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Volume 33. number 2 FEBS LETTERS July 1973

TESTOSTERONE AND IN RAT PLASMA AND TISSUES

P. ROBEL, C. CORPECHOT and E.E. BAULIEU Unit& de Recherches sur le Mtftabolisme MoKculaire et la Physio- Pathologic des Stboides de I’lnstitut National de la SantC et de la Recherche M&dicale, UniversitC Paris-&d and Ddpartement de Chimie Bio$gique, HOpital de BicCtre, 78 rue du G&&al Leclerc, 94 Bic&re,

Received 22 April 1973

1. Introduction 2. Material and methods

From studies of rat ventral explants in Normal 3 month old Wistar rats, weighing 250- culture, in which in the tar- 300 g were used throughout the experiments. They get organ can be correlated with testosterone and tes- were killed by decapitation, and plasma and organ tosterone activities [ 1,2] , the concept has samples were collected and pooled. 10% Homogen- emerged that testosterone may be largely if not exclu- ates of the organs were made by the use of an ultra- sively active through the formation of metabolic de- Turrax, then 4 vol of ethanol containing tracer rivative(s) formed in situ. Parallel investigations have amounts of radioactive testosterone and androstano- indicated that an active dihydro-metabolite of testos- lone were added. After standing at -20°C overnight, terone, androstanolone (17&hydroxy-5a-androstan-3- the precipitate was eliminated and, after evaporation one, also known as dihydro-testosterone, DHT) is ac- under vacuum, the extracts were taken up in water cumulated in bound form in ventral prostate nuclei and extracted by ethyl acetate. [3-51 whereas a prostatic cytosol binds an- The gas chromatographic electron capture tech- with a higher affinity than testosterone nique described by Van der Molen and Groen [8] for ]6,71. the measurement of testosterone in human plasma Whether or not androstanolone formation is an was modified in order to allow the measurement of obligatory intermediary step in testosterone activity testosterone and dihydro-testosterone in rat plasma in the prostate itself and in other responsive and samples. The ethyl acetate extract was structures is still undecided, and therefore a systemat- dried, redissolved in hexane: (1: 1) and ap ic measurement of testosterone and androstanolone plied to a silica column [9] , followed by a silica in selected organs of normal adult male rats has been gel thin-layer chromatography in the benzene: undertaken as a first approach for understanding the (85: 15) system (2 migrations) [2] . Testosterone and physiological situation. It is believed that the local androstanolone were located using a radiochromato- concentration of active might be related to gram scanner, and eluted. Then the heptafluorobutyr- the presence and the binding characteristics of high ates of testosterone and androstanolone were formed, affinity receptors possibly involved in action. purified by thin-layer chromatography, and measured by gas liquid chromatography with electron capture detection [lo] . 2Ofl-OH-pregn-4-ene-3-one hepta- fluorobutyrate was used as internal standard for tes- tosterone heptafluorobutyrate, and testosterone hep- tafluorobutyrate was used as internal standard for * Postal address: Lab , F 94 Bicetre, France. androstanolone heptafluorobutyrate. The limit of

218 North-Holland Publishing Company - Amsterdam Volume 33, number 2 FEBS LETTERS July 1973

Table 1 Testosterone and androstanolone in rat plasma and tissues’. of testosterone So-reductase [ 141 and the relatively high affinity of the cytosol androstanolone receptor Testosterone Androstanolone for testosterone (0.2-0.5 that for androstanolone)

