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Uptake, binding and metabolism of testosterone in rat brain tissues
Brain Research, 53 (1973) 139-150
139
© ElsevierScientificPublishing Company,Amsterdam- Printed in The Netherlands.
UPTAKE, BINDING AND METABOLISM OF TESTOSTERONE IN RAT BRAIN TISSUES
M. MONBON, B. LORAS, J. P. REBOUD ANDJ. BERTRAND Bdtiment de M~decine, Faeult~ des Sciences, 69100 Villeurbanne and Unit~ de Recherches Endocriniennes et M~taboliques chez l'Enfant, H6pital Debrousse, 69005 Lyon (France) (Accepted October 10th, 1972)
SUMMARY
Slices of cerebral cortex, pituitary and hypothalamus of one-day castrated adult male rats were incubated with tritiated testosterone (T), and the ventral prostate was used as a control target organ. Uptake seemed to be a non-specific process of exchange diffusion. Tissue specificity appeared in macromolecular binding and intracellular metabolism of the hormone. Pituitary tissue bound testosterone less and much more slowly than did prostatic tissue, but the binding was greater than that observed in hypothalamus and cerebral cortex. Moreover, the percentage of bound hormone in nuclear extracts of hypothalamus, and especially pituitary, appeared to be dependent on its concentration in the medium. The main metabolite found in the prostate was dihydrotestosterone (DHT). In the pituitary less D H T was found, but the rate of conversion of T to DHT remained far higher than in hypothalamus and cortex. In the 4 tissues studied, the nucleus bound higher percentages of radioactivity, and more D H T was found in the nucleus than in the corresponding cytosol. In addition, conversion of T to D H T increased in parallel with the degree of binding. These two observations suggest that there is no qualitative difference in general handling of T by prostatic and neural 'receptors', although large quantitative differences are found.
INTRODUCTION
Since feedback mechanisms of sex hormones have been described, various methods - - autoradiographyl, 13, injection in vivo 6,9,1°,17,zz and incubation in vitro 14,1s,21 - - have established that the hypothalamic-hypophyseal axis acts as a target organ to testosterone. We have studied in vitro some conditions of penetration and binding of tes-
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m. MONaON et al.
tosterone in the diencephalic-hypophyseal region, and its intracellular metabolism, in castrated adult male rats. The prostate was used as a control target organ in view of a possible comparison of general functioning of the 'receptors'. The cerebral cortex was taken as a control 'non-target' organ. MATERIALS
Young adult male Wistar rats weighing about 250 g were castrated 24 h before the experiment. The animals were killed by decapitation, and the following organs or parts of organs were removed: fragment of frontal cortex; the entire hypothalamus, between 1 and 2 mm in thickness, limited anteriorly by the optic chiasm, laterally by the hypothalamic fissures and posteriorly by the mammillary bodies; the entire pituitary, removed from the sella turcica after section of the stalk; ventral lobes of the prostate. Tritiated testosterone ([1,2-3H]-T) was supplied by the C.E.A. (Saclay), with a specific activity of 42.6 Ci/mmole, its purity checked by paper chromatography before use. It was stored at --30 °C in ethanol at a concentration of 34 ng/ml. Before use, the alcohol was evaporated under nitrogen and the hormone redissolved in a small quantity of propanediol and then in the incubation medium. The following media were used. Incubation media. 'Complete' Krebs medium: this is Krebs medium !II .5. 'Incomplete' Krebs medium: medium Ili lacking fumarate, glutamate and pyruvate. Homogenizing media. Medium I: Tris-HC1 (pH 7.4), 50 mM; EDTA, 1.5 mM; mercapto-ethanol, 2 mM; MgC12, 2 mM; KC1, 10 mM; saccharose, 250 mM. Medium II: Tris-HC1 (pH 7.4), 50 mM; EDTA, 1.5 mM; mercapto-ethanol, 2 mM; MgC12, 2 mM; KC1, 10 mM; NaCI, 1 M. METHODS
Removal of organs and incubation After decapitation and dissection, the removed organs were immediately placed in Krebs medium at 0 °C, then minced into tiny fragments. After preincubation for 15 min at 0 °C and for 15 min at 37 °C, the tissue was incubated with tritiated testosterone in Krebs medium at 37 °C, under continuous agitation in the presence of 99.5 % oxygen. Most incubations were done with 3.4 ng testosterone/ml of medium, which appears to be the physiological concentration for rats 15. Individual organs were pooled and incubated in a volume approximately proportional to the total weight (1 ml/50 mg). Homogenization and cellular fractionation After incubation, each tissue was carefully washed with 100 ml of medium I over a linen filter, homogenized for 1 min in a glass-teflon Potter-Elvehjem, and filtered through several layers of gauze. The homogenates were fractionated in a Beckman centrifuge (type L3 40) at
TESTOSTERONE IN RAT BRAIN TISSUES
141
800 × g during l0 rain. A pellet of crude nuclei and a cytoplasmic supernatant were obtained. The latter was centrifuged for 90 min at 105,000 × g, yielding the 'cytosol' fraction free of subcellular particles. The nuclear pellet was washed twice, suspended in medium II and agitated for 30 min. The suspension was further fractionated, after 15 min centrifugation, into a pellet (chromatin q- nuclear membranes) and a soluble fraction containing proteins and nucleic acids of low molecular weight. All these operations were performed at temperatures ranging from 0 to 4 °C.
Measurement of radioactivity An aliquot of each homogenate or cellular fraction was dissolved in a small quantity of Soluene (0.1 ml) and left at 40 °C for 24 h. Fluorescence of Soluene was neutralized by 0.4 ml of glacial acetic acid; 0.5 ml of ethanol was added, then 14 ml of a toluene-based fluor (PPO, 5 g; dimethyl-POPOP, 0.3 g; toluene 1 litre). The samples were counted in a TriCarb scintillation spectrometer (Model 33 20). Correction for quenching was made by the external standard method. All samples were counted for the time required to ensure less than 5 % error in counting.
