Androgen Synthesis and Aromatization by Equine Corpus Luteum Microsomes*

THE JOURNAL OF BIOLOGICAL CHEMISTRY @ 1989 by The American Society for Biochemistry and Molecular Biology, Inc. Vol. 264. No. 13, Issue of May 5, pp. 7161-7168 1989 Printed in d. S. A. Androgen Synthesis and Aromatization by Equine Corpus Luteum Microsomes* (Received for publication, September 9, 1988) Ihsan Al-Timimi, Jean-Luc Gaillard, Hakima Amri, Pierre and Silberzahns From the Laboratoire de Biochimie, Unit6 Associie au Centre National de la Recherche Scientifique, Universite, 14032 Caen, France Whereas mare corpus luteum does not produce an- gens have been isolated from the follicular fluid of the mare drogens or estrogens in vivo, the incubation of mare (11, 12) and sow (13). These steroids are produced in uitro by corpus luteum microsomes with progesterone and the equine follicular wall and corpus luteum (8, 14). Equine NADPH resulted in 17a-hydroxyprogesterone and es- and porcine granulosa cells are able to produce androgens and trogen production with a small yield of androstenedi- 19-norandrogens in uitro (8, 15). In the mare, the follicular one. In thepresence of anaromataseinhibitor (4- fluid concentration of 19-nortestosterone is 10 times higher hydroxyandrostenedione), 17a-hydroxyprogesterone than that of testosterone whatever the stage of follicular andandrostenedione were accumulated.aromatiza- growth (IZ), and this raises the question as to the role of 19- tion of testosterone and androstenedione occurred via norandrogens as aromatase substrate in the equine follicular stereospecific loss of the 1&28 hydrogen atoms and fluid. was inhibited by MgC12, KCl, and EDTA. The K,,, of estrogensynthetasefromequinecorpusluteumfor In the present study, we have investigated the ability of testosterone was 18.5 f 2.7 nm and for androstenedi- microsomes from the cyclic corpus luteum of the mare to one was 11.5 f 1.5 nm. 19-Norandrogens were aro- synthesize and aromatize androgens and norandrogens. matized with a slightly higher efficiency than were androgens, but the affinity of the aromatase was lower EXPERIMENTAL PROCEDURES for 19-norandrogens than for androgens. Our results ChemicaZ+"1&2~-3H]Androstenedione (50 Ci/mmol), [lfl,2fl-3h] suggest that aromatases from equine testis and corpus testosterone (50 Cilmmol), and [4-"C]progesterone (57.2 mci/mmol) luteum are closely related enzymes. On the other hand, were purchased from Du Pont-New England Nuclear. [l~u,2a-~h] the question arises as to the relationship among the cell Testosterone (60 Ci/mmol), 12,4,6,7-3H]estrone (95 Ci/mmol), origin, the synthetizing abilities, and in vivo produc- [2,4,6,7-'H]estradiol (89 Ci/mmol), 11,2,6,7-3H]testoster~ne (94 Ci/ mmol), tion of the corpus luteum in different mammalian spe- [1,2,6,7-3H]androstenedione(90 Ci/mmol), [1,2,6,7-'H]progesterone ( 84 Ci/mmol), [4-'4CC]androstenedione(59 mci/mmol), [4- cies. "C]testosterone (56.9 mci/mmol), 19-[4-"C]nortestosterone (60 mci/mmol), and [4-"C]estradiol(50 mci/mmol) were obtained from Amersham Corp. Radioimmunoassay (RIA)' reagents were obtained from biomirieux (CharbonniBres, France). Chemicals were obtained from Sigma, Merck, and Carlo Erba. Solvents of analytical grade were obtained from Merck and Carlo Erba and solvents of HPLC grade from Rathburn. Preparation of Microsomes and Mitochondria-Corpora lutea were dissected on ice, weighted, and the luteal tissue was homogenized in 0.05 M Tris maleate buffer, ph 7.4, containing 0.25 M sucrose, 1 mm P-mercaptoethanol, and 40 mm nicotinamide using a Waring Blender (3 X 20 s, full speed) followed by a further homogenization step using There are species differences in the ability of the corpus luteum to synthesize and aromatize androgens. In uitro, the bovine corpus luteum is unable to synthesize androgens (1) and estrogens because it lacks l7a-hydroxylase, 17,20-lyase, and 19-hydroxylase aromatase systems (2). In contrast, the human (3) and the porcine (4) corpora lutea synthesize in uitro estrogens from acetate or progesterone. The mare corpus luteum produces neither androgens nor estrogens in uiuo. Plasma testosterone fluctuations in the cycling mare do not seem associated with the presence of a corpus luteum (5), and there is no increase in the circulating 178-estradiol (6) or estrone sulfate (7) level during the luteal phase of the mare. Mahajan and Samuels (8) did not succeed in metabolizing progesterone into estrogens in vitro. However, these authors have reported the ability of mare corpus luteum to transform androgens into estrogens, and YoungLai (9) found a detectable amount of 178-estradiol in the mid-cycle corpus luteum of the mare. We have recently reported the existence in thequine testis of a specific aromatase system which, in contrast to human placental aromatase, is able to aromatize norandrogens with at least the same efficiency as androgens (10). 19-Norandro- * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $To whom correspondence should be addressed. 7161 an Ultra Turrax. The microsomes were prepared by differential centrifugation as described previously (10). The microsomes were either diluted in buffer or lyophilized after washing with distilled water and stored at -18 "C. Mitochondria were prepared according to the Canick and Ryan procedure (16). Protein concentrations were measured by the procedure of Lowry et al. (17). Progesterone, testosterone, androstenedione, and 17P-estradiol were extracted from microsomal preparations by 4 X 3 ml of diethyl ether. RIA was performed as described previously (10, 18). The cross-reactions of the antisera have been published previously (18). Androgen Synthesis-Incubations were performed using a I-ml reaction volume of 50 mm Tris maleate buffer, ph 7.4 (buffer I), brought to 5 p~ [4-"C]progesterone and containing 0.5 mg/ml microsomal protein. The beginning of the incubation time was set at the addition of 0.3 mm NADPH. The incubation was performed for 30 min at 37 "C in air. The control incubations did not contain NADPH. In some experiment 4.5 p~ 4-hydroxyandrostenedione was added as The abbreviations used are: RIA, radioimmunoassay; HPMES, microsomal estrogen synthetase from human placenta; ETMES, microsomal estrogen synthetase from equine testis; ECLM, equine corpus luteum microsomes; ECLMES, microsomal estrogen synthetase from equine corpus luteum; HPLC, high performance liquid chromatography.

