Biochemical Characterization of the Nadph : 4-Ene-3-Ketosteroid %Oxidoreductase in Rat Ovarian Suspension Cultures

Jour~l o/Steroid Biochemistry. Vol. IO. pp. 325 lo 333. Pcrgamon Press Ltd. 1979. Printed in Great Britain

BIOCHEMICAL CHARACTERIZATION OF THE NADPH : 4-ENE-3-KETOSTEROID %OXIDOREDUCTASE IN RAT OVARIAN SUSPENSION CULTURES

P. KONINCKX, G. VERHOEVEN, W. HEYNS and P. DE MOOR Laboratory of Experimental Medicine, Department of Developmental Biology, Kathoheke Universiteit Leuven, Rega Instituut, Minderbroedersstraat 10, B-3000 Leuven, Belgium

(Received 26 June 1978)

SUMMARY

A method was devised to measure the NADPH:4-ene-3-ketosteroid Sa-oxidoreductase of ovarian cells, in suspension or in monolayer culture. The enzymatic activity was assayed by measuring initial conversion rates of [sH]-17epitestosterone to its 5a-reduced metabolita. With this substrate no Ill-keto derivatives were formed, thus facilitating the chromatographic separation of the reaction products. Before and during the incubation with the tritiated substrate, aminoglutethimide (10m4 M) was added to the incubation medium in order to block the endogeneous steroid production. The characteristics of the Sa-reductase in living cells were investigated and compared with those found in ovarian homogenates. In living cells the apparent K, approximated 0.78 FM. The Sa-reductase of l’lepitestosterone was inhibited by 4-ene-3-keto-steroids without an oxygen substitution on carbon 11 but not by Sa-reduced steroids. 17/%atradiol in high concentration functions as an uncompetitive inhibitor. In homogenates an apparent K M of 0.57pM was measured. The enzyme exhibited a pH optimum around 7 and required NADPH as cofactor. Inhibition of the enzyme activity by 17/?-estradiol could not be demonstrated under these conditions. The characteristics of the Sa-reductase in the rat ovary are thus comparable to those described in androgen target cells. The activity of the Sa-reductase in living cells was comparable with the activity found in homogenates supplemented with NADPH. Cofactor availability is thus not a limiting factor for Sa-reductase activity in ovarian cells.

INTRODUCTION Since in a steroidogenic tissue both the function of the enzyme and the local steroid hormone concen- The Sa-reductase is a widespread enzyme activity trations are different from those in the liver or in transforming 4-ene-3-keto steroids into Su-reduced androgen target tissues it seemed important to deter- steroids. Originally Sa-reduction was studied in the mine the characteristics of the Sa-reductase in the rat liver where it plays a catabolic role. Later on it was ovary. In order to mimic the in uivo situation as shown to be an activating mechanism in the androgen closely as possible this was done on living cells. The target cell and more recently this enzyme activity was results are compared with those found in ovarian also detected in steroidogenic organs such as the homogenates and with the biochemical characteristics testis, the ovary and the adrenal [l]. Evidence is described in the liver [S], and in androgen target accumulating that Sa-reductase contributes to the organs such as the prostate [9], the kidney [lo], the regulation of steroidogenesis in the adrenal [Z] and brain [ll, 123 and the skin [13]. that Sa-reduced metabolites are involved in follicular maturation [3] and steroid hormone production [44] in the ovary. MATERIALS AND METHODS In order to study the hormonal regulation of the Animals products and incubation media. Wistar rats rat ovarian Sa-reductase activity in oitro [7] a method (Pfd: Wist l), 60-90 days old, were obtained from the had to be developed to assay this enzyme activity breeding center of the Catholic University of Leuven. in living cells. They were housed in an environment of constant Trivial names and abbreviations used: Dihydrotestoster- light-dark regimen and temperature, and they one (DHT): 17@hydroxy-5a-androstan-3-one; 17-Epidihy- received a constant pellet diet. drotestosterone, (17-epiDHT), 17a-hydroxy-5a-androstan- [1,2-3H]-17-Epitestosterone (50 Ci/mmol) and [1,2- 3-one; 17-Epitestosterone, 17a-hydroxy-4-androsten-3-one; testosterone, 17/I-hydroxy-4-androsten-3-one; Sa-Reduc- ‘HI-testosterone (50 Ci/mmol) were purchased from tase, NADPH 4-ene-3-ketosteroid-Sa-oxidoreductase (EC New England Nuclear and repurified on silica gel t.1.c. 1.3.1.99). (chloroform-acetone, 35 :5, V/V) before use. Unlabeled S.B.10/3--F 326 P. KONINCKX et al.

