View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Volume 144, number 2 FEBS LETTERS August 1982 Norethisterone does not inhibit rat testis A5- 3/?-hydroxysteroid dehydrogenase J. Spona Endocrine Research Unit, First Department of Obstetrics and Gynecology, University of Vienna, A 1090 Vienna, Austria Received 10 June 1982 Norethisterone as-3P-hydroxysteroid dehydrogenase Rat testis 1. INTRODUCTION and stored at - 20” C. The cyanoketone used was 2~ cyano- 4,4,17cu~trimethylandrost-5-en-17fi-ol-3-one. A key step in the biosynthesis of progesterone /3-Nicotinamide adenine dinucleotide was pur- and of androgens is the production of A4-3-keto- chased from Sigma Chem. Co. (St Louis MO). All steroids from A5-3/I-hydroxysteroids [ 11. This enzy- other chemicals were of analytical grade and were matic conversion is a two step process in which obtained from Sigma, Boehringer or Merck AG dehydrogenation is followed by isomerization 121. (Darmstadt). The first step is rate limiting, and the enzyme which mediates the dehydrogenation is A5-3&hydroxy- 2.1. Enzyme extraction from rat testes steroid dehydrogenase (EC 1.1.1.145); it can be Testes of adult male rats of the Sprague-Dawley found in extracts of Pseudomonas testosteroni [3] strain (Himberg; Oncins, specific-pathogen free, and is localized in the microsomes and mitochon- 200-250 g body wt were obtained from For- dria of all steroid synthesizing organs such as schungsinstitut fur Versuchstierzucht, University of adrenal, ovary, placenta and testis [4]. Vienna. The animals were used 2 weeks after ship- The enzymes from mammalian and bacterial ment. The rats were housed in a temperature-con- sources were reported to exhibit similar properties trolled room lit for 12 h/day and unrestricted access [5] and were found to be inhibited by estrogens, was provided to food and water. The rats were progestins, androgens and steroidal analogues killed by decapitation and the testes were dissected, [5,16]. Genital defects such as hypospadias in male decapsulated and rinsed with ice-cold buffer 1 (50 infants were reported to be associated with the ad- mM phosphate buffer, 0.25 M sucrose, 7 mM di- ministration of progestins and estrogens to preg- thiothreitol, 0.5 mM EDTA, pH 7.4). All further nant women or female animals 191. Inhibition by procedures were done at 0-4°C. Tissue (1 g/ml these steroids of A5-3/3-hydroxysteroid dehydro- buffer 1) was homogenized by 10 strokes at 1000 genase has been assumed to cause hypospadias rev./min in a glass-Teflon Potter Elvehjem-type [9,10,17]. homogenizer. The homogenate was centrifuged at Here, we have studied the effects of some proges- 800 x g for 10 min. The crude homogenate was tins and estrogens on rat testis A5-3/%hydroxy- centrifuged at 10 000 X g for 20 min and the steroid dehydrogenase activity using both de- supernatant was spun at 100 000 x g for 1 h to ob- hydroepiandrosterone and pregnenolone as sub- tain the microsomal pellet. The microsomes were strate. washed with buffer 1 and recentrifuged at 100 000 x g for 1 h. The pellet was resuspended by homo- genizing it in 1 ml buffer l/pair of testes to obtain 2. MATERIALS AND METHODS 5-10 mg protein/ml. The suspension was stored at All steroids used in this study were obtained from -20°C until used for enzyme assays. Schering AG (Berlin) and were dissolved in ethanol Published by Elsevier Biomedical Press OO145793/82/ooOO-0000/$2.75 0 1982 Federation of European Biochemical Societies 283 Volume 144. number 2 FEBS LETTERS August 1982 2.2. Enzyme assay as dehydroepiandrosterone and pregnenolone has The enzyme preparation (300 ~1) containing been studied in many laboratories. The present in- 1.5-4 mg protein was preincubated at 37°C with vestigation was done to delineate possible differ- 750 ~1 buffer (0.2 M pyro hosphate, pH 8.9) 200 ~1 ences between C- 19 and C-2 1 testicular deydrogen- 4 NAD solution (5 X 10 - M, pH 7) and 1550 ~1 ases and to investigate whether A5-3fi-hydroxy- water. The reaction was started with dehydro- steroid dehydrogenase is inhibited by estrogens and epiandrosterone or pregnenolone as substrate dis- progestins. Data on the Michaelis constants (table solved in ethanol. The total volume of ethanol did 1) add further evidence to the suggestion [21-241 not exceed 100 ~1, and the blank contained all that A5-3P-hydroxysteroid dehydrogenases medi- components except for the substrate. For the deter- ating the dehydrogenation of C- 19 and C-2 1 steroids minations of inhibition constants the reaction in the testes are identical: equimolar mixtures of mixture was preincubated with the steroid to be dehydroepiandrosterone and pregnenolone did not tested prior to the addition of substrate. Absorbance give additive rates, but maximal velocities