7162 Estrogen Synthesis aromatase inhibitor (19). Steroids were extracted five times by 4 ml of ethyl acetate/diethyl ether (l:l, v/v) (solvent I). The organic phases werepooled, evaporated to dryness, and redissolved in 100 pl of methanol; the radioactivity of 10 pl was measured to calculate the recovery, and 80 pl was applied on 0.25-mm Silica Gel 60 Fzs4 plates (20 X 20 cm, Merck) and run in a cyclohexane/ethyl acetate (1:1, v/ v) solvent system (solvent 11). The metabolites were identified and quantified by HPLC (LC 5060, Varian) coupled with a radioactive flow detector (Flo-onebeta, Radiomatic Instruments & Chemical Co., Inc.). Aromatase Assay by 3H20 Release-Aromatization of androgens was assessed according to the method of Thompson and Siiteri (20). Mare corpus luteum mitochondrial, cytosolic (150,000 X g supernatant), or microsomal protein was incubated in the presence of [la,za- 3H]testosterone, [l&2&3h]testosterone, or [l/3,26-3h]androstenedione with 0.3 mm NADPH in 1 ml of 50 mm Tris maleate buffer, ph 7.4, for 5-40 min; the beginning of the reaction was set when NADPH was added. Control reactions contained no NADPH. Chloroform (2 ml) was added to the reaction mixture to terminate the reaction and to extract partially estrogens and the remaining androgens from the aqueous phase. Following centrifugation, 0.5 ml of the aqueous phase was treated with an equal volume of 10% aqueous suspension of activated charcoal with 1% dextran-t 70. The mixture was allowed to sit on ice for 2 min prior to the addition of 0.25 ml of protamine sulfate (8 mg/ml). After sedimentation by centrifugation (2,700 X g for 10 min), the 3H20 released was assayed by measuring the radioactivity of 0.5 ml of supernatant. Arornatase Assay by RIA-Onemg of microsomal protein was incubated in 1 ml of 50 mm Tris maleate buffer, ph 7.4, at 37 "C with 8 p~ unlabeled testosterone and 0.5 mm NADPH. The reaction was stopped by adding 2 ml of solvent I. Traces of 3H-estrogen (5000 cpm) were added to calculate the recovery of steroid extraction. The aqueous phase was submitted to four other extractions by 3 ml of the same solvent system. The organic phases were evaporated and dissolved in bovine serum albumin-phosphate buffer. l7p-estradiol and estrone were assessed by RIA. Arornatase Assay with 4-'4C-Androgens-Incubations using 1 ml of reaction volume brought to 1.5 pm [4-14C]testosterone, 19-[4-"C] nortestosterone, or [4-"C]androstenedione, 0.3 mm NADPH and containing 0.5 mg of pooled microsomal protein were performed at 37 "C for 15 min in air; control incubations did not contain NADPH. Extraction was performed by 5 X 3 ml of solvent 1. Estrogens were analyzed by HPLC after TLC. After autoradiography, spots corresponding to estradiol and estrone were scraped off, and their radioactivity was measured. Thin Layer Chromatography and HPLC-TLC was performed after the above incubations with [la,2a-3h]testosterone,[l(3,2/3-3h]testos- terone, [lp,2p-3h]androstenedione,[4-14c]androstenedione,[4-14c] testosterone, 19-[4-14C]nortestosterone, [4-I4C]progesterone. After extraction of steroids, the organic phases were evaporated. The dry residues were dissolved in methanol, applied on 0.25-mm silica gel plates (Merck), and run in solvent 11. Autoradiography was performed when I4C-substrates were used. Spots corresponding to 17P-estradiol and estrone were scraped off and eluted by 8 ml of chloroform; the radioactivity of the eluates was measured when [3H]testosterone was used as substrate. After autoradiography, "C-steroid spots were scraped off, and their radioactivity was eluted by 8 ml of chloroform. Each of the dried eluates was solubilized in methanol and chromatographed by HPLC (LC 5060, Varian) to obtain a complete separation of progesterone (P), 1701-hydroxyprogesterone (17a-OHP), androstenedione (A), 19- norandrostenedione (NA), testosterone (T), 19-nortestosterone (NT), estrone (E1), and 17P-estradiol (E?). Steroids were eluted from a C18 reversed-phase 4-bm column (15 X 0.4 cm, Merck) at a flow rate of 1 ml/min with acetonitrile/methanol/water (34:6:60, v/v). The flow was monitored with a variable wavelength UV detector (UV100, Varian) set at 230 nm, and the radioactivity of each peak was measured with the radioactive flow detector. RESULTS Androgen Production-After incubation of corpus luteum microsomes with [4-'4C]progesterone and NADPH, the products were first analyzed by TLC (Table I). Autoradiography showed several radiolabeled zones on the plates. The spots corresponding to [E1], [Ez + PI, [T + NT], and [A + NA + 17a-OHPI were scraped off, eluted, and their constituents by Equine Corpus Luteum TABLE I Rf and retention time of some steroids in TLC and HPLC TLC was performed on 0.25-mm silica gel plates (Merck) and run in cyclohexane/ethyl acetate (l:l, v/v). Steroids were separated by HPLC on a reverse-phase C18 Merck column (15 X 0.4 cm) with acetonitrile/methanol/water (34:660, v/v) at flow rate of 1 ml/min. Steroid RF time Retention min Progesterone 62 0.58 17a-Hydroxyprogesterone 17.6 0.45 Androstenedione 15.7 0.48 19-Norandrostenedione 0.42 11.9 Testosterone 0.37 13 19-Nortestosterone 0.33 9.7 Estrone 15.6 0.66 176-Estradiol 11.5 0.53 analyzed by HPLC. Androstenedione was detected in small