ENDOCRINE RESEARCH, 14(1), 93-107 (1988) TESTOSTERONE MEIABoLISM AND Ils leslosterone-d[pendenl ACIIVAIION IN IHE UROPYGIAL GLAND OF QUAIL. J.Y. FLoCHo, R.F. MORFIN2, J.Y. DANIEL3 and H.H. FLoCHo Services de Biochimie (0) et de Biologie Cellulaire (3) de la Facullé de Médecine, Laboratoire de Biochimie (2) de la Facullé des Sciences, UA 598 du CNRS, 29279 BREST Cédex, FRANCE. Abstract Ihe ill vitro metabolism in the uropygial gland of lhe male quail resuitslnlo large yields of 5B-reduced and/or 17rt-hydroxylaled melaboliles. This melabolism was sludied in glands of sexually quiescent quails five days after a slngle intra-musculal' injection of lestoslerone lo the birds. This lrealment led lo an increased production of inaclive melaboliles (epitesloslerone and ils 58 reduced metaboliles) and to a decrease of unmelabollzed leslosterone. Thus testoslerone controls ils own metabolism and by UlIS way means to modulate ils action in lhe uropygial gland of quai!. Inlroduclion We have proposed the uropygial gland of adull male quail as a model for study.1ng lhe mechanism of action of androgens in sebaceous-like glands. In facl, we have shown in lhis organ the presence of androgen receptors (1) whose inactivalion stale (2) mld cellular concentration (2,3) were androgen-dependenl. We have also established that of quai!, the alcoholic moiely of the waxes secreled by uropygial gland testosterone was varied according lo lhe bird's androgenic slalus. 1hus proved to increase lhe relative concenlration of lhe dodecane-2,3-diol in the secretory products of lhe qlalld (4). Consequently, the dodecane-2,3-diol concenlration was suggesled to be used as a convenient index of quai! androqenicity (4,5). Moreover, by using electron-microscopic lechniques, we have recen 93 Copyright 1988 by Marcel Dekker, Inc.
94 FLOCH ET AL. tly shawn that little morphological differences existed between the uropygial gland of quail and the sebaceous gland of rat, and that testosterone stimulation induced the same ultraslructural modifications in both glands (6). Finally, we have also proved experiments that the uropygial gland of quail produced large yields of 170-hydroxylated and 5~reduced testosterone metabolites (7). ln numerous target organs to androgens, testosterone metabolism is a decisive step for hormonal action, and the plasma lestosterone levels may modulate the conversion of testosterone into active metabolites at the cellular levei (8). This study is a first. attempt to prove the androgen dependency of testost~rone metabolism in the uropygial gland of quail. For this purpose, several new testosterone metabolites were identified and their formation was measured five days after a single injection of testosterone to sexually quiescent quail. Ihe yields were compared with those measured in glands of sexually quiescent controls. Materials and methods Experiments were carried out with lhree groups of nine adult male quails purchased from a poultry farm (P. JOUAN, 22000 Plérin, FRANCE). The birds were obtained when six weeks oid, and reared at 18 C in controlled environment ct~mbers with tap water and industrial food 76650 Pelite Couronne,rRANCE) available ad libitum. were maintained under a short (6L: 1 1 ting schedule until ail quail were stabilized in sexual quiescence (evaluated by the area of lhe cloacal gland). One group was llsed as control. The birds of the next group received in the pectoral muscle a single injection of 100 1-19 testosterone dissolved in ethanol. The birds of the last group were injected with 10 mg of testosterone. Five days later, ail the birds were killed by decapitation. Their uropygial glands were excised, pooled in groups of three glands and immediately placed on crushed iee. Ihe glands were sliced with scissors and mineed with an arbor tissue press