steroids were obtained from Sigma and Steraloids; A purified nuclear fraction was prepared as described Sa-androstane-3B,17a-diol was received from Schering by Verhoeven and De Moor[lS]. Co., Belgium. Solvents were analytical grade from Assay of the Sa-reductase. The Sa-reductase was Merck and aminoglutethimide was a gift from Hoff- assayed by measuring the initial conversion rate of mann-La Roche. Bovine pancreatic trypsin, soya [‘HI-17-epitestosterone to its Sa-reduced metabolites. bean trypsin inhibitor, deoxyribonuclease (DNAse), Standard solutions of labeled and unlabeled steroids collagenase and glucose-6-phosphate dehydrogenase were evaporated to dryness under a nitrogen stream were purchased from Boehringer. Bovine serum albu- and solubilized in the incubation medium in a shak- min (BSA), NADPH and NADH were purchased ing water bath at 37°C for at least 10 min. Before from Sigma and minimum essential medium (MEM) the assay the cells were incubated with 10m4 M and foetal calf serum from Flow laboratories. aminoglutethimide for 2 h, and during the incubation The media used for the preparation of a cell sus- 10e4 M aminoglutethimide was present in the incuba- pension and for the assay of the Sa-reductase acti- tion mixture also. For the assay of the Sa-reductase vity were Hanks buffered saline solutions pH 7.2, lo-’ M 17-epitestosterone and about 0.05 PCi supplemented with potassium penicillin G (100 U/ml) [3H]-17-epitestosterone were used. When cell homo- and streptomycin (2OO&ml): For tissue blocks BSA genates were used, the medium was supplemented and Ca2 + were present at a concentration of 3 mg/ml with an NADPH generating system [15] consisting and 1 mM respectively; for dissociated cells, the BSA of glucose-6-phosphate (5 mM), NADP (3.10m4 M) concentration was raised to 5 mg/ml and the Ca2+ and glucosed-phosphate dehydrogenase (0.87 HI/ml). concentration was reduced to 0.1 mM. To prepare The volume of the incubation mixture was made up Ca2+ and Mg2+ free media, these salts were simply to 2ml. The incubation was performed in a shaking omitted. water bath at 37°C and the reaction was stopped Monolayer cultures were performed in MEM sup- by two drops of HCl 6N. Steroids were extracted plemented with potassium penicillin G (lOOU/ml), immediately with 5 vol. of a mixture of cyclohexane streptomycin (2OOyglml) and 10% foetal calf serum. and ethylacetate (1: 1, V/V). The extract was washed All.glassware was siliconized and all media were milli- twice with HCl 1 N and twice with distilled water pore filtered (GSWP, 0.22 p pore diameter). and about 4Opg of 17-epitestosterone, 17-epiDHT Cell dispersion; suspension and monolayer cultures. and 5a-androstane-3/?,17adiol were added. After the Ovarian cell suspensions were prepared using the addition of reference steroids the extracts were technique described by Hopkins and Farquehar[14] evaporated to dryness under a nitrogen stream and with some modifications. Animals were killed by ether chromatographed twice on precoated Silicagel F-254 anaesthesia, the ovaries were removed aseptically, glass plates in chloroform-acetone (35: 5). Reference chilled immediately on ice and freed of fat tissue. steroids were localized under U.V. at 24Opm or at After an incubation with collagenase (0.25x, 15 min) 350pm after spraying the plates with primulin, [16]. the corpora lutea and the largest follicles were Radiometabolites were localized by means of a Pack- removed by microdjssection. The remaining tissue ard radiochromatogram scanner. The zones corre- (mainly stromal tissue) was chopped with a scalpel sponding to the unreacted substrate and the metabo- into