amounts, while 17a-hydroxyprogesterone and estrone were the principal metabolites formed by microsomes from a pool of 25 equine corpus luteum; 18% of the substrate was metabolized. Their ability to synthesize androgen was confirmed after blockage of aromatase by 4-hydroxyandrostenedione. Under these conditions, more than 24% of the progesterone was metabolized into 17a-hydroxyprogesterone and androstenedione (Fig. 1). The results of incubations with eight different preparations of equine corpus luteum microsomes (ECLM) are shown in Table 11. In the absence of an aromatase inhibitor, despite the high variation in the amounts of 17ahydroxyprogesterone and estrone synthesized from one corpus luteum to another, androstenedione was found at about the same low values. In half of the experiments, the amount of (A + El) or (17a-OHP + A + El) synthesized was greatly enhanced in the presence of an aromatase inhibitor (4-hydroxyandrostenedione). 17P-Estradio1, 19-nortestosterone, 19-norandrostenedione, and testosterone were not identified in any of the above experiments. Experimental Conditions of the Aromatase Assay-The RIA results (Table 111) show that the mare corpus luteum micro- somes contained detectable amounts of endogenous androstenedione, testosterone, estradiol, and a large amount of progesterone. The rate of estrogen formation during the incubation of 0.5 mg/ml corpus luteum microsomal protein with 3 p~ testosterone, as measured by 3Hz0 release and RIA, was linear for at least 30 min at 37 "C (Fig. 2). To determine whether the rate of 3H-estrogen formation correlated well with 3Hz0 formation, 1.5 p~ [1~-2fi-3H]testosterone was incubated with a 0.5 mg/ml pool of microsomal protein and 0.3 mm NADPH. Aliquots of the reaction mixture were withdrawn at various time intervals. One set was assessed for 3H20 release. In a second set, the product and remaining substrates were extracted and purified by TLC. The rates of t3h]estradiol and 3H20 formation are compared in Fig. 3 which shows that the ratio of 3Hz0/[3H](Ez + El) was always 2.13. After incubation, 3Hz0 contained 68% of the radioactivity of the aromatized androgens, while 3H-estrogen retained 31.3% of this radioactivity. The activity of the microsomal estrogen synthetase from equine corpus luteum (ECLMES) was not affected by preincubation of ECLM at 30 "C for 30 min but was lost (84%) by preincubation at 45 "C. The reaction rate increased with the reaction temperature up to 37 "C for a 15-min incubation and stabilized between 37 and 40 "C after which a drastic loss of activity was observed (Fig. 4). Aromatization was linear with protein concentration up to 1.37 mg/ml with 1.5 pm testos-

f Estrogen Synthesis by Equine Corpus Luteum 7163 b, I t 5 10 15 20 Time(r TABLE II Steroid production by different preparations of ECLM After incubation of 0.5 mg/ml microsomal protein with 5 PM [4- %]progesterone and 0.3 mm NADPH, the 17a-[4- CJhydroxyprogesterone (17~0HP), androstenedione (A), and estrone (E,) formed were purified and quantified by TLC and HPLC with a radioactive flow detector. The experiments were done either in the presence (+) or absence (-) of 4-hydroxyandrostenedione (4-OHA). ND, not detected. Steroid identified Corpus luteum 4-OHA 17a-OHP A E1 pmol/tube 1-148.2 2.5 59 + 165.8 111.3 4 2 155.7 9.7 57.3 + 161 49.6 13.7 3 108.9 24.2 56 + 99 80.3 9.6 4 188 21.8 62.3 -I- 260 213 6.2 5 35.8 21.3 81.9 -I- 19.5 141.5 ND 6 45.8 19.6 25.5 + 69.2 46.5 2.5 7 84.6 11.8 11.2 + 108.4 40 ND 8 80.4 23.5 30.6 + 129.3 42.2 ND t 0 5 10 15 20 L5 Time(i ln) FIG. 1. HPLC identification of 4-4C-steroids. HPLC elution of TLC-purifiedlabeled (a) 17a-hydroxyprogesterone (I7n-OHP) and androstenedione (A) and (b) estrone (El) obtained by the incubation of 0.5 mg/ml microsomal protein with 5 pm [4- C]progesterone and 0.3 mm NADPH. The flow was monitored at 230 nm, and its radioactivity was measured with a radioactive flow detector. terone, and up to 1.5 mg/ml with 1.5 PM androstenedione (Fig. 5). The enzyme activity was inhibited by the addition of MgC12, KCl, and EDTA (Table IV). The addition of 0.5% Triton X- 100 or sodium dodecyl sulfate inhibited the aromatase activity by 95 and 93%, respectively, while 2 PM 4-hydroxyandrostenedione inhibited the aromatase activity by 88% when 0.5 mg/ml protein was incubated with 1.5 pm [l&2/3-3h]testosterone for 15 min at 37 C. To determine the apparent K, for NADPH, 0.5 mg/ml protein was incubated with 1.5 pm androstenedione and O- 1000 PM NADPH for 15 min at 37 C. The apparent & for NADPH, measured from a double-reciprocal plot, was about TABLE Endogenous steroid content (pmol/mg of protein) of ECLM 10 mg of microsomal protein was extracted by 4 X 3 ml of diethyl ether. Prior to RIA, steroids were separated by a Sephadex LH-20 column, 6 X 0.5 cm (Pharmacia LKB Biotechnology Inc.) with benzene/methanol (85:5, v/v). ECLM pool Progesterone Androstenedione Testosterone 17/3-E&radio1 916 0.29 0.58 1 360 0.20 0.40 0.31 2 300 0.24 0.14 0.50 3 237 0.22 0.18 0.15 4 689 0.30 0.42 5 539 0.29 0.17 0.19 6 412 0.36 0.22 0.33 85 iflm (Fig. 6). Under these experimental conditions, at 6 and 40 ELM, 1.7 and 6.3%, respectively, of androstenedione was aromatized, and 1.2 and 0.63%, respectively, of NADPH was consumed