TESTOSTERONE t/letabolism 95 (Harvard Apparatus, Boston, USA). Such procedure provided minces with a large proportion of ruptured cells. [4J4 C] Testosterone (50 mci/mmole) was from NEN (Boston, Mass., USA). The minces (600 mg) were incubated in a shaking water bath at 37 C with 0.5 [4-14 Cl-testosterone substrate (81 x 10 4 dpm) in 15 ml of phosphate buffer (0.067 M ; ph 7.4) containing 0.5 mm NADPH. After 3D min, incubations were stopped by 10 ml acetone, extracted with ethyl acetate (3 x 10 ml), and delipidated with hexane : melhanol : water partitioning (25 50 10, v/v). A bulk separation of the radiosteroids contained in the ex tracts illvojved a thin layer chromatography on silicagel F254 ates (Merck, Darmstadt, FRG) developped in benzene - 95 % ethano] (9 1, ). The metabolites were located on the chromatograms autoradiography, recovered by f abbreviations of steroids with 5a-A-3a,17a-diol :: ~-a-androstane-3a, 17a-diolCsynthetized in Lahoratory) 5a-A-3B,17a-diol :: 5a-androstane-3B, 17a-dioHsynthetized in Laboratory) 5a-A-3B,17B-diol 5a-androstane-3B, 17~-dioJ(Merck) 5a-A-3a,17B-diol :: 5a-androstane-3a, 17f3-cliol(Merck) 5R-A-3a,17a-cliol :: 5(3-androstane-3a, 17a-dioHsynthetized in Lahoratory) 5A.-A-3a,17B-diol :: 5B-androstane-3a, 17B-dioHsynthetized in Laboratory) 58-A-3j3,17a-diol :: 5B-androstane-3B, 17a-diol(synthetized in Laboratory) 58-A-38,17R-diol :: 5R-androstane-3R, 17R-diol(Mal<or-chemical) Epitestosterone :: 17a-hydroxy-4-androslen-3-one(Sigma) Testosterone :: 17R-hydroxy-4-androsten-3-one(Rou5sel-Uclafl 5a-A-3a-ol-17-one 3a-hydroxy-5a-androstane-17-one (t>\erck) 5a-A-17a-ol-3-one :: 17a-hydroxy-5a-androstane-3-one(Steraloids) 5a-A-17B-ol-3-one :: 178-hydroxy-5a-androstane-3-one (Merck) 5B-A-17a-ol-3-one 17a-hydroxy-5fl-androsLane-3-one (Steraloids) 5R-A-3a-ol-17-one :: 3a-hydroxy-5fl-androsbme-17-one (Merck) 58-A-17B-ol-3-one :: 17R-hydroxy-5R-androslane-3-one(Merck) 5B-A- 3B-ol-17-one :: 3B-hydroxy-5Randrostanp.-17-one (Merck) Androstenedione :: 4-androsten-3,17-dione (Sigma) 5a-A-3,17-dione :: Sa-androstane-3,17-dione 50-A-3,17-dione :: 5S-androstane-3,17-dione (Merck) scrapping the radioactive gel and eluted with 3 x 10 ml acetate (7). Recovery yields were computed from radioactivity measurements in portions of these eluates which were then dried
96 FLOCH ET AL. and subjected to a second thin layer chromatography on silicagel F. Developments of the chromatoqrams were carried out either in etherethyl acetate (95 5, v/v) or in dichlorometllane-ether (9 : 1, v/v) for separation of steroid epimers. Dicilloromethane-ether (7 : J,v/v) was also used for analysis of androstane-diols epimers. Detection and recovery of the separaterl radiosteroids was carried out as above. A Packard 300 liquid scintillation spectrometer was used for radioactivity measurements. Iderltifications hy crystallization to constant activity and by gas chromatography-mass spectrometry were carried out as previously described (7,8). RESULTS of testosterone n~tabolites testosterone melabolites recovered from the incubations was carried out by thin layer chromatography. This involved a two step procedure which is outlined in tahle I. After elution from the first chromatography in henzene-ethanol (9 : 1, v/v), four eluates containing the non-separated mixtures of steroid epimers were applied on second thin layer plates which were developped in different solvent mixtures. Thus, this procedure applied to reference steroids (Table 1) showed reasonably good ion of 8 androstanediol epimers with ether-ethyl acetate (95 : 5, v/v). Testosterone was resolved from its 17a-epimer, and androstane-3,17 dione epimers were separated in dichloromethane-ether (9:1, v/v) after two developments of the plates. Separation of 7 androstanolone epimers was obtained with the same solvent mixture but with three of the The of unknown radiosteroid more than androstanediols was not tried because of lhe lack of reference steroids. Identification of radiotestosterone metaboliles We have already reported identification of epitesloslerone and 5B-A-17a-ol-3-one as metabolites of testosterorle by the uropygial