Measurement of hormone incorporation The cytosol and nuclear supernatant fractions were filtered at 4 °C on Sephadex gel columns tested against Dextran Blue. The cytosol was eluted with medium I lacking saccharose, and the nuclear supernatant with medium II. An aliquot of each fraction was counted to give the elution profile. Radioactivity bound to the excluded peak was used as the criterion of incorporation.
Androgen analysis* Isolation of testosterone, D H T and A4-androstenedione was carried out by reverse isotopic dilution, as indicated in the diagram (Fig. 1). Since the level of radioactivity (R.A.) of the total extract was low, specific activity could not be measured by systematic crystallization of isolated steroids. Therefore non-labelled- or 14C-carrier was added and the purity of steroids was verified by the constancy of the respective ratios aH :#g of non-labelled carrier, or 3H: 14C in the course of purification. Paper chromatography (PC) was done at 27 °C, the first on Whatman 3MM and the second on Whatman no. 2 paper. Silica gel thin-layer plates (TL), as purchased, were washed once in ethanol and once in methanol and stored in a desiccator. Elution was done with a toluene-methanol mixture (97:3), and acetylation performed in the usual way with acetic anhydride in pyridine. Saponification by potassium hydroxide in methanol frequently caused a decrease in aH:14C or aH:/zg of carrier ratios, which was more marked for testosterone than for DHT. However, the presence of tritiated metabolites of these two steroids could not be established. Acetylation followed by saponification of mixtures of standard [1,2-all] - and [4-14C]testosterone, * Testosterone, 17fl-hydroxy-4-androsten-3-one; DHT, 17fl-hydroxy-5a-androstan-3-one; A4-androstenedione, 4-androsten-3-17-dione; epitestosterone, 17a-hydroxy-4-androsten-3-one; isoandrosterone, 3/~-hydroxy-5a-androstan-17-one; fl-DHT, 17fl-hydroxy-5fl-androstan-3-one;etiocholanolone, 3a-hydroxy-5fl-androstan-17-one.
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M. M O N B O N e t
al.
ISOLATION OF STEROIDS
Sample Addition of non-labelled carrier + [14C]testosterone and DHT 1. Extraction: 3 × 7-8 v CHCh. 2. Measurement of total R.A. on 1/50.
I
PC hexane-MeOH-water 100.90:10 6 h Steroids of Rtestosterone = 0-1 TL CHC13-EtOH 95: 5 twice
Steroids of g testosterone = 2.3-3.1
less polar
TL CHCl3-ether - 90:10 twice --3
A4
Testosterone 1st S.A.*
DHT 1st S.A.
PC hexane-benzene-MeOH-water 66: 33 : 80:10
Ether twice
CHCIa-EtOH 95:5
2nd S.A.
2nd S.A.
2nd S.A.
Acetylation
Acetylation
TL CHCIa-EtOH 95: 5 twice
TL CHCla-ether 90:10 twice
3rd S.A.
3rd S.A.**
Saponification
Saponification
TL
TL
1st S.A.
* S.A., Specific activity measured by the 3H:a4C or 3H:pg non-labelled carrier ratios. ** Only if [laC]DHT added. Fig. 1.
a n d o f s t a n d a r d [1,2-3H]- a n d [4-14C]DHT, resulted in a decrease o f the ratio 3H." 14C. I t a p p e a r e d , therefore, t h a t p a r t o f the t r i t i u m present in the 1 o r 2 p o s i t i o n was able to exchange with h y d r o g e n ions present in solution d u r i n g this reaction. F o r this reason, only the values o b t a i n e d before this e x p e r i m e n t a l stage were used for the calc u l a t i o n o f results. Isolation of testosterone. The first thin-layer c h r o m a t o g r a p h y ( T L C ) s e p a r a t e d testosterone f r o m the m o s t p o l a r c o m p o u n d s : androstan-17fl diols (3a a n d 3fl), A4-androsten-17fi diols (3a a n d 3fl) a n d 5fl-androstan-3a, 17t3 diol. The following P C s e p a r a t e d testosterone f r o m epitestosterone and 5fl-androstan3fl, 17-fl diol. Isolation of A4-androstenedione. The first T L C separated A4-androstenedione
TESTOSTERONEIN RAT BRAINTISSUES
143
from known steroids eluted from the first chromatogram: DHT, isoandrosterone, fl-DHT and etiocholanolone. Isolation of DHT. The first T L C separated D H T from all the above-mentioned steroids. The closest was isoandrosterone (RDnT = 0.85-0.90). If the first T L was overloaded or migration less successful, the second TL completed the separation of D H T from isoandrosterone. Because of variability in the different batches of TL, the best separation of D H T from isoandrosterone was achieved either in a CHCla-ether system (90:10), or in pure ether, or occasionally in a CHCla-ethanol (95:5) system. RESULTS
Hormone uptake in tissue The amount of radioactivity present in the homogenate after the tissue had been washed with medium was taken as the criterion of penetration. Testosterone uptake by the 4 organs studied was investigated in relation to time (Table I). With cerebral cortex and prostate, virtually maximal uptake resulted after a 60-min incubation period. In contrast, in pituitary and hypothalamus, radioactivity was still increasing after 150 min of incubation, indicating a slow penetration and a hormone concentration below saturation. In the 4 organs studied, the proportionality between the measured amount of intra-tissular hormone and hormone concentration, up to 10 times plasma concentration, revealed no factor limiting hormone penetration. However, each organ showed a specific ratio of R.A./mg of tissue to R.A./ml of medium (cortex, 5.0; pituitary, 6.0; hypothalamus, 4.8; prostate, 3.3). When tissues were frozen before incubation, these ratios were not significantly changed.
Cellular fixation of hormone Radioactivity bound to the macromolecular peak excluded from a Sephadex G-25 column was taken as the criterion of fixation.
Effect of time of incubation (Table II) During the measurement of fixation by cytosol macromolecules, clear difTABLE I Radioactivity (R.A.) of the homogenate is expressed as disint./min/mg wet weight. The tissues were incubated in incomplete Krebs medium containing 3.4 ng tritiated testosterone/ml.