by the reaction. Subcellular Distribution of Aromatase and Stereospecificity of Hydrogen Elimination-Aromatase activity, as measured by 3H~0 release, was found to be only associated with the microsomal fraction; no significant activity was measured in the cytosol or in the mitochondria. When microsomes were incubated with [lcu,2cy-3h]testosterone, the 3H20 release was about 24% of that released after incubation with [l&2p-3h] testosterone (Fig. 7). After incubation with [1&2P-3H]androstenedione, 3H-estrogens contained 23.7 of the radioactivity of aromatized androstenedione. Aromatization of Androgens by Different Corpora Lutea- Table V shows that the microsomal fractions from different corpora lutea were all able to produce estrogens from testosterone or androstenedione. Nonetheless, a marked disparity III

7164 Estrogen Synthesis by Equine Corpus Luteum RIA / time (min) FIG. 2. Comparison of estrogen formation by RIA and Hz0 release. Incubations were carried out using 1 mg/ml mare corpus luteum microsones with 8 FM nonlabeled testosterone (MA) or [lfl,2p-3h]testosterone (3H20 release). Results are corrected for H in (Y position at 37 C (n = 2). 0-l m n B I n n 0 10 20 30 40 50 60 preincubation temperature C FIG. 4. Effect of temperature on estrogen formation by mare corpus luteum microsomes. Microsomal protein was preincubated without any substrate for 30 min. The aromatase activity was measured by 3Hz0 release after incubation of 0.5 mg/ml microsomal protein with 1.5 pm [1@,2fi-3H]testosterone and 0.3 mm NADPH for 10 min at 30 C. Inset, effect of temperature of incubation on aromatase activity. 0.5 mg/ml microsomal protein was incubated with 1.5 pm [lp,2/s3h]testosterone and 0.3 mm NADPH for 15 min. time (min) FIG. 3. Aromatization as a function of time. Results are expressed in cpm as the rate of formation of 3HZ0 and 3H-estrogen from [lfl,2fl-3h]testosterone at 37 C. The reaction mixture contained 1.5 pm [l@,28-3h]testosterone, 0.3 mm NADPH, and 0.5 mg/ml protein (n = 2). in aromatizing ability was observed among the 10 corpora lutea examined. In the absence of the necessary data (histology and date of ovulation), we were unable to determine whether a correlation existed between these results and the age of the corpus luteum. Aromatization of Androgens and 19-Norundrogens-When 1.5 pm [4-14C]androstenedione, [4-4C]testosterone, or 19-[4-14C]nortestosterone and 0.3 mm NADPH were incubated with 0.5 mg/ml pooled microsomal protein for 15 min, estrone and estradiol were synthesized, as identified by TLC and HPLC. The corpus luteum microsomes were able to aromatize both testosterone and 19-nortestosterone to estradiol-170, while estrone was obtained from androstenedione. The reaction velocities were 23.93 f 1.3, 16.45 -t- 1.87, and 16.06 + 0.67 pmol/min/mg of protein for 19-nortestosterone, testosterone, and androstenedione, respectively. In these experiments, 19- norandrogens were not synthesized from testosterone or androstenedione either in the presence or absence of NADPH. The K, of ECLMES for testosterone (18.5 f 2.7 nm, n = 12) was greater than that for androstenedione (11.5 +- 1.5 nm, 0 0.5 1.0 1.5 2.0 protein (mgfml) FIG. 5. Estrogen formation by mare corpus luteum microsomes as a function of protein concentration. Microsomal protein in the amount indicated was incubated with 1.5 pm [1&213-~H] androstenedione (A) or [1&2&3H]testosterone (T) and 0.3 mm NADPH for 15 min at 37 C. Results are not corrected for 3H in a position. n = 8) (Fig. 8, a and b). The incubation temperature (20-37 C) and the protein concentration (20-40 pg/ml) had no measurable effect on the apparent K,,, values. The V,., was calculated to be 40 pmol/min/mg of protein for androstenedione and 38 pmoi/min/mg for testosterone at 37 C. In these experiments, less than 12% of the androgens were metabolized into estrogens. Steroid Inhibition of Aromutase-Androstenedione, testosterone, and 19-norandrostenedione were very effective inhibitors of the aromatization of [ lp,2p-3h]testosterone, whereas dehydroepiandrosterone and 19-nortestosterone were less effective. Epitestosterone and 16a-hydroxytestosterone were weak inhibitors of ECLMES (Table VI). The inhibition kinetics of [l/3,2fi-3h]testost.erone aromatization by 100 nm androgens or 19-norandrogens shows that androstenedione, testosterone, 19-norandrostenedione, and 19-nortestosterone

TABLE IV Effect of some chemicals on aromatization by ECLMES The reaction mixture contained 0.5 mg/ml microsomal protein, 1.5 p~ [l/3,2@-3h]testosterone, and 0.3 mm NADPH. Incubations were carried out for 15 min at 37 "C in the presence or absence of EDTA, MgC12, or KCl. Aromatase activity was estimated by 3H20 release. Results (n = 4) are expressed as velocity of aromatization (H.D.) and as percentage of inhibition of control aromatization (labeled testosterone alone). Additive Concentration mm Control EDTA 10 20 40 MgCL 50 100 200 KC1 125 250 500 Estrogen Synthesis by Equine Corpus Luteum 7165 Velocity of aromatization 27.46 k 1.02 19.89 f 0.64 13.89 f 0.47 08.05 f 0.26 19.15 f 0.80 15.64 f 1.40 09.48 f 0.48 21.94 f 1.90 19.08 f 0.48 12.38 f 0.92 Inhibition % 27.56 49.42 70.68 30.26 43.04 65.48 20.10 30.51 54.91 TABLE V Estrogen formation from testosterone and