TESTOSTERONE METABOLISM 97 TABLE l Reference Steroids TLe on Si F254 (Merck) ~-- 1( *) 2(*) 3(*) 4(*) 5(*) Polar unknowns 58-A-3a,17a-diol 58-A-3a,17$-diol 5a-A-3a,17a-diol 5a-A-3B,17a-diol 5a-A-3B,17~-diol 5B-A-3B,17a-diol 5a-A-3a,17R-diol 5B-A-3B,17B-diol epitestosterone testosterone 5B-A-17a-ol 3-one 5B-A- 3a-ol-17-one 5B-A-17B-ol- 3-one 5a-A- 3a-01-17-one 5a-A-17B-ol-3-one 5a-A-17B-01-3-one 5B-A- 3B-01-17-one androstenedione 5B-A-3,17-dione 5a-A-3,17-dione 1 0-0,33 0,33-0,37 0,37-0,42 0,42-0,60 0,60-0,67 U,67-0,78 0,30 U,40 U,49 0,53 0,56 0,49 0,59 0,43 0,61 0,66 0,33 0,43 U,32 0,34 0,45 0,49 0,5U 0,57 0,59 0,8U 0,86 ~~~~----~~- ~ (*) RF are given for the following solvent systems: 1. Benzene- 95 % ethanol (9:1, v/v) ; 2. Ether-ethyl acetate (95:5, v/v) ; 3. Dichloromethane-ether (9:1, v/v) developped twice ; 4. Dichloromethane-ether (9:1, v/v) developped 3 times ; 5 - Dichloromethaneether (7 : 3, v/v) developped twice.
98 FLOCH ET AL. TABLE Il ------- Specifie activity (dpm/mg) Steroid carrier dilution with carrier lst crystal!. 2nd crystal!. 3rd crystal!. 5a-A-~ol-17-one 221 MLQ* CR* 229 206 209 178 177 187 5B-A-3B-ol-17-one 1288 MLQ* CR* 1302 1264 1225 1284 1241 1296 5B-A-3B-ol-17B-diol 5814 MLQ* CR* 5858 5667 5426 5892 5199 5633 F~k~t, ~econd and th~kd cky~tall~za~on~ weke cakk~ed out ~n ethyl acetate-hexane, acetone-hexane and ethyl acetate-heptane, ke~pec~vely. * MLQ = Mothek l~quok~ 6kac~on~ - * CR = Cky~tal~ 6kac~on. gland of male quail (7). We have now evidences for the formation of four other testosterone metabolites : 5a-A-3a-ol-17-one (androsterone), 5B-A-3R-ol-17-one, 5B-A-3B,17B-diol and 5B-A-3B,17a-diol. Identifications of radiolabelled androsterone and 5R-A-3S-01-17-one and 5S-A-3B,178-diol with authentic carriers were proved when constant specifie activity was reached after three crystallizations in different solvent pairs of the carrier-diluted radiometabolites (Table Il). Since the quantities of 5R-A-3B,17a-diol carrier required for crystallization to constant specifie activity were not available, identification of the radiolabelled testosterone metabolite was based upon chromatographie mobilities and mass spectra identical with those of authentic 5R-A-3R,17a-diol.
TESTOSTERONE METABOLISM 99 TABLE III TMS derivative of 5a-A-3!3,17B-diol 5a-A-3a, 17B-diol 5B-A-3R,17a-diol 5R-A-3a, 17a-diol Retention time 9.07 min 7.24 min 6.02 min 5.40 min Thus thin layer chromatography on silica gel f 254 developped in ethyl acetate-ether (95 : 5, v/v) gave a Rf of 0,59, and two developments in dichloromethane-ether (7 : 3, v/v) gave a Rf of 0,43. Gas chromatography at 260 0 [ on a 30 m long open tubular glass capillary column (0.3 mm Ld.) coated with OV-101 separated the TMS derivative of 58-A-3B,17a-diol from those of other epimers (Table III) Mass spectra taken from the chromatographie peak at 6.02 min after injection of the TMS derivative of the isolated radiometabolite gave 3B,17a-diol. The identification of these four testosterone-metabolites, and our previous report of androstenedione, epitestosterone and mie fragments at 436-346-331-256-241 in a mass spectrum identical with that obtained from the TMS derivative of authffiltic 5B-A 5B-A-17aol-3-one formation in uropygial glands of quails, lead to the characterization of corresponding enzymatic activities. Testosterone metabolic pathway in uropygial glands of quails may then be depicted as in Fig.l. Testosterone metabolism in uropygial glands of lestosterone-treated guails Based on the identified testosterone metabolites and their separa tion by thin layer chromatographies, the testosterone transforma tions were measured in vitro with minces of uropygial glands of the testosterone treated and control quails.