Number of rats
10 8 10 9 8
Time (min)
5 10 30 60 150
Organ Cerebralcortex Pituitary
Hypothalamus Prostate
616 1200 1601 2742 3070
472 946 1627 1865 2960
814 1350 2169 2440 4150
802 1010 1570 2460 2370
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M. MONBON et al.
TABLE II Radioactivity bound to the peak excluded from Sephadex G-25 is expressed as disint./min/mg wet weight of tissue incubated in incomplete Krebs medium containing 3.4 ng tritiated testosterone/ml. Number of rats
Time (min)
Cytosols Cerebral cortex
Pituitary
Hypothalamus Prostate
10 8 10 9 8
5 10 30 60 150
0.5 0.6 1.0 0.7 1.0
11.9 12.2 12.0 28.0 34.0
0.4 0.9 1.0 1.1 1.4
38.8 28.6 45.4 48.6 45.0
Nuclei. Prostate
4.2 10.9 11.3 -
TABLE III Radioactivity bound to the peak excluded from Sephadex G-25 is expressed as disint./min/mg wet weight. The organs were incubated for 60 min in complete Krebs media containing 3 concentrations of [all]testosterone. Organ
Prostate
Pituitary
Hypothalamus
Cerebral cortex
Cellular fraction
Cytosol Nuclear supernatant (SNx) Ratio SNx: cytosol Cytosol SNx Ratio SNx: cytosol Cytosol SNx Ratio SNx: cytosol Cytosol SNx Ratio SNx: cytosol
[ 3Hi testosterone in ng/ml of medium 3.4*
10.9"*
31.6"**
96 19 0.20 52 < 1 2 ~ 1 1 < 1 -
178 126 0.71 67 9 0.13 17 9 0.53 16 5 0.31
272 119 0.44 128 15 0.12 24 6 0.25 -
* 8 rats. ** 16 rats. *** 11 rats.
ferences were seen, in r e l a t i o n to time and to the o r g a n considered. Indeed, cytosolic ' r e c e p t o r s ' o f the cortex, h y p o t h a l a m u s and p r o s t a t e seemed to have reached saturation after 30 min, whereas hypophyseal ' r e c e p t o r s ' were not saturated even after 150 rain o f incubation. In the p r o s t a t e the cytosolic and nuclear ' r e c e p t o r s ' reached s a t u r a t i o n at the same rate. Quantitatively the radioactivity fixed by cytosols o f the cortex and h y p o t h a l a m u s was c o m p a r a b l e and far inferior to that in the prostate, while the pituitary was intermediate. H o r m o n e fixation by cytosolic m a c r o m o l e c u l e s o f the different organs depended greatly on the state o f the tissue: indeed, m a c r o m o l e c u l a r peaks derived f r o m cytosol
TESTOSTERONEIN RAT BRAIN TISSUES
145
fractions of organs which had been frozen before incubation were devoid of radioactivity, however long the time of incubation, whereas in fresh samples radioactivity was present.
Effect of hormone concentration (Table III) Cellular fixation of the hormone differed according to the cellular fraction under consideration: in general the cytosol fixed more hormone than the nuclei; and in addition the nuclear 'receptors' appeared to reach saturation more quickly than the cytosolic 'receptors', except in the pituitary. Whatever the concentration, the pituitary fixed less than the prostate and far more than the hypothalamus or cortex, which scarcely differed. Distribution of free and protein-bound radioactivity in relation to (all]testosterone concentration (Table IV) Filtration of subcellular fractions (cytosol and nuclear supernatant) on Sephadex G-25 revealed two radioactive peaks: one eluted with the proteins and the other corresponding to free steroids. The relation between these peaks indicated the distribution of radioactivity between two 'compartments' within the cellular fraction. This ratio was studied in each fraction and organ in relation to hormone concentration. In general, relatively more radioactivity was bound by the nuclei than by the cytosol, thus revealing greater specificity in nuclear fixation. But only nuclei of the prostate showed a ratio greater than I. In the prostate, this ratio appeared to be independent of hormonal concentration, whereas in hypophyseal and hypothalamic nuclei the former increased faster than the latter. Metabolism o f androgens The amount of radioactivity in these cellular fractions did not permit crystallizaTABLE IV Ratio of peaks of bound to free radioactivity after filtration of cellular fractions (cytosol and nuclear supernatant) on Sephadex G-25. The organs were incubated for 60 min in complete Krebs medium with 3 different concentrations of [SH]testosterone.
Organ
Cellularfraction Ratio of bound to free radioactivity ( [3H]testosterone in ng/ml of medium) 3.4 (8 rats)
10.88 (16 rats)
31.55 (11 rats)
Cytosol Nuclear SN
0.097 1.640
0.131 1.380
0.132 1.650
Hypophysis
Cytosol Nuclear SN
0.009 0.001
0.017 0.031
0.005 0.385
Hypothalamus
Cytosol Nuclear SN
0.001 0.001
0.004 0.033
0.002 0.118
Cerebral cortex
Cytosol Nuclear SN
0.001 0.001
0.002 0.025
-
Prostate
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M. MONBON et al.