androstenedione by different corpora lutea Comparison of estrogen formation (values represent mean f S.D., n = 4), as expressed by 3Hz0 release from [l@,2j3-3h]testosterone or [ 1@,2@-3H]androstenedione, by different mare corpora lutea. Results are not corrected for 3H in 01 position. Microsomes (0.5 mg/ml protein) were incubated with 1.5 p~ l@,2@-3h-androgens and 0.3 mm NADPH for 15 min at 37 "C. Formation of estrogens Corpus luteum Testosterone Androstenedione pmollminlmg 1 18.55 f 0.33 21.26 f 0.20 2 15.31 f 0.20 17.10 f 0.44 3 13.30 f 0.19 14.88 f 0.35 4 05.17 f 0.41 07.37 f 0.07 5 07.28 f 0.24 09.43 f 0.20 6 09.16 f 0.19 10.48 f 0.29 7 20.81 f 21.27 0.8 f 0.17 8 17.12 f 0.42 19.56 f 0.56 9 27.29 f 1.15 27.85 f 1.75 10 22.51 f 0.58 23.78 f 1.68 /. I. I. I. I. I 100 200 100 200 1 1INADPH pm FIG. 6. Effect of NADPH concentration on initial velocity. 0.5 mg/ml microsomal protein (n = 2) was incubated with 1.5 p~ [1@,2@-3H]androstenedione at 37 "C for 15 min with different concentrations of NADPH. t 0 11 8.2 8 -'HI testosterone I [l a.2 a-'h] testosterone mitochondria cytosol microsomes FIG. 7. Stereospecific removal of hydrogen atoms and subcellular distribution of aromatase. 3H20 resulted from incubation of 0.5 mg/ml protein of subcellular fraction with 8 p~ [l@,2p-3h]- or [1~~,2a-~H]testosterone and 0.5 mm NADPH. The mixtures were incubated for 15 min at 37 "C (n = 2). 100 200 1IA p"' FIG. 8. Androgen dependence of aromatase. Double-reciprocal plots of the aromatization of several concentrations of l@,2p3handrogens in the presence of 0.3 mm NADPH. 40 pg/ml protein was incubated with [l~,z@-3h]testosterone for 5 min at 25 "C (a), or with [l@,2@-3h]androstenedione for 5 min at 20 "C (b). Results are corrected for 3H in 01 position. were competitive inhibitors (Fig. 9). While the maximal velocity in the presence of inhibitors was unchanged, the apparent K,,, increased from 18 to 44, 71, 125, and 250 nm, respectively, when 100 nm 19-nortestosterone, 19-norandrostenedione, testosterone, or androstenedione was added to the reaction mixture.

7166 Estrogen Synthesis by Equine Corpus Luteum TABLE VI Znhibitory effect of some steroids on [l~,2@-3h]testosterone aromatization 250 Ng/ml microsomal protein of mare corpus luteum was incubated with 1 NM [Ip,2P-3H]testosterone and 0.3 mm NADPH for 10 min at 37 "C in the presence or absence of three different concentrations of nonradioactive steroids. Results are expressed as percentage of inhibition f S.D. (n = 4) of control aromatization (labeled testosterone alone). DHA, dehydroepiandrosterone; Epi-T, epitestosterone; 16- OHT, 16a-hydroxytestosterone. Additive Concentrations 0.5 #M 1 #M 2 #M Androstenedione 44.3 f 5.1 63.5 f 3.6 76.3 f 1.1 Testosterone 36.5 f 2.1 48.7 f 1.1 65.7 f 1.7 19-Norandrostenedione 25.3 f 1.0 32.0 f 5.8 51.2 f 0.4 19-Nortestosterone DHA Epi-T 16-OHT 15.2 f 2.4 18.2 f 4.2 25.2 f 3.7 14.0 f 1.9 14.3 f 4.1 22.7 f 1.1 8.8 f 2.6 10.8 f 0.4 9.7 f 0.8 5.2 f 0.4 12.0 f 0.8 13.7 f 2.3 100 200 1fI pm.' FIG. 9. Kinetic analysis of the inhibition of aromatase activity by androgens and 19-norandrogens. Double-reciprocal plots of reaction velocity showing competitive inhibition of testosterone aromatization in the presence of 100 nm androstenedione (A), testosterone (T), 19-norandrostenedione (NA), or 19-nortestosterone (NT). Enzyme activity was measured by 3H20 release after a 5-min incubation (n = 4) at 25 "C with 40 Ng/ml microsomal protein, 0.3 mm NADPH, and varied concentrations of [lfl,2&3h]testosterone. Results are corrected for 3H in a position. DISCUSSION According to YoungLai (9), the mid-cycle corpus luteum of the mare contains progesterone, 17a-hydroxyprogesterone, 20a-dihydroprogesterone, pregnenolone, and a detectable amount of 17P-estradiol. Our results show that the microsomal fraction has, in addition, detectable amounts of androstenedione and testosterone, which suggests that the mare corpus luteum possesses the capacity to produce androgens and estrogens. These findings contrast with the report of Mahajan and Samuels (8) who obtained neither androgens nor estrogens after the incubation of mare corpus luteum homogenates with progesterone and thus concluded that the more corpus luteum, deprived of 17,20-lyase, does not produce estrogens. Our study clearly shows that mare corpus luteum microsomes possess the enzymatic equipment necessary for the transformation of progesterone to androstenedione (17ahydroxylase and 17,20-lyase) and are able to aromatize androgens. The 17a-hydroxylase activity of the mare corpus luteum was one-third of that of the aromatase (5.8 f 2 versus 17.3 f 6.3 pmol/min/mg). The 17a-hydroxylase of ECLM was found to be 10 times less active than that of neonatal pig testicular microsomes (21). As in the human placenta and ovary (22) and equine testis (lo), the aromatase activity of the mare corpus luteum was located in the microsomal fraction and was NADPH-dependent. The mean ECLMES activity was found to be about 45% of the microsomal estrogen synthetase from human placenta (HPMES) activity determined in a single experiment (data not shown). The 23.7 and 31.3% of the radioactivity of aromatized [ l(3,2(3-3h]androstenedione and [l&2(3-3h]testosterone found in estrogens when using ECLMES may arise from the 24% (23) and 32% (24) tritium in the a position of [1(3,2(3-3H] androstenedione and [ l&2(3-3h] testosterone, respectively. The 3H20 release from [la,2a-3h]testosterone correlated well with the 16% tritium in the (3 position of [la,2a-3h]testosterone (indicated by Du Pont-New England Nuclear). Thus, in all likelihood, the aromatization of androgen occurred via the stereospecific loss of the lp,2(3 hydrogen atoms, as in the human placenta (25, 26), equine testis (lo), and mare granulosa cells (27). 