100 FLOCH ET AL. o tio" o OH OH ph o ~ OH o OH o OH G)~ o... <D HO OH : FIGURE 1 Hetabolic pathway of testosterone in the uropygial gland of male quail. Enzymic activities are : 17B-hydroxysterold dehydrogenase 5a-reductase 17~hydroxysteroid oxidoreductase 3B-hydroxysteroid oxidoréductase 5 B-reductase ~hydroxysteroid oxidoreductase ~ ~ ~ Sterolds Identifled by crystallization to constant S.A. or gas chromatographymass spectrometry.
TESTOSTERONE METABOLISM 101 TABLE IV 14C-testosterone transformations by minces of uropygial glands of control and testosterone-injected sexually quiescent quails (mean percent of total extracted radioactivity ± sd). 14C_steroids Control 100 ~g Testosterone 10 mg festosterone n (3 x 3) n = (3 x 3) n (3 x 3) sd sd sd Testosterone 42,18 1,46 33,00 1,33 * 5,47 0,24 * Androstènedione 15,94 1,27 21,75 3,72 20,09 1,83 5a-A-178-ol-3-one 1 1 1 5a-A-3 ~ol-17-one 0,45 0,02 0,74 0,55 1,22 0,34 * 5a-A-3,17-dione 0,26 0,06 0,29 0,08 0,36 0,04 Epltestosterone 16,83 2,18 15,55 2,91 36,13 2,17 * 5a-A-17~ol-3-one 0,47 0,24 0,62 0,03 1,08 0,09 * 58-A-l~ol-3-one 0,51 0,22 0,91 0,20 2,14 0,51 * 58-A-38, 17~diol 2,72 0,59 3,30 0,93 11,09 1,11 * 1,29 0,36 1,71 0,31 0,16 0,02 * 58-A-38-178-diol 7,29 2,19 8,09 0,32 1 58-A-3S-ol-17-one 3,15 0,18 4,36 1,12 8,85 1,62 * 5S-A-3,17-dione 0,64 0,03 0,95 0,15 * 1,65 0,12 * unidentified polar 5,26 0,39 5,49 0,27 7,15 0,63 * metabolites sd standard deviation * : siqnificantlv different from controis Table IV shows that intra muscular injection of 10 mg testosterone to sexually quiescent quail resulted in a urnmatic alteration of the rate of testosterone transformations by minces of uropygial glands when measured 5 days later. Non-transformed 14C-testosterone was found to be elght fold decreased wlth treated birds when comwith controls.