tion of isolated steroids. However, the ratio c o u n t s / m i n :/~g of carrier steroid, or the ratio 3H:14C, remained c o n s t a n t in successive chromatographies, once k n o w n interfering steroids were separated from testosterone and D H T . Acetylation in particular did n o t modify the ratio. Therefore the purity of isolated steroids was considered acceptable. Results are shown in Tables V a n d VI. Table V gives the results of a 60-min i n c u b a t i o n . Testosterone, D H T and A4_ a n d r o s t e n e d i o n e were isolated from the cytosol a n d from the whole nuclei (pellet of centrifugation at 800 × g, washed twice). The prostate metabolized testosterone more completely t h a n did the neural tissues, with considerable f o r m a t i o n of D H T . Hyp o t h a l a m u s a n d cortex metabolized only a small part of the testosterone (less than 20 %) a n d little D H T was formed. The pituitary was intermediate between these two groups. F o r m a t i o n of A4-androstenedione was slight in all 4 tissues. Nuclei contained less testosterone a n d more D H T t h a n corresponding cytosols. However, the difference between prostate a n d neural tissues r e m a i n e d large. TABLE V Distribution of radioactivity among isolated androgens of the cytosol and whole nuclei, after cellular fractionation of organs incubated for 60 min in Krebs medium in the presence of 3.4 ng/ml [3H]testosterone. Results expressed as % of extracted radioactivity. Organ
Pituitary Hypothalamus Prostate Cerebral cortex
Cytosol
Nucleus
T
DHT
A4
T
DHT
/[4
63.9 91.0 2.5 77.9
12.1 5.2 46.1 1.8
1.6 0.4 0.1 0.2
42.7 2.0 62.8
27.6 9.0 82.9 6.9
3.7 0.9 0.1 0.5
TABLE VI Distribution of radioactivity between testosterone and dihydrotestosterone isolated from cytosol and nuclear extract of organs incubated in Krebs medium in relation to time, in the presence of tritiated testosterone (3.4 ng/ml). Results expressed as % of extracted radioactivity. Cellular fraction
Organ
Incubation period ( min ) 5 T
Cytosol
10 DHT T
Pituitary 100 Hypothalamus 99.4 1.2 Prostate 21.7 40.5 Cerebralcortex 100 -
Nuclear extract Pituitary 100 Hypothalamus 83.2 < 3 % Prostate 15 82 Cerebral cortex 80.6 -< 3
60
30
DHT T
DHT T
93.7 8.0 50.0 97.0 4.6 8.8 77.8 2.5 95.6 1.5 87.1 -
150 DHT T
DHT
6.0 6 4 . 5 14.8 46.0 31.0 1.6 90.0 6.2 78.0 6.5 33.6 3.7 55.7 - 48.7 1.1 89.0 3.3 90.8 1.4
90 10 76.9 4.9 4 87 78.4 < 3 ~
15 70 3 77
41 18 88 <3~
TESTOSTERONE IN RAT BRAIN TISSUES
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Table VI shows the results of incubation carried out under the same conditions as before, but for varying lengths of time. No analysis for A4-androstenedione was done since it constituted an insignificant part of the total R.A. Nuclear R.A. was analysed, not as previously in the whole nuclei, but in the soluble nuclear extract obtained as described in the 'cellular fractionation' section. As in the preceding incubation, the prostate metabolized a great deal, the pituitary an appreciable amount, and the hypothalamus and cortex very little testosterone. After 30 min of incubation no further increase of testosterone metabolism in the cytosols was observed in any of the 4 tissues. The amount of DHT obtained increased with time in the pituitary, up to 31 70 after 150 min, but in the prostate the maximum (78 ~) was reached after 10 min of incubation, as in the hypothalamus where the amount reached a low maximum of 6.5 70. Nuclear extract, like whole nuclei, was richer in DHT than was the corresponding cytosol, and metabolism was slower, particularly in the hypothalamus and pituitary. DISCUSSION
This study, which aimed at comparing the behaviour of testosterone in the diencephalon and the prostate on the one hand, and in the diencephalon and the cerebral cortex on the other, permitted us to make several observations. Hormone uptake occurs easily in all the organs studied in vitro but at various rates (Table I). The proportion between rate of uptake and amount of hormone present, together with the observation that rate of uptake is not modified by freezing - - which, however, prevents fixation - - suggests a penetration by diffusion. The ratios (R.A./g of tissue: R.A./ml of medium) we found for the pituitary (6.0), hypothalamus (4.8) and cerebral cortex (5.0) are smaller than those reported by Samperez e t al. la in rats castrated for 40 days. In addition, we found only slight variation within the 4 tissues studied. Thus, under the experimental conditions used, hormone uptake seems rather non-specific to the tissue. In contrast with penetration, cellular binding of the hormone in the different organs studied indicates very clear specificity (Table II), although we only studied the whole protein fraction and did not try, in this study, to isolate the specific molecule. In the prostate, despite relatively low uptake, binding is considerable and rapid, reaching its maximum after 30 min in cytosol and nuclear supernatant. At the same time the cytosolic metabolism of testosterone is intense (Table VI). The principal metabolite is DHT, which accounts for almost 80 ~ of the radioactivity after 10 min. The subsequent lower rate can probably be attributed to slower formation of other metabolites, since our results are expressed as percentages. These other metabolites constitute a smaller percentage of the nuclear extract where DHT remains the major metabolite: 88 70 in 150 min. Analysis of whole nuclei confirms these results (Table V), which are in agreement with the results from other laboratories~,3,7, 23. The rate of hormone binding by cytosolic macromolecules of the pituitary is much slower than for the prostate: even in the 150-min incubation, fixation is not maximal. At the same time, DHT accumulates in cytosol and nuclei, suggesting either
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M. MONBONet al.