4-Hydroxyandrostenedione has been shown to be an inhibitor of aromatase activity in human placenta and mammary tumor (28,29) and granulosa cells (30). Khalil et al. (15) have reported that 4-hydroxyandrostenedione completely inhibits the synthesis of 19-norandrostenedione from androstenedione in porcine granulosa cells. In our experiments, it also inhibited ECLMES but not the synthesis of androgen from progesterone. The activity of ECLMES, like that of microsomal estrogen synthetase from equine testis (ETMES) (18), was reduced by Mg+ and inhibited by EDTA. Although Ryan (31) reported that EDTA had little or no inhibitory effect, this substance has been found to stimulate the aromatization of testosterone by HPMES by 10% (24) and of 19-norandrostenedione by 50% (32). Reed and Ohno (24) reported that HPMES was stimulated by K+, and they optimized their reaction medium with the addition of 100 mmkc1. In contrast, ETMES was unaffected by the presence of KC1 at less than 100 mm (18) and like ECLMES, was inhibited when higher concentrations were added (data not shown), With HPMES, which is the most frequently studied model, the different K, values (14, 30, or 40 nm) reported (33-35) for androstenedione may result from the method of enzyme preparation or from the assay technique used. With microsomes, Kellis and Vickery (33) found a K,,, of 14 nm which was increased to 60 nm after purification of the aromatase. Yoshida and Osawa (36) found a K, of 10 nm with microsomes and 12 nm with aromatase purified by immunoaffinity chromatography using monoclonal anti-human placental aromatase cytochrome P-450 antibody. These values are very similar to the apparent K,,, we found with ECLMES (11 nm) and with equine testicular microsomes (13 nm) (18). With microsomes from sow ovary freed from corpora lutea, the apparent K, for androstenedione is 800 nm (37). Thus, the affinity of ECLMES for androstenedione resembles that found in the human placenta but seems to be very different from that in the porcine ovary. Using human placental microsomes, Kellis and Vickery (33) measured maximal velocities of 94 pmol/min/mg of protein, while Miyairi and Fishman (34) found a VmaX of 30-50 pmol/ min/mg of protein for the aromatization of androstenedione at 37 "C. With the equine testis, a V, of 21 pmol/min/mg of protein at 20 "C may be calculated for testosterone from our data obtained with a 16-year-old stallion (10). Similar velocities were observed with the equine corpus luteum. In contrast, the aromatization bysow ovary freed from corpus luteum aromatase (38) shows a maximal velocity which is 2 orders of

magnitude lower. There seems to be a correlation between the maximal velocities observed and the level of steroidogenic ability in the different tissues, since human placenta and equine testis and corpus luteum have a much more active endocrine function than the stroma of the sow ovary. 19-Norandrogens appear to be poorly aromatized by HPMES (25, 39). The conversion of 19-norandrogens is 5-20% of that of C19 androgens at concentrations ranging from 20 to 700 p~ (31, 40-42). Androgens are converted into estrogens in 50-100-fold greater yields than 19-norandrogens by partially purified human placental aromatase (43). Kellis and Vickery (44) reported that the V,,, for 19-norandrostenedione of the purified aromatase was 1/10 of that observed for androstenedione. In contrast, ECLMES aromatized androgens at a concentration of 3 p~ (200 X K,) with a slightly lower velocity (-30%) than the corresponding 19-norandrogens. Since these results are similar to those obtained previously with the aromatase of the stallion testis, they may provide evidence for a species specificity of the equine aromatase. Our results show that the inhibition of [ 1/3,2P-3H]testosterone aromatization by androstenedione, testosterone, 19-nortestosterone, and 19-norandrostenedione exhibits competitive kinetics. 19-Norandrostenedione (35,42) and 19-nortestosterone (39, 45) are competitive inhibitors of the aromatization of androstenedione by human placental microsomes, and 19- nortestosterone competitively inhibits the aromatization of testosterone by equine testicular microsomes (18). Similarly, with HPMES, androstenedione and testosterone are mutually competitive inhibitors (34). These results suggest that these steroids share a common binding site and possible a single identical site of aromatization. In our study, the kinetics of competitive inhibition showed that the sequence of increasing affinity for the active site of ECLMES was 19-nortestosterone, 19-norandrostenedione, testosterone, and androstenedione. To explore further this sequence and to compare it with those published for HPMES (46) and ETMES (10, 