102 FLOCH ET AL. ln contrast, 14C-epitestosterone and 5~-A-17a-ol-3-one were increased two and four times, respectively. As a general rule, an increase of the 5~-reduced-17a-hydroxylated derivatives of testosterone was observed with the treated birds. The effects of a testosterone were not as as above and 20 % decrease of non metabolized 14C-testosterone was observed in minced glands of treated birds when compared with those of controls. Each of the enzymatic activity involved in 14C-testosterone transformations may be quantified if based on total corresponding radiometabolites. (hus total 17S-oxidation of 14 C-testosterone corresponds with androstenedione + 5a- A-3a-ol-17-one + 5a -A-3,17-dione + 5S-A-3B-ol-17 one + 5B-A-3,17-dione radiometaholites. The total 17a-reduction is given by addition of epitestosterone, 5a-A-17a-ol-3-one, 5B-A-17a 01-3-one and 5B-A-38, 17a-diol quantities. Total 5B-reduction is the sum of 5B-A~17a-ol-3-one, 5B-A-3B,17a-diol, 5B-A-17B-ol-3-one, 5R-A-3S~17R-diol, 5B-A-3B-ol-17-one and 5~-A-3,17-dione. Total 5areduction is figured from addition of 5a-A-3a-ol-17-one with 5a-A 3,17-dione and 5a-A-17a-ol-3-one quantities.total 3B-reduction of 14C-testosterone metabolites corresponds with 5B-A-3R,17a-diol + 5B-A-3B,17S-diol + 5R-A-3B-ol-17-one transformation products. The 3a-redudion is only shown by 5a-A-3a-ol-17-one. ResuHing data are shown in table V. When compared wjth control animais, ail enzymatic activities involved in 14C-testosterone metabolism are signiincreased in the glands five days after injection of a single 10 mg dose of testosterone. Much less ficant increase occurs five days after injection of a single 100 ~] dose of testosterone, the 17S-hydroxysteroid desl1ydrogenase activity being the most significantly increased. Discussion ln mammals, according to species and tissues, androgen action is mediated by testosterone itself or by its active metabolites among which 5~dihydrotestosterone i8 the most potent (11). Thus, the metabolic conversion of testosterone in target organs to androgens
TESTOSTERONE METABOLISM 103 l ABLE V Expression of Testosterone-transforming enzyme activities in minces of uropygial glands of control and lestosterone-injected sexually quiescent quails. Data were obtained from addition of radioactivity percent values of 14C-Testosterone metabolites relevent to each enzymatic activity. 100 J.lg 10 mg Enzymac transformations control Testosterone Testosterone sd sd sd 17 8- oxidation 20,44 1,23 28,10 3,26 * 32,17 1,94 * 17 0.- reduct10n 20,53 1,35 20,38 3,00 50,55 1,35 * 5 13- reduction 15,60 3,34 19,33 1,70 23,89 3,07 * 5 0.- reduction 1,18 0,28 2,09 0,71 2,66 0,25 * 3 13- reduction 13,17 2,86 15,75 2,26 19,95 2,65 * 3 0.- reduction 0,45 0,02 0,74 0,55 1,22 0,34 sd standard deviation * : signiflcantly different from controls. is generally involved for both potentialization and modulation of biological responses and is also found to be associated with testosterone itself or with 50.-dihydrotestosterone (11).ln androgen target organs of birds however, 5B-reduction is a major pathway in testosterone metabolism. This was found in the cloacal gland of quail (12,13) and in the brain, the hypothalamus and the pituitary gland of various bird species (14,15). lt is weil known that 5R
104 FLOCH ET AL. reduced steroids are ail devoid of androgenic activity and consetheir production may only rerleet a process for testosterone inactivation (13,14,15).ln Lhe uropygial gland of quail, we have already shown thal testosterone metabolism involves little 5areduction but a very large conversion to lesloslerone Followed 5R-reduction (7) (figure 1). Our presenl identification of 5a-A-3a-ol-17-one, 5B-A-3Rol-17-one, 5R-A-3B,17R-diol and 5B-A-3B,17a-diol I~es our previous (7) where androstenedione, epilestoslerone and 5R-I\-17a-ol-3-one were identified From 14C-testosterone with minces of uropygial of quails. Among the major and minor metaholites of lestoslerone,17r-hydroxysteroids are markedly absent (except for 5R-A-3R,17B-diol) but replaced by 17-keto- and 17a-hydroxysteroids. Ihe presence of large amounts of androstenedione, produced in vitro from lesloslerone in te of Lhe NADPH supplementation of the digests SlH1l]ests that larger quantities may be formed ~len t ion with the reduced cofactor is omitted, and that 17R-oxidatiorl occurs prior to 17a-reduction. Except for androstenedione, about mie percent 01 the melabolites correspond wilh 17-ketosteroids, ail other transformation being l~hydroxylated or 5B-reduced. It is obvious that even 5 after ion of a 10 my dose of testosterone sorne of the male hormone must yet he present in the exarnined uropygial glands. \~e know that the plasma of sexual active quail carries 1.8 nq/ml of testosterone (10). This normal level was brouqht to 6.4 ± 1.5 ny/ml on the day followiny injection of tire single 10 mg dose fo testosterone but was back ta 1.8 on the FiFth day when the birds were killed. In contrast, the testosterone level was that of sexual active birds one day after injection of the 100 ~g dose but was hack to that of sexually quiescent controls (0.16 ± 0.03 ny/ml) Five days later. Therefore, after 5, quails of the 10 mg-injecled group correswith sexually active birds which had eliminated most of the massive injection. Our reported evidences for active transforma