slow D H T formation or an insignificant subsequent metabolism. The percentage of A4-androstenedione, even if greater than in the other organs studied, remains very low (Table V). The rate of hormone binding in hypothalamic cytosol is indistinguishable from that in the cortex. There is a distinctly smaller accumulation of D H T than in the pituitary, particularly in the cytosol. On the other hand, hypothalamic nuclei form D H T more effectively after a longer period of time (18 ~ after 150 min) than do cortical nuclei. An investigation of binding in relation to concentration of hormone in solution (Table III) shows that the physiological amount used was non-saturating; even a concentration about 10 times greater (31.6 ng/ml) did not saturate the cytosolic macromolecules binding testosterone or its metabolites. The determination of the binding capacity would need a competitive study. A relatively small quantity of radioactivity is bound within the nuclear fraction; the percentage of total nuclear radioactivity remaining in the pellet after extraction is approximately 50 for all tissues studied. Mainwaring et al. 8 have studied this unextractable radioactivity in the prostate: it corresponds to hormonal fixation by nuclear chromatin. In the different organs (Table IV), the ratio of bound to free radioactivity is greater in the nuclei than in the cytosol, which suggests greater specificity of fixation, mainly in the prostate. In this organ the ratio of bound to free radioactivity seems relatively independent of hormonal concentration. In contrast, it increases sharply with hormonal concentration in nuclear supernatants of the hypothalamus and especially the pituitary. This indicates a displacement of the equilibrium in favour of bound radioactivity. This displacement suggests modifications of the 'receptor' activity as hormonal concentration increases. If this phenomenon is effective in vivo, it could play a role in the feedback regulatory mechanisms characteristic of these tissues. In all 4 tissues studied, the nucleus is richer in D H T than is the cytosol. This may equally well suggest either formation in situ, or preferential transport of D H T formed elsewhere. It appears that in the prostate 5a-reductase exists in all subcellular fractions 2°. In neural tissues this enzyme seems to be mainly present in microsomes, and in relatively larger quantity in the hypothalamus 16. Testosterone and D H T represent a large proportion of the radioactivity in nuclear extract. However, other metabolites exist in this fraction as in the whole nuclei, since the sum of the two steroids does not account for 100K of total radioactivity. Most of these other metabolites are more polar than testosterone, and more abundant in the neural tissues than in the prostate. Investigating the same neural tissues, Jouan et al. 4 found that after incubation the main metabolite bound to cytosolic proteins was a slightly polar compound. Little D H T was formed - - never more than 10700. In contrast with the high affinity of brain for oestradiol, the low affinity of these tissues for testosterone makes difficulties in attempts to demonstrate specific protein 'receptors' at physiological concentration. Moreover, in the entire hypothalamus only certain groups of neurones form the releasing factor for gonadotrophins; hence it is logical to assume that these cells are very active in D H T formation and in androgen
TESTOSTERONE IN RAT BRAIN TISSUES
149
binding, while the other neurones behave like those of e.g. the frontal cortex. Then, comparing our results with the study in vivo of Stern and Eisenfeld22, it seems difficult to extrapolate results obtained in vitro to the situation in vivo. However, the presence at a relatively high proportion of metabolites other than DHT in neural tissues, and the possible presence of an aromatizing system of androgens in the diencephalonn, 12, may be responsible for qualitative differences found between 'receptors' of the central nervous system and the prostate. In addition, since we used 3-month-old adult rats only, this study is limited to a static image of what is in fact an extremely dynamic system of regulation. Research into the evolution of androgenic receptors in the course of sexual maturation would define more clearly the overall function of this regulatory system.
REFERENCES 1 APPELGREN,L. E., The distribution of labelled testosterone in mice, Acta endocr. (Kbh.), 62 (1969) 505-512. 2 BRUCHOVSKY,N., AND WILSON, I. D., The conversion of testosterone to 5 a-androstan-17 fl-ol-3one by rat prostate in vivo and in vitro, J. biol. Chem., 243 (1968) 2012-2021. 3 HARPER, M. E., FAHMY,A. R., PIERREPOINT,C. (~., GROOM, M., AND GRIFFITHS,K., Some aspects of steroid metabolism in the canine prostate. In V. H. T. JAMESAND L. MARTINI(Eds.), 3rd Int. Congr. Hormonal Steroids, Hamburg, 1970, Excerpta Medica, Amsterdam, 1971, abstract 84. 4 JOUAN,P., SAMPEREZ,S., THIEULANT,i . L., El" MERCIER,L., Etude de la composition des stdroides lids aux 'r~k:epteurs' cytoplasmiques de l'hypophyse antdrieure et de rhypothalamus apr6s incubation en prdsence de testostdrone all, C.R. Acad. Sci. (Paris), 272 (1971) 2368-2371. 5 KREBS,H. A., Body size and tisssue respiration, Biochim. biophys. Acta (Amst.), 4 (1950)249-269. 6 LOBL, R. T., AND SHAPmO, S., Alterations in the binding of 3H-testosterone during development in the rat, Neuroendocrinology, 8 (1971) 107-115. 7 MAINWARING,W. I. P., A soluble androgen receptor in the cytoplasm of rat prostate, J. Endocr. 45 (1969) 531-541. 8 MAINWARING,W. I. P., AND PETERKEN, B. M., A reconstituted cell-frce system for the specific transfer of steroid-receptor complexes into nuclear chromatin isolated from rat ventral prostate gland, Biochem. J., 125 (1971) 285-295. 9 McEWEN, B. S., PFAFF, D. W., AND ZIOMOND,R. E., Factors influencing sex-hormone uptake by rat brain regions. II. Effects of neonatal treatment and hypophysectomy on testosterone uptake, Brain Research, 21 (1970) 17-28. 10 McEwEN, B. S., PEAFF, D. W., AND ZIGMOND, R. E., Factors influencing sex-hormone uptake by rat brain regions. III. Effects of competing steroids on testosterone uptake, Brain Research, 21 (1970) 28-38. 11 NAFTOLIN,F., RYAN, K. J., AND PETRO, Z., Aromatization of androstenedione by the diencephalon, J. clin. Endocr., 33 (1971) 368-370. 12 NAFTOLIN, F., RYAN, K. J., AND PETRO, Z., Aromatization of androstenedione by the anterior hypothalamus of adult male and female rats, Endocrinology, 90 (1972) 295-298. 13 PFAFF, D. W., Autoradiographic localisation of radioactivity in rat brain after injection of tritiated sex hormones, Science, 161 (1968) 1355-1356. 14 PI-IUONO, N. T., uND SAUER, G., Die spezifische Aufnahme yon markiertem Testosteron im Hypothalamus, Acta biol. med. germ., 26 (1971) 1247-1249. 15 RIVAROLA, i . A., SNIPES, C. A., AND MIGEON, C. J., Concentration of androgens in systemic plasma of rats, guinea pigs, salamanders and pigeons, Endocrinology, 82 (1968) 115-121. 16 ROMMERTS,F. F. G., AND VAN DER MOLEN, H. J., Occurence and localisation of 5a-steroid reductase, 3a- and 17 fl-hydroxysteroid dehydrogenases in hypothalamus and other brain tissues of the male rat, Biochim. biophys. Acta (Amst.), 248 (1971) 489-502. 17 RoY, S. K., AND LAUMAS,K. R., 1,2 all-testosterone: distribution and uptake in neural and genital tissues of intact male, castrate male and female rats, Acta endocr. (Kbh.), 61 (1969) 629-640.