18), the ability of different steroids to inhibit the aromatization of [ 1/3,2P-3H]testosterone was tested. The most interesting finding was the difference in the potency of 19-norandrogens to inhibit the aromatization of androgens observed between HPMES and ECLMES. Similar to ETMES and in contrast to HPMES (which aromatizes 19- norandrogens very slowly while its aromatization of androstenedione is inhibited to the same extent by testosterone and 19-nortestosterone), ECLMES had a lower affinity for norandrogens than for androgens but aromatized the former at a higher velocity than the latter. The differences with HPMES and the similarities with ETMES which we have observed for ECLMES (effects of K and EDTA, efficiency of the inhibition of androgen aromatization by 19-norandrogens, and above all, the ability to aromatize 19-norandrogens) raise the possibility of the existence of aromatase isozymes among the human and equine species, while indicating that within the equine species, estrogen synthetase would not exhibit tissue specificity. The presence of aromatase isozymes among different species (human, baboon, horse, bovine, pig, and rat) has already been indicated by Osawa et al. (23), who used monoclonal antibody against human placental aromatase to suppress aromatase activity. Brodie et al. (47) have also reported finding subtle differences between aromatase enzymes from human placenta and rat ovary. It has long been believed that the ability of the corpus luteum to produce androgens and estrogens is directly related to the extent to which thecal cells contribute to its composition (48, 49). Accordingly, the ovarian androgens would be Estrogen Synthesis by Equine Corpus Luteum 7167 synthesized by theca-derived cells and aromatized by granulosa-derived cells or by a theca-granulosa cooperation. Previous histological studies have shown that in the mare, the corpus luteum originates from granulosa cells alone (50, 51). Both small and large luteal cells were described by Mossman and Duke (52) in the mare corpus luteum. Nevertheless, the ultrastructural study of the mare corpus luteum by Levine et al. (53) confirmed that only granulosa lutein cells were present in the mare cyclic corpus luteum up to luteolysis. Thus, although the mare corpus luteum appears to result from the luteinization of granulosa cells alone, we have shown that it is able to produce and to aromatize androgens in vitro. The participation of the thecal cells in the formation of corpora lutea and their synthesizing abilities in different species still appear controversial. Theca interna as well as granulosa cells contribute to the formation of the corpus luteum in women and sows (53-56), and both of these corpora lutea synthesize 17/3-estradiol(3,4). However, in humans, the fate of the theca interna cells in the young corpus luteum is still not clear (56). Mossman and Duke (52) have postulated that the theca interna cells degenerate and that the cells later found at the periphery of the corpus luteum are paraluteal cells induced by the granulosa lutein cells from the surrounding stroma. The early bovine corpus luteum is composed of both theca and granulosa lutein cells (57,58). The luteal cells which bind anti-granulosa cell antibody are progressively replaced by cells which bind anti-thecal cell antibody. However, despite the participation of thecal cells in its formation, the bovine corpus luteum, lacking 17a-hydroxylase, 17,20-lyase, and 19-hydroxylase aromatase systems, appears to be unable to synthesize androgens or estrogens in vitro (1,2,59). We have found (data not shown) that cow corpus luteum microsomes have very low 17a-hydroxylase and 17,20-lyase activities when incubated with labeled progesterone and 17a-hydroxyprogesterone and that the subcellular fractions (microsomes, mitochondria, and cytosol) were not able to aromatize [la,2a-3h]testosterone, [ lp,2/3-3h]testosterone, or [ l/3,2/3-3h]androstenedione. The theca interna cells of rodent, cow, and human ovaries show immunoreactivity to the anti-aromatase I1 cytochrome P-450; granulosa cells show this only in the large preovulatory follicle; and all corpora lutea react with the aromatase antibody (60). Our results, together with the above data, lead us to con- clude that in vivo production does not reflect the in vitro synthetic abilities and that the origin (theca or granulosa) of the corpus luteum cells does not account for their synthetic abilities. Moreover, the origin of the corpus luteum cells may vary during the different developmental phases of the corpus luteum life (early, mid-, or late cyclic corpus luteum or early, mid-, or late pregnancy corpus luteum). It has already been noted by Bjersing (61) that the apparent differences between primates and several other mammals regarding estrogen synthesis in the corpus luteum may be a difference of quantity rather than quality. The species differences in corpus luteum steroid production would thus depend on differences in the regulation of synthesis rather than on histological origin. Acknowkdgments-we are grateful to Dr. A. M. Brodie for generously supplying the 4-hydroxyandrostenedione and to Dr. Merle Shore for correcting the English text. REFERENCES 1. Zimmerman, R. C., Wun, W. S., Tcholakian, L. J., Rodriguez- Rigu, L. J., Braendle, W., and Steinberger, E. (1985) Horm. Metab. Res. 17, 458-463