Plasma 2.5 f o.32 < o.23 [7] . In addition, it is not known whether both ster- 2.2 f 0.3 3.0 f 0.64 oids are evenly distributed throughout different Ventral prostate 2.0 + 0.4 2.8 + OS4 types. Levator ani muscle 7.9 f OS4 Q 0.2 In muscles no androstanolone is found, including Thigh muscles 2.9 * 0.5 Q 0.4 in the Zevatorani, a classical androgen dependent or- 13.7 f 2.04 2.0 ?r o.44 gan. Testosterone concentration is higher than in the 60.5 f 5.54 <6 plasma, a finding to be correlated with the presence parietal cortex 1.3 ?I 0.15 Q 0.4 of a cytosol receptor in the levator ani muscle, having 12.5 f 0.54 3.0 * 1.14 an affinity for testosterone greater than for andros- Small intestine =z o.24 < 0.2 tanolone [ 151. In skeletal muscles, display ’ rig/g (Organs) or ml (plasma). a well known anabolic action, responsible for the - : Mean f SEM. ual difference in their development even they are usu- G Values too low to be measured accurately. Their upper ally not considered as typical secondary sex organs. limit is given. The concentration of testosterone in thigh muscles is 4 Significantly different from corresponding plasma values P G 0.01. of the order of that found in the plasma, whereas in ’ Significantly different from corresponding plasma values control tissues as intestine or brain cortex the value P < 0.2. is definitely lower. Therefore it might be possible that skeletal muscles contain a testosterone binding pro- sensitivity of the method is 0.4 ng of . The tein, possibly of feeble abundance and/or difficult to overall recovery of added tracers is 20-30%. Thus solubilize and which may be therefore very difficult the initial 5 g (ml) of tissue (plasma) sample should to demonstrate [ 151 . It is noteworthy that in muscle contain at least 2 ng of steroid for processing through cells which exhibit a purely hypertrophic response to the entire procedure. All measurements were done in testosterone, no androstanolone is found, whereas triplicate. The coefficient of variation for plasma tes- this latter compound has an hyperplastic activity pre- tosterone samples was found to be 10% [lo] . viously documented in prostate experiments [ 11. In several other organs, kidney, hypothalamus and pituitary, there is also testosterone accumulation, 3. Results and discussion whereas androstanolone is present in a relatively small concentration. As in the muscle, testosterone The concentrations of testosterone and dihydrotes- itself may also be implicated in androgen action on tosterone were measured in rat plasma, seminal vesi- (mice) kidney [ 161. In the hypothalamus and pitu- cles and prostate, Zeuatorani and thigh muscles, hypo- itary, the nature of the steroid molecule(s) regulating thalamus, pituitary gland and brain parietal cortex, the synthesis and secretion of releasing kidney and small intestine (table 1). hormone(s) and is (are) still controver- In the plasma, testosterone is present at a concen- sial. The present results suggest a role for testosterone tration similar to those previously reported [ 1 l- 131, itself, whereas arguments have been brought recently whereas androstanolone is not detected. in favor of reduced [ 17-191 or oestrogen [20] me- In ventral prostate and seminal vesicles, androstano- tabolites of testosterone. lone is found at a slightly greater concentration than There is possibly some interest that a regulatory testosterone, which is itself in amount similar to that molecule has to proceed through several transforma- in the plasma. The fact that testosterone concentra- tion steps before being active, and the formation of tion approaches that of androstanolone is rather unex- different active metabolite(s) in target organs may un- pected, although testosterone accumulation in pros- dergo a subtle regulation. This mechanism offers the tate cells might be favored by the relatively high K, possibility of selective pharmacodynamic or therapeu-

219 Volume 33, number 2 FEBS LETTERS July 1973

tic approaches. Testosterone might be a “hormone” [ 21 P. Robel, I. Lasnitzki and E.E. Baulieu, Biochimie 53 in the muscles and in some cells of the kidney, the (1971) 81. [ 31 K.D. Anderson and S. Liao, Nature 219 (1968) 277. hypothalamus and the pituitary, whereas it would be [4] N. Bruchovsky and J.D. Wilson, J. Biol. Chem. 243 a “prehormone” elsewhere, the active molecule being (1968) 2012. androstanolone in the prostate and the seminal vesi- [S] I. Jung and E.E. Baulieu, Biochimie 53 (1971) 807. cle and possibly also an oestrogen in the hypothala- [6] W.I.P. Mainwaring, J. Endocrinol. 45 (1969) 333. mus. Therefore selective analogues or antagonists [7] E.E. Baulieu and I. Jung, Biochem. Biophys. Res. Commun. 38 (1970) 599. could be devised in order to act upon the correspond- [ 81 H.J. Van der Molen and D. Groen, in: Gas-liquid ing target tissues, thus using the metabolism of testos- chromatography of steroids, ed. J.K. Grant, Memoir of terone as a molecular basis to dissociate its overall ef- Society for , Nr. 16 (1967) p. 155. fects. [ 91 P. Rot&, These Doctorat &s-Sciences, Paris (1967) p. 11. [lo] C. Corpechot and P. Robel, in preparation. [ll] B.J. Lloyd, J. Endocrinol. 54 (1972) 285. [ 121 K.J. Tveter and A. Aakvag, Acta Endocrinol. 65 (1970) Acknowledgements 723. [ 131 L. Buric, H. Becker, C. Peterson and K.D. Voigt, Acta The authors acknowledge the financial help of the Endocrinol. 69 (1972) 153. Ford Foundation, the Centre National de la [ 141 A.B. Roy, Biochimie 53 (1971) 1031. [ 151 I. Jung and E.E. Baulieu, Nature New Biology, 237 Recherche Scientifique, the Ligue Nationale (1972) 24. Franqaise contre le and Roussel-UCLAF. The [ 161 L.P. Bullock and C.W. Bardin, J. Clin. Endocrinol. skillful assistance of B. Leclaire, L. Helie, S. Rouzaud Metab. 35 (1972) 935. and P: Toubkis is acknowledged. [ 171 F.F.G. Rommerts and H.J. Van der Molen, Biochim. Biophys. Acta 248 (1971) 489. [ 181 C. Beyer, R.B. Jaffe and V.L. Gay, Endocrinology 91 (1972) 1372. References [ 191 Z. Knievald, R. Massa and L. Martini, Excerpta Medica Inter. Congress Series no. 219 (1970) 784. [l] E.E. Baulieu, I. Lasnitzki and P. Robe], Nature 219 [ 201 F. Naftolin, K.V. Ryan and Z. Petro, J. Clin. (1968) 1155. Endocrinol. Metab. 33 (1971) 368.

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