blocks of about 1 mm’. The pooled blocks were lites were scraped off, eluted directly in the scintilla- washed three times and treated sequentially with col- tion fluid and counted in a Packard Tri-Carb liquid lagenase (0.25x, 60 mink trypsin (O.l%, 10 min) tryp- scintillation counter. The total radioactivity of each sin inhibitor (O.l%, 10 min), DNAse (O.l%, 1 min), chromatogram was calculated by adding the radio- and Ca2+ and Mg2+ free medium. All incubations activity of the individual zones, and the radioactivity were performed in a shaking water bath at 37°C. of a particular zone was expressed as a percent of Finally the tissue was mechanically dispersed using the total. In this way more than 90”/, of the radio- three coniti glass pipettes of decreasing tip diameter activity of the extracts was routinely recovered. (about 1 to 0.1 mm diameter). The cell suspension was Acetylation and oxidation of steroids; chromato- freed of cell debris by centrifugation through a 4% graphic systems used. For acetylation, steroids were BSA solution (25Og, 10 min, OOC) and cell clumps evaporated to dryness under a nitrogen stream, 4 were removed by filtration through a Nitex filter of drops of pyridine and 1 drop of glacial acetic acid 36pm pore diameter. Routinely 2.6 x lo6 + were added, and the mixture was left overnight in 0.6 x lo6 (n = 43) cells were obtained from one ovary the dark at room temperature under an atmosphere and viability, as evaluated by trypan blue exclusion of nitrogen. For oxidation steroids were evaporated [14] was 85.6 + 4% (n = 37). Monolayer cultures to dryness, 0.1 ml of 2.5% solution of CrO, in glacial were performed in Petri dishes under an atmosphere acetic acid was added for 10 min at 0°C; the reaction of 5% CO2 in air. was stopped by 1 ml of a 20”/, ethanol solution in Homogenates and preparation of nuclei. Ovarian tis- water and the oxidized steroids were extracted with sue after removal of the largest follicles and the cor- 10 ml of methylene chloride. pora lutea (about 30mg) was homogenized at 0°C Chromatography was performed on precoated F 254 by 10 strokes of a Potter-Elvehjem homogenizer in silica-gel thin layer chromatography glass plates of 10 ml of the medium used for the suspension cultures. 0.25 mm thickness. The chromatographic systems Sa-Reductase activity in rat ovary 321 used were (V/V): t.1.c.t: chloroform-acetone (355, 5a-androstane-3,17dione on t.1.c. in systems I, II, III V/V); t.l.c.,, : methylene chloridwther (70: 30, V/V); and IV. No Sfl-reduced metabolites were detected t.1.c.,,,: chloroform-ethanol (95: 5, V/V); t.l.c.tv : ben- after separation of 5a- and 5/I-androstane_3,17dione zene-methanol (85: 15, V/V); t.l.c., : hexane-ethylace- on t.l.c.,. tate (75:25, V/V). Acetylation and t.l.c.,, yielded a AR,,, of 1.2, 1.4, 2.5 The effect of acetylation on chromatographic and 2.8 for respectively peaks I, II, III and IV. Peak mobilities was evaluated by calculating the AR,,, I and II thus consisted of monohydroxylated and value, which is the difference between the R, peaks III and IV of bihydroxylated products since (R, = In [l/RF - l] ) value of the non acetylated and peak II is known to contain only one hydroxyl group. the acetylated molecule. The AR,,, value has the pro- Peak I was identified as 1FepiDHT since it was perty to depend only on the number of acetate groups a Sa-reduced product of 17-epitestosterone with only introduced in the molecule and the chromatographic one hydroxyl group, and since the polarity of peak system used [17]. I was comparable with that of 17-epiDHT both before and after acetylation on t.l.c.,, t.l.c.,, and t.1.c.,,,.

