogestrel was con- Garmendia, F., E. Kesseru, U. Enrique and V. spicuous, suggesting that these steroids may Manuel (1976). Luteinizing hormone and inhibit P4 production at the ovarian level. progesterone in women under postocoital con- traception with D-. Fertil. Steril. Only desogestrel and danazol inhibited 17ƒ¿- 27, 1250-1255. hydroxylase. Johansson, E. D. B.(1971). Luteolytic effect of

gestagens. Acta Obstet. Gynec. Scand. 50, 75. Johnson, D. C. and T. Griswold (1983). The Acknowledgements C17,20-lyase, 17a-hydroxylase and aromatase activity in rat ovarian granulosa cells stimulated

in vivo by . Steroids 42, 565- This work was partly supported by Grant- 574. in-Aid No. 63570799 from the Ministry of Education, Science and Culture, Japan. Johnson, D. C. and T. Griswold (1984). Ovarian C17,20-lyase: Changes in intact and hypophy-

sectomized immature rats treated with pregnant mare's serum gonadotropin. J. Steroid Bio-

References chem. 20, 733-739.

Lieberman, S., N. J. Greenfield and A. Wolfson

Barbieri, R. L., J. A. Canick, A. Makris, R. B. (1984). A heuristic proposal for understanding Todd, I. J. Davies and K. J. Ryan (1977). steroidogenic process. Endocr. Rev. 5, 128- Danazol inhibits steroidogenesis. Fertil. Steril. 148.

28, 809-813. Lineweaver, H. and D. Burk.(1934). The Barbieri, R. L., R. Osathanondh and K. J. Ryan determination of enzyme dissociation constants. (1981). Danazol Inhibition of steroidogenesis J. Am. Chem. Soc. 56, 658-666. in the human corpus luteum. Obstet. Gynecol. Matsumoto, K. and L. T. Samuels (1969). In- 57, 722-724. fluence of steroid distribution between micro- Bradford, M. M.(1976). A rapid and sensitive somes and soluble fraction on steroid meta- method for the quantitation of microgram bolism by microsomal enzymes. Endocrinology Endocrinol. Japon. 394 ARAKAWA et al. June 1989

85, 402-409. Nygrren, K. and E.D.B. Johansson (1975). The McDonald, P. G. and Di P. Gilmore (1971). effect of norethisterone and some other synthe- The effect of norethindrone on hypothalamic tic gestagens upon the peripheral plasma levels and pituitary thresholds for the induction of of progesterone and estradiol during early, in the rat. Neuroendocrinology 7, human . Acta. Obstst. Gynec. Scand. 46-53. 54, 57-63. Menon, M., S. Azhar and K. M. J. Menon (1980). Shinada, T., Y. Yokota and M. Igarashi (1978). Evidence that danazol inhibits gonadotropin- Inhibitory effect of various gestagens upon induced ovarian steroidogenesis at a point the pregnenolone 313-ol-dehydrogenase-45-4- distal to gonadotropin-receptor interaction and isomerase system in human corpora lutea of adenosine 3', 5' cyclic monophosphate forma- menstrual cycles. Feral. Steril. 29, 84-87. tion. Am. J. Obstet. Gynecol. 136, 524-530. Weiss, M. and B. Eckstein (1984). Periovulatory Mukherjee, T.K., S.W. Wright, N.J.H. Davidson changes in steroid C17,20-lyase activity in and K. Fotherby (1972). Effect of norgestrel ovaries of immature rats treated with pregnant on corpus luteum function. J. Obstet. Gynecol. mare serum gonadotropin. Endcrinology 114, 79, 175-182. 1912-1916.