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18 SAMPEREZ, S., THIEULANT, M. L., ET JOUAN, P., Mise en 6vidence d'une association macromol6culaire de la testost6rone 1,2-3H dans l'hypophyse ant6rieure et rhypothalamus du rat normal et castr6, C.R. Acad. Sci. (Paris), 268 (1969) 2965-2967. 19 SAMPEREZ,S., THIEULANT, M. L., ET JOUAN, P., Recherches compl6mentaires sur les modalit6s de la p6n6tration in vitro de la testost6rone-3H dans l'hypophyse ant6rieure, l'hypothalamus et le cortex c6r6bral du rat normal et castr6, Ann. Endocr. (Paris), 32 (1971) 31-42. 20 SHIMAZAKI,J., HORAGUCHI, T., OHKI, Y., AND SHIDA, K., Properties of testosterone 5 a reductase of purified nuclear fraction from ventral prostate of rats, Endocr.jap., 18 (1971) 179-187. 21 SHORE, L. S., AND SNIPES, C. A., Metabolism of testosterone in vitro by hypothalamus and other areas of the brain, Fed. Proc., 30 (1971) 363-367. 22 STERN,J. M., AND EISENFELD, A. J., Distribution and metabolism of all-testosterone in castrated male rats. Effects of cyproterone, progesterone and unlabeled testosterone, Endocrinology, 88 (1971) 1117-1125. 23 UNHJEM, O., Binding of androgens to components of rat ventral prostate nuclei in vitro, Acta endocr. (Kbh.), 63 (1970) 69-78.
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© ElsevierScientificPublishing Company,Amsterdam- Printed in The Netherlands.
UPTAKE, BINDING AND METABOLISM OF TESTOSTERONE IN RAT BRAIN TISSUES
M. MONBON, B. LORAS, J. P. REBOUD ANDJ. BERTRAND Bdtiment de M~decine, Faeult~ des Sciences, 69100 Villeurbanne and Unit~ de Recherches Endocriniennes et M~taboliques chez l'Enfant, H6pital Debrousse, 69005 Lyon (France) (Accepted October 10th, 1972)
SUMMARY
Slices of cerebral cortex, pituitary and hypothalamus of one-day castrated adult male rats were incubated with tritiated testosterone (T), and the ventral prostate was used as a control target organ. Uptake seemed to be a non-specific process of exchange diffusion. Tissue specificity appeared in macromolecular binding and intracellular metabolism of the hormone. Pituitary tissue bound testosterone less and much more slowly than did prostatic tissue, but the binding was greater than that observed in hypothalamus and cerebral cortex. Moreover, the percentage of bound hormone in nuclear extracts of hypothalamus, and especially pituitary, appeared to be dependent on its concentration in the medium. The main metabolite found in the prostate was dihydrotestosterone (DHT). In the pituitary less D H T was found, but the rate of conversion of T to DHT remained far higher than in hypothalamus and cortex. In the 4 tissues studied, the nucleus bound higher percentages of radioactivity, and more D H T was found in the nucleus than in the corresponding cytosol. In addition, conversion of T to D H T increased in parallel with the degree of binding. These two observations suggest that there is no qualitative difference in general handling of T by prostatic and neural 'receptors', although large quantitative differences are found.
INTRODUCTION
Since feedback mechanisms of sex hormones have been described, various methods - - autoradiographyl, 13, injection in vivo 6,9,1°,17,zz and incubation in vitro 14,1s,21 - - have established that the hypothalamic-hypophyseal axis acts as a target organ to testosterone. We have studied in vitro some conditions of penetration and binding of tes-
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tosterone in the diencephalic-hypophyseal region, and its intracellular metabolism, in castrated adult male rats. The prostate was used as a control target organ in view of a possible comparison of general functioning of the 'receptors'. The cerebral cortex was taken as a control 'non-target' organ. MATERIALS
Young adult male Wistar rats weighing about 250 g were castrated 24 h before the experiment. The animals were killed by decapitation, and the following organs or parts of organs were removed: fragment of frontal cortex; the entire hypothalamus, between 1 and 2 mm in thickness, limited anteriorly by the optic chiasm, laterally by the hypothalamic fissures and posteriorly by the mammillary bodies; the entire pituitary, removed from the sella turcica after section of the stalk; ventral lobes of the prostate. Tritiated testosterone ([1,2-3H]-T) was supplied by the C.E.A. (Saclay), with a specific activity of 42.6 Ci/mmole, its purity checked by paper chromatography before use. It was stored at --30 °C in ethanol at a concentration of 34 ng/ml. Before use, the alcohol was evaporated under nitrogen and the hormone redissolved in a small quantity of propanediol and then in the incubation medium. The following media were used. Incubation media. 'Complete' Krebs medium: this is Krebs medium !II .5. 'Incomplete' Krebs medium: medium Ili lacking fumarate, glutamate and pyruvate. Homogenizing media. Medium I: Tris-HC1 (pH 7.4), 50 mM; EDTA, 1.5 mM; mercapto-ethanol, 2 mM; MgC12, 2 mM; KC1, 10 mM; saccharose, 250 mM. Medium II: Tris-HC1 (pH 7.4), 50 mM; EDTA, 1.5 mM; mercapto-ethanol, 2 mM; MgC12, 2 mM; KC1, 10 mM; NaCI, 1 M. METHODS
Removal of organs and incubation After decapitation and dissection, the removed organs were immediately placed in Krebs medium at 0 °C, then minced into tiny fragments. After preincubation for 15 min at 0 °C and for 15 min at 37 °C, the tissue was incubated with tritiated testosterone in Krebs medium at 37 °C, under continuous agitation in the presence of 99.5 % oxygen. Most incubations were done with 3.4 ng testosterone/ml of medium, which appears to be the physiological concentration for rats 15. Individual organs were pooled and incubated in a volume approximately proportional to the total weight (1 ml/50 mg). Homogenization and cellular fractionation After incubation, each tissue was carefully washed with 100 ml of medium I over a linen filter, homogenized for 1 min in a glass-teflon Potter-Elvehjem, and filtered through several layers of gauze. The homogenates were fractionated in a Beckman centrifuge (type L3 40) at
TESTOSTERONE IN RAT BRAIN TISSUES
141
800 × g during l0 rain. A pellet of crude nuclei and a cytoplasmic supernatant were obtained. The latter was centrifuged for 90 min at 105,000 × g, yielding the 'cytosol' fraction free of subcellular particles. The nuclear pellet was washed twice, suspended in medium II and agitated for 30 min. The suspension was further fractionated, after 15 min centrifugation, into a pellet (chromatin q- nuclear membranes) and a soluble fraction containing proteins and nucleic acids of low molecular weight. All these operations were performed at temperatures ranging from 0 to 4 °C.