RESULTS Moreover, in time course experiments it was shown to be an intermediate product (Fig. 2). Identijication of C3H]-1 7-epitestosterone metabolites Peaks III and IV were identified as .17a-5a-andros- The incubation of [‘HI-17epitestosterone with an tanediols since they were k-reduced, bihydroxylated ovarian cell suspension or with an ovarian homo- products. Peak III was identified as Sa-androstane- genate yielded four peaks on a radiochromatogram 38,17a-diol by crystallization to constant specific (Fig. 1) after extraction and two runs on t.1.c.t. activity (Table 1) and peak IV was tentatively identi- No Ill-keto derivatives were formed. Indeed no fied as 5a-androstane-3a.l7a-diol. Indeed, peak IV radioactivity was found to be associated with andros- was more polar than peak III on t.l.c., and t.1.c.,,, tenedione (RF = 0.57) or 5a-androstane-3,17dione and on t.l.c., 5a-androstane-3a,l7adiol is known to (Rr = 0.66). Incubation with [3H]-testosterone on the be more polar than 5a-androstane-3j?.17adiol [18]. contrary yielded large amounts of 17-keto derivatives. Crystallizations to constant specific activity or to con- All three metabolites of l’l-epitestosterone were stant 14C/3H ratio were not performed for 17-epi- Sa-reduced. After CrO, oxidation the radioactivity of DHT and for .5a-androstane-3a,l7a-diol since the peaks I, III and IV had the same mobility as necessary products could not be obtained.