7168 Estrogen Synthesis by Equine Corpus Luteum 2. Savard, K., and Telegdy, G. (1965) Steroids 5, (suppl.) 205-210 34. Miyairi, S., and Fishman, J. (1986) J. Biol. Chem. 261, 6772-3. Hammerstein, J., Rice, B. F., and Savard, K. (1964) J. Clin. 6777 Endocrinol. Metab. 24, 597-605 35. Kelly, W. G., Judd, D., and Stolee, A. (1977) Biochemistry 16, 4. Watson, J., and Leask, J. T. S. (1975) J. Endocrinol. 64, 163-140-145 173 36. Yoshida, N., and Osawa, Y. (1988) 8th International Congress of 5. Silberzahn, P., Quincey, D., Rosier, C., and Leymarie, P. (1978) Endocrinology, Japan, 15-19-001 (abstr.) J. Reprod. Fertil. 53, 1-5 37. Kautsky, M. P., and Hagerman, D. D. (1980) J. Steroid Biochem. 6. Pattison, M. L., Chen, C. L., Kelley, S. T., and Brandt, G. W. 13,1283-1290 (1974) Bwl. Reprod. 11, 245-250 38. Kautsky, M. P., and Hagerman, D. D. (1976) Steroids 28, 247-7. Makawiti, D. W., Allen, W. E., and Kilpatrick, M. J. (1983) J. 259 Reprod. Fertil. 68, 481-487 39. Thompson, E. A., Jr., and Siiteri, P. K. (1973) Ann. N. Y. Acad. 8. Mahajan, D. K., and Samuels, L. T. (1974) Steroids 24,713-730 Sci. 212,378-391 9. YoungLai, E. V. (1971) J. Endocrinol. 50,589-597 40. Gual, C., Morato, T., Hayano, M., Gut, M., and Dorfman, R. I. 10. Gaillard, J.-L., and Silberzahn, P. (1987) J. Biol. Chem. 262, (1962) Endocrinology 71,920-925 5717-5722 41. Meigs, R. A., and Ryan, K. J. (1971) J. Biol. Chem. 246,83-87 11. Short, R. V. (1962) J. Endocrinol. 24,59-63 42. Ganguly, M., Cheo, K.L., and Brodie, H. J. (1976)Biochim. 12. Silberzahn, P., Dehennin, L., Zwain, I., and Reiffsteck, A. (1985) Biophys. Acta 431,326-334 Endocrinology 117,2176-2181 43. Fronckowiak, M. D., and Osawa, Y. (1983) Endocrinology 112, 13. Khalil, M. W., and Walton, J. S. (1985) J. Endocrinol. 107,375-304 (abstr.) 281 44. Kellis, J. T., Jr., and Vickery, L. E. (1987) J. Bid. Chem. 262, 14. Ryan, K. J., and Short, R. V. (1965) Endocrinology 76,108-114 8840-8844 15. Khalil, M. W., Morley, P., Glasier, M. A., and Armstrong, D. T. 45. Thompson, E.A., Jr., and Siiteri, P. K. (1974) J. Biol. Chem. (1987) Aromatase, future perspectives, Miami, 32 (abstr.) 249,5373-5378 16. Canick, J. A., and Ryan, K. J. (1978) Steroids 32, 499-509 46. Siiteri, P. K., and Thompson, E. A., Jr. (1975) J. Steroid Biochem. 17. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. 6,317-322 (1951) J. Biol. Chem. 193,265-275 47. Brodie, A.M. H., Coombes, R. C., and Dowsett, M. (1987) J. 18. Silberzahn, P., Gaillard, J.-L., Quincey, D., Dintinger, T., and Al- Steroid Biochem. 27,899-903 Timimi, I. (1988) J. Steroid Biochem. 29,119-125 48. Henderson, K. M., and Swanston, I. A. (1978) J. Reprod. Fertil. 19. Brodie, A. M. H., and Longcope, C. (1980) Endocrinology 106, 52, 131-134 19-21 49. Rotchild, I. (1981) in Recent Progress in Hormonal Research 20. Thompson, E. A., Jr., and Siiteri, P. K. (1974) J. Biol.Chem. (Greep, R. O., ed) Vol. 37, pp. 185-298, Academic Press, New York 249,5364-5372 50. Harrison, R. J. (1946) J. Anut. 80,160-166 21. Nakajin, S., and Hall, P. F. (1981) J. Biol. Chern. 256, 3871-51. van Niekerk, C. H., Morgenthal, J. C., and Gerneke, W. H. (1975) 3876 J. Reprod. Fertil. 23, (suppl.) 171-175 22. Thompson, E. A., Jr., and Siiteri, P. K. (1979) Horm. Res. (Basel) 52. Mossman, H. W., and Duke, K. L. (1973) in Handbook of Physi- 11, 179-185 ology (Greep, R. O., and Astwood, E. B., eds) Vol. 2, pp. 389-23. Osawa, Y., Higashiyama, T., Fronckowiak, M., Yoshida, N., and 401, American Physiological Society, Bethesda, MD Yarborough, C. (1987) J. Steroid Biochem. 27, 781-789 53. Levine, H., Wight, T., and Squires, E. (1979) Biol. Reprod. 20, 24. Reed, K. C., and Ohno, S. (1976) J. Biol. Chem. 251,1625-1631 492-504 25. Fishman, J. (1982) Cancer Res. 42, (suppl.) 3277-3280 54. Corner, G. W., Jr. (1956) Am. J. Anat. 98, 377-401 26. Brodie, H. J., Kripalini, K. J., and Possanaza, G. (1969) J. Am. 55. Cavazos, L. F., Anderson, L. L., Belt, W.D., Henricks, D. M., Chem. SOC. 91,1241-1242 Kraeling, R. R., and Melampy, R. M. (1969) Biol. Reprod. 1, 27. YoungLai, E. V., and Jarrell, J. F. (1983) Acta Endocrinol. 104, 83-106 227-232 56. Crisp, T. M., Dessouky, D. A., and Denys, F. R. (1970) Am. J. 28. Brodie, A. M. H., Schwarzel, W. C., Shaikh, A. A., and Brodie, H. J. (1977) Endocrinology 100, 1684-1695 29. Brodie, A. M. H., Marsh, D. A., and Brodie, H. J. (1980) J. Steroid Biochem. 13, 1395-1401 30. Koos, R. D., LeMarie, W. J., Hung, T. T., and Brodie, A. M. H. (1985) Steroids 45, 143-150 31. Ryan, K. J. (1959) J. Bwl. Chem. 234,268-272 32. Townsley, J. D., and Brodie, H. J. (1968) Biochemistry 7,33-40 33. Kellis, J. T., Jr., and Vickery, L. E. (1987) J. Biol. Chem. 262, 4413-4420 Awt. 127,37-70 57. Gier, H. T., and Marion, G. B. (1961) J. Dairy Sci. 44, 1187 58. Alila, H. W., and Hansel, W. (1984) Biol. Reprod. 31, 1015-1025 59. Ursely, J., Darbon, J-M., and Leymarie, P. (1981) Steroids 38, 271-279 60. Matsuda, H., Fujita, H., Ishimura, K., and Osawa, Y. (1984) Acta Histochem. Cytochem. 17,311-322 61. Bjersing, L. (1981) in Functional Morphology of the Human Ovary (Coutts, J. R. T., ed) pp. 119-136, MTP Press Ltd., Lancaster, United Kingdom