Measurement of radioactivity An aliquot of each homogenate or cellular fraction was dissolved in a small quantity of Soluene (0.1 ml) and left at 40 °C for 24 h. Fluorescence of Soluene was neutralized by 0.4 ml of glacial acetic acid; 0.5 ml of ethanol was added, then 14 ml of a toluene-based fluor (PPO, 5 g; dimethyl-POPOP, 0.3 g; toluene 1 litre). The samples were counted in a TriCarb scintillation spectrometer (Model 33 20). Correction for quenching was made by the external standard method. All samples were counted for the time required to ensure less than 5 % error in counting.
Measurement of hormone incorporation The cytosol and nuclear supernatant fractions were filtered at 4 °C on Sephadex gel columns tested against Dextran Blue. The cytosol was eluted with medium I lacking saccharose, and the nuclear supernatant with medium II. An aliquot of each fraction was counted to give the elution profile. Radioactivity bound to the excluded peak was used as the criterion of incorporation.
Androgen analysis* Isolation of testosterone, D H T and A4-androstenedione was carried out by reverse isotopic dilution, as indicated in the diagram (Fig. 1). Since the level of radioactivity (R.A.) of the total extract was low, specific activity could not be measured by systematic crystallization of isolated steroids. Therefore non-labelled- or 14C-carrier was added and the purity of steroids was verified by the constancy of the respective ratios aH :#g of non-labelled carrier, or 3H: 14C in the course of purification. Paper chromatography (PC) was done at 27 °C, the first on Whatman 3MM and the second on Whatman no. 2 paper. Silica gel thin-layer plates (TL), as purchased, were washed once in ethanol and once in methanol and stored in a desiccator. Elution was done with a toluene-methanol mixture (97:3), and acetylation performed in the usual way with acetic anhydride in pyridine. Saponification by potassium hydroxide in methanol frequently caused a decrease in aH:14C or aH:/zg of carrier ratios, which was more marked for testosterone than for DHT. However, the presence of tritiated metabolites of these two steroids could not be established. Acetylation followed by saponification of mixtures of standard [1,2-all] - and [4-14C]testosterone, * Testosterone, 17fl-hydroxy-4-androsten-3-one; DHT, 17fl-hydroxy-5a-androstan-3-one; A4-androstenedione, 4-androsten-3-17-dione; epitestosterone, 17a-hydroxy-4-androsten-3-one; isoandrosterone, 3/~-hydroxy-5a-androstan-17-one; fl-DHT, 17fl-hydroxy-5fl-androstan-3-one;etiocholanolone, 3a-hydroxy-5fl-androstan-17-one.
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M. M O N B O N e t
al.
ISOLATION OF STEROIDS
Sample Addition of non-labelled carrier + [14C]testosterone and DHT 1. Extraction: 3 × 7-8 v CHCh. 2. Measurement of total R.A. on 1/50.
I
PC hexane-MeOH-water 100.90:10 6 h Steroids of Rtestosterone = 0-1 TL CHC13-EtOH 95: 5 twice
Steroids of g testosterone = 2.3-3.1
less polar
TL CHCl3-ether - 90:10 twice --3
A4
Testosterone 1st S.A.*
DHT 1st S.A.
PC hexane-benzene-MeOH-water 66: 33 : 80:10
Ether twice
CHCIa-EtOH 95:5
2nd S.A.
2nd S.A.
2nd S.A.
Acetylation
Acetylation
TL CHCIa-EtOH 95: 5 twice
TL CHCla-ether 90:10 twice
3rd S.A.
3rd S.A.**
Saponification
Saponification
TL
TL
1st S.A.
* S.A., Specific activity measured by the 3H:a4C or 3H:pg non-labelled carrier ratios. ** Only if [laC]DHT added. Fig. 1.