Fig 1. t.1.c. Radiochromatogram of [‘HI-I7-epitestosterone metabolites. [3H]-17-Epitestosterone and 3 x 10’ cells were incubated for 3 h at 37°C in 2 ml of Hanks medium supplemented with 5 mg BSA/ml and 10m4 M aminoglutethimide. The extract was chromatographed twice in chloroform-acetone (35:5, V/V). Peaks I, II, III and IV were identified as respectively 17-epiDHT, unreacted 17-epitestosterone, Sa-androstane-3/?,17a-diol and 5a-androstane-3a.17ar-diol. The markers (M) and the origin (0) are indicated, together with the localization of 5a-androstane-3.17-dione (A), androstenedione (B), 17-epiDHT (C) 17-epitestosterone (D) and 5a-androstane-3/?.17a-diol (E). P. KONINCKX et al.

Total 5a-Reduced so .*

1 2 3 L TIME (hours)

Fig. 3. 17-Epitestosterone metabolism by monolayer cultures (0) and by ovarian homogenates (0) as a function of time. 3 x 10” Cells, in monolayer culture for 24 h or 1 2 3 L an homogenate (about 0.3 mg wet wt.) were incubated with TIME (hours) 17-epitestosterone as described in Fig. 1. Together with Fig. 2. 17-Epitestosterone metabolites as a function of time. the homogenate an NADPH generating system was added Cells (3 x 10’) were incubated with l’lepitestosterone as to the incubation mixture. The mean of duplicate deter- described in Fig. 1. The individual 5a-reduced metabolites mination (0) and the mean & S.D. of triplicate determina- (0) (0) and the sum (0) of the 5a-reduced metabolites are indi- tions are indicated. cated. Each point represent the mean of duplicate determinations. serum albumin was included in the incubation mixture in order to prevent aggregation of cells. As Assay conditions of the 5a-reductase expected this slowed down the formation of In all three systems used, suspension cultures, k-reduced products, probably by aspecific binding monolayer cultures and homogenates, the total of 17-epitestosterone. At the concentrations used this amount of Sa-reduced metabolites formed was pro- effect, however, was only minimal (Fig. 5). portional to the duration of the incubation until The use of aminoglutethimide to block endogeneous about 30”/, of the 17-e.pitestosterone (10m7 M) incu- steroid formation bated was reduced. With living cells the duration of Aminoglutethimide in concentrations up to 10e4 M incubation could be extended at least up to 3-4 h. had no effect on the amount of Sa-reduced metabo- With homogenates, however, the rate of formation of k-reduced products declined after one hour of incubation (Fig. 3). This could be due to thermal denaturation of the enzyme [9, lo]. The total amount of Sa-reduced metabolites formed was proportional to the number of cells incubated, irrespective of the amount of l’l-epitestosterone incubated and of the duration of incubation at least up to 4 h (Fig. 4). Since the half maximal conversion rate was obtained at about 10e6 M, lo-’ M 17-epitestosterone was routinely used to assay Su-reductase. Bovine *__--- _. ___--- __--

Table 1. Specific activity of crystals during four subsequent 1 , crystallisations of [3H]-51x-androstane-3B,17adiol with 3105 authentic 5a-androstane-3B,l7adiol NUMBER “b”: CELL55’o5

Specific activity Fig. 4. 17-Epitestosterone metabolism by different numbers Crystal (c.pWmg) of freshly dispersed ovarian cells. From 0.125 x 10’ to 5 x lo5 cells/ml were incubated with 10Wsh4 17epitestos- 1. Ethanol-HI0 6980 terone for 1 h (O---O) and from 0.25 x 10’ to 1.5 x 10’ 2. Methanol-H10 8307 cells/ml were incubated with lo-’ M 17-epitestosterone for 3. Cyclohexan-thylacetate 8457 respectively 1, 2, 3 and 4 h (C---O) as described in Fig. 4. Acetone-Hz0 7856 1. Each point represents the mean of duplicate determinations. Sa-Reductase activity in rat ovary 329

tosterone. All 4-ene-3-keto steroids investigated inhibited this Sa-reductase activity, except those with an oxygen substitution on carbon 11. No marked product inhibition was found with various Sa-reduced steroids, while phenoiic compounds inhibited the Scr- reductase activity at higher concentrations (Table 2). This effect of 17/?-estradiol could be characterized as an uncompetitive inhibition [19] since on a Line- weaver-Burk plot the slopes obtained with different concentrations of 17fi-estradiol remained constant (Fig. 8a). In homogemztes an apparent K, of 0.1$7yM was found (Fig. 8b), while 17&estradiol had no effect upon t 1 i 1 1 1 L the %-reduction of ~7~pit~t~te~one. A pH opti- 0 20 LO 60 80 100 BSA concentration (mg/ml) mum was determined in the presence of OSmM NADPH using a Tris-citrate buffer (50 mM) with 1 M Fig. 5. Influence of BSA concentration upon the percentage NaCI, 2SmM CaCI,, 25mM MgCI, and 0.25mM of 5a-reduced metabolites formed. Ovarian homogenates were incubated Fur M min with varying concentrations of EDTA. The pH varied from 4-5 to 9 and an optimum BSA and with lir-epitestosterone as described in Fig. 3, was found around pH 7 iFig. 9). The cofactor speci- Means & SD. of triplicate experiments are depicted. ficity was evaluated using purified nuclei since in total homogenates the endogeneous NADPH would inter- lites formed in cell homogenates and in freshly dispersed cells. When amino~uteth~mide was added to monofayer cultures, however, the amount of 5a- reduced metabolites formed was increased when concentrations of 1O-4-1O-5 M were used and was de- creased at concentrations exceeding lo-’ M (Fig. 6a). When monuIayer cuItures were preincubated with 10m4 M ~i~og~uteth~mide for different periods of time, the initial conversion rate increased with the duration of pre-incubation and reached a plateau after 2 h of preincubation (Fig. 6b). This increase in conversion rate of 17-epitestosterone, however, was quantitatively variable and was not seen in ail experiments. The absorption spectrum of aminoglutethimide dis- kk 0 10-T 10-s to- s lo-‘ IO-’ plays a peak at 205 and 238 nm, both in H&l and w” ~~lNOGL~TETH~Ml0E CONCENTRATION (Mf in ethanol. Am~nog~utethimide co&d thus be located easily by U.V. fluorescence on t.1.c. When e3H]-17- ii epitestosterone (2.10-lo mol) was chromatographed g 201 directly in the presence of aminoglutethimide ,” (2.10-‘mol) on t.1.c. in system I (R, = 0.15) and II (RP = 0.15) respectively 18-7 and 13.9”/, of the radioactivity was associated with the ~inog~utethimide. This non-specific adsorptian of [‘H]-17-epitestosterone to aminoglutethimide on t.l.c., which was also “_r15 seen after incubation, could be prevented completely by washing out the ~inogIutethimide from the extracts by 1 N HCi. fbf