interme- was recorded at 340 nm by a Beckman spectro- diate between those of the separate substrates. How- photometer model DU-8 kinetic analysis system ever, multiple A5-3fl-hydroxysteroid dehydrogen- (Beckman Instr., Irvine CA) at 37°C. AAlmin was ases in bovine ovarian microsomes were proposed registered 3 times through 3 min intervals and cal- in [21]. Experiments on hydride-ion transfer from culated as means of 3 such determinations. the 2 substrates to NAD+ suggested the presence of 1 enzyme which metabolized only dehydroepi- 2.3. Other techniques androsterone and 1 enzyme which used both pregnen- Michaelis constants were determined by linear olone and dehydroepiandrosterone [2 11. Evidence regression analysis of Lineweaver Burk [ 181 data for multiple dehydrogenases may raise many im- and inhibition constants were calculated from Dixon-plot [ 191 data by linear regression on a Dee Data System 34/l 1 (Digital Equipment Corp., Table 2 Maynard, MA). Protein measurements were done Determination of apparent inhibition constants (K,) of as in [20] using bovine serum albumin as standard. A5-3P-hydroxysteroid dehydrogenase for various steroids using dehydroepiandrosterone (5.78 X 10 -6 M or 46.24 10e6 M) or pregnenolone (5.27 10e6 M or 3. RESULTS AND DISCUSSION x x 42.12 x 10e6 M) as substrate Whether or not a single A5-3P-hydroxysteroid dehydrogenase metabolizes various substrates such Steroid K, (M x 10-6) Dehydroepi- Pregnen- androsterone olone Table 1 Determination of Michaelis constants of d5-3fi-hydroxy- Norethisterone n.i. n.i. steroid dehydrogenase from rat testes microsomes for Norethisterone acetate 150 175 various substrates Diethylstilbestrol 63 29” Progesterone n.i. n.i. Steroid K,(M x 10-6) Cyproterone acetate 47 ni. Cyanoketone 0.03 0.03a Dehydroepiandrosterone 5.1 Androstenedione 30 40 Pregnenolone 2.3 17/3-Estradiol n.i. n.d. Dehydroepiandrosterone plus 17a-OH-Progesterone n.d. n.i. pregnenolone (equimolar) 3.4 a non-competitive Rat testes microsomal enzyme preparation (300 ml) con- n.i., not inhibitory; n.d., not determined taining 1.5-4 mg protein was incubated with various Steroids were preincubated with 300 ~1 enzyme prepara- concentrations of different substrates in the presence of tion containing 1.5-4 mg protein in the presence of 5 x 10 -3 M NAD. Data are given as means of 3 different 5 x 10 -3 M NAD. Data are given as means of two dif- experiments ferent experiments 284 Volume 144, number 2 FEBS LETTERS August 1982 portant questions concerning the distribution of the sexual organogenesis was thought to be analogous enzymes in different cell types and the possibility of to that observed in infants born with a severe form different effects of trophic hormones. These data of congenital adrenal hyperplasia due to genetic suggest a single enzyme but examination of stereo- deficiency of A5-3/?-hydroxysteroid dehydrogenase. specificity of hydride transfer may yield a more In addition, injection of cyanoketone into preg- definitive answer to the question. nant rats was found to inhibit adrenal and testicular Of major interest is the observation that nor- A5-3fi-hydroxysteroid dehydrogenase as noted by ethisterone does not inhibit testicular A5-3p- histochemical determinations and was registered hydroxysteroid dehydrogenase, when dehydroepi- to produce hypospadias [lo]. But a recent review androsterone or pregnenolone is used as substrate [25] concludes that adverse effects of synthetic pro- (table 2). Inhibition by progestins of this enzyme gestins on the fetus had been shown in some, but not was proposed for the development of hypospadias all, of the reports, and that any positive effects reg- in male infants born from females who were treated istered were small. Inhibition by norethisterone of with norethisterone or other synthetic progestagens testicular of A5-3/I-hydroxysteroid dehydrogenase 1171. This assumption was made on grounds of activity could not be detected by the present assay results of experiments in which male fetuses of system when dehydroepiandrosterone or pregnen- pregnant rats treated with these steroids were found olone were used as substrates (table 2). In addi- to develop hypospadias. The effect of progestins on tion, no great differences were noted between the I/V 7576- 6818- 6061- 5303- 4546 - 3788- 3030- 2273- I I I I I I t MxlCP 13.3 26.6 39.9 53.2 66.5 79.8 Fig. I. Dixon plot of inhibition by androstenedione of testicular A5-3jShydroxysteroid dehydrogenase: (o) 5.2 x 10P6 M pregnenolone; ( .cJ 42.18 X 10 -6 M pregnenolone: V = velocity in mU . min -I . mg protein -I. 285 Volume 144. number 2 FEBS LETTERS August 1982 4802 - 4268- 3135 - 3201- 2668 - 2134- 1601 - 1067 - 534 - 0 0’ -?/ 1 1 I I , I 1 W- MxlO-" 17.8 35.6 53.4 71.2 89.0 106.8 124.6 142.5 Fig.2.