TESTOSTERONE METABOLISM 105 tions of testosterone in uropygial glands of quails imply thal dosage of the male hormone in Lhese glands is of liltle inlerest, the measurement of ail its endoqeneous metabolites being nol yet feasable. Nevertheless, the use of in vitro incubation procedures with radiolabelled testosterone allowed an Recurate enouyh measurement of enzymatic activities involved in teslosterone lransformations. Aside of 50:- and5b-reduclions of testoslerone, the production of 17a-hydroxysteroids has not been reported so far in other target organs to androgens of birds. Thus, Lestosterone metabolism appears to proceed as an inaclivation step in the uropygial gland of quail. We have already shown that in vivo, lestosterone itself is the most potent androgen as evaluated by dodecane-2,3-diol production (5) as weil as by androgen receptor activation (2). In contrast, epitestosterone is unable to induce the production of dodecane-2,3-diol as weil as the activation of androgen receptors (10). As yet, other more polar metabolites of testostprone were not identified, but the characterized 3~reduclion of the testosterone metabolites could lead to further inactivation and excretion of the steroids by specific hydroxylations as demonstraled in human and canine target tissues (9,16). The rate of testosterone transformalions in larget organs to androgens was sometimes found to be dependenl on the plasma testoslerone level in mammals (10) and in birds (17). However, until now most results outlined lhat a high level of plasma testosterone resulted in a high production rate of active metabolites (Sa -dihydrolestosterone), and conversely a low level of plasma testosterone resulted in a low production rate of active metabolites. In other words, in testosterone-treated animais, the effects of the hormone are potentiated by the concomitant increase of the production of active metabolites at the level of the target cells. The situation is quite different in the uropygial gland of the quail : testosterone stimulates the synthesis of androgen receptors (2) and thus prepares the gland to respond. Our evjdences show now that it also gives rise to a metabolic shift which results in an inactivation of the hormonal stimulus.
106 FLOCH ET AL. The 100 ~ dose is able to activate the androgen receptors in sexually-quiescent quail, but one day later, plasma testosterone concentration is back to that of controis and the ratio of activated versus inactivated androgen receptors is not different From that found in controls. However five days after the injection of 10 mg testosterone, 14C-testosterone is still significantly more metabolized in treated than in untreated birds. This indicates that the effect of testosterone on its own metabolism persists after cessation of the stimulation. During sexual activity, the circulating testosterone is increased about twenty fold when compared with sexually-quiescent quail. Concomitantly the weight of the cloacal gland is increased ten fold but no weight increase is observed for the uropygial gland and its secretion of dodecane-2,3-diol is only increased by 20 % (4,13). Such discreapancies suggest that testosterone metabolism protects the uropygial gland of quail from an overstimulation by the male hormone during sexual activity. REFERENCES 1 - Amet, Y., Abalain, J.H., Daniel, J.Y., Thieulant, M.L. and Floch, H.H., C. R. Acad. Sei. Paris, 295 : 523, 1982. 2 - Amet, Y., Abalain, J.H., Di Stéfano, S., Daniel, J.Y., Tea, K. Floch, H.H. and Robel, P., J. Endocr., 109 : 299, 1986. 3 - Amet, Y., Abalain, J.H., Daniel, J.Y., Di Stéfano, S. and Floch H.H., Gen. Comp. Endocr., 62 : 210, 1986. 4 - Abalain, J.H., Picart, O., Berthou, F., Ollivier, R., Amet, Y., Daniel, J.Y. and Floch, H.H., J. Chromatogr., 274 : 305, 1983. 5 - Abalain, J.H., Amet, Y., Daniel, J.Y. and Floch, H.H., J. Endocri., 103 : 147, 1984. 6 - Abalain, J.H., Amet, Y., Lecaque, O., Secchi, J., Daniel, J.Y., and FLoch, H.H., Cell. Tissue Res., 346 : 273, 1986. 7 - Floch, J., Morfin, R., Picart, O., Daniel, J.Y. and Floch, H.H. Steroids, 45 : 391, 1985.
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