Partial characterization of the Sa-reductase Using a living celf suspension, the Sa-reductase was measured at different substrate concentrations (Fig. 7a). The half maximal conversion rate was estimated from Fig. 5. Influence of aminoglutethimide. Ovarian cell mono- a Lineweaver-Burk plot, at 0.78 PM. With all restric- layer cultures (3 x lo5 cells, 24 h of culture) were incu- tions imposed by the very low conversion rates, the bated for 1.5 h as described in Fig. 1 with 17-epitestoster- Sa-reductase activity could not be saturated and a one and various concentrations of aminoglutetbimide (a) or were preincubated with lo-’ M arninoglut~thirn~e for second apparent K, of over 100 PM could be calcu- various durations of time and incubated with ITepitestos- lated (Fig. 7b). Various steroids were tested for an terone and 10e4 M aminogiutethimide for 1 h (b). Each inhibitory effect upon the Sa-reduction of 17-epites- point represents the mean -I: S.D. of triplicate experiments, 5 12.5

Table 2. Percentual inhibition of the 5a-reduction of 17-epitestosterone by various steroids

% Inhibition of the Sa-reduction of [“HI-17-epitestosterone Steroid lO-6 M 5 x 10-6M

4-ene-3-keto steroids 17-Epitestosterone 51.1 f 2.5 75.2 f 1.3 Testosterone 21.9 + 6.1 41.1 + 3.6 4-Androsten-3,17dione 11.1 + 2.7 22.9 f 2.8 4-Pregnen-3,2Odione 47.4 f 3.7 69.3 + 3.7 2Oa-Hydroxy4pregnen-3-one 12.3 + 0.3 43.3 f 1.6 a 17a-Hydroxy4pregnen-3,2Odione 49.0 + 4.9 76.4 f 8.0 7: 11-Deoxy-cortisol 29.0 + 0.5 37.1 * 3.1 s Cortisol 0.2 + 3.9 2.3 + 0.3 2 Corticosterone 0.6 &- 1.3 0.2 + 0.5 E Sa-reduced Steroids !2 DHT -5.0 f 8.0 - 1.0 + 7.6 cl!- Sa-Androstane-3a,l7Bdiol 13.3 + 2.0 12.0 &- 1.0 5a-Pregnane-3,2Odione 0.4 f 1.0 0.2 f 3.7 3a-Hydroxy-Sa-pregnan-20-one 0.3 rf: 1.5 2.9 + 1.7 5a-Pregnane-3a.20adiol 17.2 k 11.4 25.5 rt 9.7 Phenolic Steroids 17/&estradiol 7.0 + 7.3 31.6 + 3.2 Estrone 18.7 &- 1.7 33.9 f. 6.7

Aher 1 h of recovery 3 x 10’ cells were incubated with lo-’ M 17epitestos- terone and lO-6 and 5 x 10m6 M of various steroids as described in Fig. 1. The mean f S.E. of triplicate experiments is indicated.

17-Epitestosterone concentration ( M 1

Fig. 7. Influence of l’l-epitestosterone concentration. Cells (3 x 10’) were incubated with lo-’ to 5 x lo-’ M 17epitestosterone as described in Fig. 1. The % Sa-reduced metabolites (0) and the amount of 5a-reduced metabolites (0) formed are indicated. The Lineweaver-Burk plots are shown in the insert. Two apparent K, values of respectively 0.78 PM (a) and more than 1OOpM (b) are depicted.

- -i-.- - - _ -. .^ . .^ - - - - _ - . _. _ 5a-Reductase activity in rat ovary 331

2 L 6

la)

I! I t a I I I I I / , 2.10-7 &JO-7 6.10-? 8.10-7 10-s 2.10-7 L.lO-7 6.10-7 8.W7 10-G 17-EPITESTOSTERONE CONCENTRATION (MI

Fig. 8. Influence of I7j-estradiol concentration. In two separate experiments approximately 3 x lo5 cells (a) or homogenates thereof (b) were incubated with different concentrations of 17-epitestosterone with (open symbols) and without i’@-estradiol (closed symbols) as described in Figs. 1 and 3. The intact cells (a) were incubated with respectively 10e6 M (A), 5.106 M (Cl) and IO-$ M (0) 17/I-estradiol. The homogenates (b) were incubated with (0, 0) and without (U, Cl) an NADPH generating system. The Lineweaver-Bulk plots, depicted in the insert, show an uncompetitive inhibition of 17/?-estradiol in intact cells (a) and an apparent K, for 17-epitestosterone of 0.57pM in homogenates (b). Each point represents the mean f SD. of triplicate experiments.

fere. The incubation of about 350,000 nuclei for 1 h with 10m7 M 17-epitestosterone in the presence of OSmM NADH, OSmM NADPH and an NADPH generating system yielded respectively 0.32 + 0.09%, 3.78 + 1.56% and 5.52 + 0.60% Sa-reduced metabolites, demonstrating that NADPH was used as cofactor. In order to compare the activity of the Sa-reductase in a living cell with the activity of the Sn-reductase in an homogenate about 3 x 10’ cells were incubated with 17-epitestosterone, with and without prior

Table 3. The Su-reductase activity was assayed in 3 x 10’ cells with and without prior homogenisation as described in Fig. 1

% Sa-Reduced metabolites Living cell Homogenate

Experiment 1 17.3 * 0.4 20.0 f 1.1 Experiment 2 14.0 * 1.5 15.6 + 0.6 Experiment 3 16.4 + 0.03 17.2 k 1.2 Fig. 9. Influence of pH upon See-reductase activity. Ovarian 15.9 * 1.7 17.6 & 2.2 homogenates were incubated in a Tris-citrate buffer together with 0.5 mM NADPH as described in Fig. 3. The Together with the homogenates an NADPH generating pH varied from 4.5 to 9. Each point represents the system was incubated. The mean + S.D. (triplicates) of mean f S.D. of triplicate experiments. three separate experiments are indicated. 332 P. KONINCKX et al. homogenization. The percentage of Sa-reduced meta- The liver See-reductase activity has a higher K, value bolites formed was comparable in both systems, but and a broader substrate specificity. This led to the slightly higher in an homogenate (Table 3). This in- hypothesis that several Sa-reductases with different crease in activity of the Sa-reductase in homogenates properties are operating in the liver [S]. was comparable to the y0 trypan blue positive cells: The characteristics of the rat ovarian Sa-reductase respectively 18% in the first experiment, 13% in the are comparable with those from target tissues such second and 10% in the last experiment. as prostate and kidney. The role of the ovarian Sa-reductase could however be completely different. In androgen target tissues the formation of active DISCUSSION androgens as DHT depends completely on the A system has been developed to assay Sa-reductase Sa-reduction of testosterone. In the ovary, on the con- activity in living ovarian cells. This procedure offers trary, most of the Su-reduced androgens formed could the advantage that it permits the selection and culture derive from Sa-reduced CZ1 steroids. Indeed, a path- of specific cells. Moreover, it permits the study of the way metabolizing SE-reduced CZ1 steroids to Cl9 regulation of the Sa-reductase activity by various hor- steroids has been described [23]. Moreover, pro- mones and humoral factors secreted by the pituitary gesterone and 2Oa-hydroxy-pregn4-en-3-one, which or locally, in adjacent ovarian compartments, and the must locally be present in very high concentrations study of the role of the Sa-reductase activity in the and which are better substrates for the Sa-reductase regulation of steroidogenesis [Z]. may be expected to compete actively for the enzyme and could even “protect” testosterone and andros- The assay of 5u-reductase tenedione against 5u-reduction. The use of [3H]-17-epitestosterone to measure the The inhibition of the Scz-reduction of 17-epitestos- 5x-reductase in the rat ovary is a definite improve- terone by various steroids was comparable both ment over the methods described up to now. Since qualitatively and quantitatively in living ovarian cells no 17-keto derivatives are formed the number of and in prostate [9] or kidney [lo] nuclei, except for metabolites is largely reduced. Furthermore, the inhibition of the Sa-reductase activity by 17/3- 5a-androstane-3aJ7adiol and 5a-androstane-3/?,17a- estradiol in intact cells (Table 1). Since this inhibition diol are easily separated on t.1.c. was uncompetitive [19] and since it was not found Theoretically in steroidogenic cells endogeneously in homogenates, we suggest that 17@stradiol inter- formed 4-ene-3-keto steroids could inhibit competi- feres with the membrane transport of 17-epitestoster- tively Sa-reductase. Therefore steroidogenesis should one or becomes cytotoxic in high concentration. be inhibited when Sa-reductase activity is assayed. Although the estrogen concentrations used are very Aminoglutethimide was used for this purpose since high, this could have physiologic implications since it is an effective inhibitor of cholesterol side chain in the follicular fluid estrogen concentrations up to cleavage [20] and since it has no direct effect upon 5 x 10m6 M have been described [24]. Sa-reductase as tested in homogenates. No endogeneous inhibition of Sa-reductase could The activity of the Sa-reductase be demonstrated in freshly dispersed cells. In mono- The activity of the ovarian 5x-reductase is very high layer culture, however, aminoglutethimide caused (l-10 nmol/mg protein/h) which is compatible with sometimes an increase in Sa-reduction of 17-epi- a regulation of steroidogenesis [Z]. The activity in testosterone which is compatible with endogeneous living ovarian cells was not substantially different inhibition. The inclusion of aminoglutethimide in the from that found in cell homogenates supplemented incubation mixture could thus be omitted in freshly with optimal concentrations of NADPH. The slight dispersed cells, but becomes necessary in monolayer increase in Sa-reduction of 17-epitestosterone in culture especially when the steroid hormone produc- homogenates can be accounted for by the number tion is stimulated in order to study the hormonal of death cells in the cell suspension. Thus, in uiuo, regulation of the Sa-reductase activity. the ovarian Sa-reductase is maximally active and the intracellular NADPH availability is not a limiting The biochemical characterization of Lx-reductase factor. The biochemical characteristics of the Sa-reductase have been described in the liver [8] and in androgen Acknowledgements-The skilful technical assistance of Mrs. target tissues such as the prostate [9], the kidney M. Mentens-Oost, Miss G. Lemmens and Mrs. B. Willems- Minten are greatly appreciated. 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