a n d o f s t a n d a r d [1,2-3H]- a n d [4-14C]DHT, resulted in a decrease o f the ratio 3H." 14C. I t a p p e a r e d , therefore, t h a t p a r t o f the t r i t i u m present in the 1 o r 2 p o s i t i o n was able to exchange with h y d r o g e n ions present in solution d u r i n g this reaction. F o r this reason, only the values o b t a i n e d before this e x p e r i m e n t a l stage were used for the calc u l a t i o n o f results. Isolation of testosterone. The first thin-layer c h r o m a t o g r a p h y ( T L C ) s e p a r a t e d testosterone f r o m the m o s t p o l a r c o m p o u n d s : androstan-17fl diols (3a a n d 3fl), A4-androsten-17fi diols (3a a n d 3fl) a n d 5fl-androstan-3a, 17t3 diol. The following P C s e p a r a t e d testosterone f r o m epitestosterone and 5fl-androstan3fl, 17-fl diol. Isolation of A4-androstenedione. The first T L C separated A4-androstenedione
TESTOSTERONEIN RAT BRAINTISSUES
143
from known steroids eluted from the first chromatogram: DHT, isoandrosterone, fl-DHT and etiocholanolone. Isolation of DHT. The first T L C separated D H T from all the above-mentioned steroids. The closest was isoandrosterone (RDnT = 0.85-0.90). If the first T L was overloaded or migration less successful, the second TL completed the separation of D H T from isoandrosterone. Because of variability in the different batches of TL, the best separation of D H T from isoandrosterone was achieved either in a CHCla-ether system (90:10), or in pure ether, or occasionally in a CHCla-ethanol (95:5) system. RESULTS
Hormone uptake in tissue The amount of radioactivity present in the homogenate after the tissue had been washed with medium was taken as the criterion of penetration. Testosterone uptake by the 4 organs studied was investigated in relation to time (Table I). With cerebral cortex and prostate, virtually maximal uptake resulted after a 60-min incubation period. In contrast, in pituitary and hypothalamus, radioactivity was still increasing after 150 min of incubation, indicating a slow penetration and a hormone concentration below saturation. In the 4 organs studied, the proportionality between the measured amount of intra-tissular hormone and hormone concentration, up to 10 times plasma concentration, revealed no factor limiting hormone penetration. However, each organ showed a specific ratio of R.A./mg of tissue to R.A./ml of medium (cortex, 5.0; pituitary, 6.0; hypothalamus, 4.8; prostate, 3.3). When tissues were frozen before incubation, these ratios were not significantly changed.
Cellular fixation of hormone Radioactivity bound to the macromolecular peak excluded from a Sephadex G-25 column was taken as the criterion of fixation.
Effect of time of incubation (Table II) During the measurement of fixation by cytosol macromolecules, clear difTABLE I Radioactivity (R.A.) of the homogenate is expressed as disint./min/mg wet weight. The tissues were incubated in incomplete Krebs medium containing 3.4 ng tritiated testosterone/ml.
Number of rats
10 8 10 9 8
Time (min)
5 10 30 60 150
Organ Cerebralcortex Pituitary
Hypothalamus Prostate
616 1200 1601 2742 3070
472 946 1627 1865 2960
814 1350 2169 2440 4150
802 1010 1570 2460 2370
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M. MONBON et al.
TABLE II Radioactivity bound to the peak excluded from Sephadex G-25 is expressed as disint./min/mg wet weight of tissue incubated in incomplete Krebs medium containing 3.4 ng tritiated testosterone/ml. Number of rats
Time (min)
Cytosols Cerebral cortex
Pituitary
Hypothalamus Prostate
10 8 10 9 8
5 10 30 60 150
0.5 0.6 1.0 0.7 1.0
11.9 12.2 12.0 28.0 34.0
0.4 0.9 1.0 1.1 1.4
38.8 28.6 45.4 48.6 45.0
Nuclei. Prostate
4.2 10.9 11.3 -
TABLE III Radioactivity bound to the peak excluded from Sephadex G-25 is expressed as disint./min/mg wet weight. The organs were incubated for 60 min in complete Krebs media containing 3 concentrations of [all]testosterone. Organ
Prostate
Pituitary
Hypothalamus
Cerebral cortex
Cellular fraction
Cytosol Nuclear supernatant (SNx) Ratio SNx: cytosol Cytosol SNx Ratio SNx: cytosol Cytosol SNx Ratio SNx: cytosol Cytosol SNx Ratio SNx: cytosol
[ 3Hi testosterone in ng/ml of medium 3.4*
10.9"*
31.6"**
96 19 0.20 52 < 1 2 ~ 1 1 < 1 -
178 126 0.71 67 9 0.13 17 9 0.53 16 5 0.31
272 119 0.44 128 15 0.12 24 6 0.25 -
* 8 rats. ** 16 rats. *** 11 rats.
ferences were seen, in r e l a t i o n to time and to the o r g a n considered. Indeed, cytosolic ' r e c e p t o r s ' o f the cortex, h y p o t h a l a m u s and p r o s t a t e seemed to have reached saturation after 30 min, whereas hypophyseal ' r e c e p t o r s ' were not saturated even after 150 rain o f incubation. In the p r o s t a t e the cytosolic and nuclear ' r e c e p t o r s ' reached s a t u r a t i o n at the same rate. Quantitatively the radioactivity fixed by cytosols o f the cortex and h y p o t h a l a m u s was c o m p a r a b l e and far inferior to that in the prostate, while the pituitary was intermediate. H o r m o n e fixation by cytosolic m a c r o m o l e c u l e s o f the different organs depended greatly on the state o f the tissue: indeed, m a c r o m o l e c u l a r peaks derived f r o m cytosol
TESTOSTERONEIN RAT BRAIN TISSUES
145
fractions of organs which had been frozen before incubation were devoid of radioactivity, however long the time of incubation, whereas in fresh samples radioactivity was present.
Effect of hormone concentration (Table III) Cellular fixation of the hormone differed according to the cellular fraction under consideration: in general the cytosol fixed more hormone than the nuclei; and in addition the nuclear 'receptors' appeared to reach saturation more quickly than the cytosolic 'receptors', except in the pituitary. Whatever the concentration, the pituitary fixed less than the prostate and far more than the hypothalamus or cortex, which scarcely differed. Distribution of free and protein-bound radioactivity in relation to (all]testosterone concentration (Table IV) Filtration of subcellular fractions (cytosol and nuclear supernatant) on Sephadex G-25 revealed two radioactive peaks: one eluted with the proteins and the other corresponding to free steroids. The relation between these peaks indicated the distribution of radioactivity between two 'compartments' within the cellular fraction. This ratio was studied in each fraction and organ in relation to hormone concentration. In general, relatively more radioactivity was bound by the nuclei than by the cytosol, thus revealing greater specificity in nuclear fixation. But only nuclei of the prostate showed a ratio greater than I. In the prostate, this ratio appeared to be independent of hormonal concentration, whereas in hypophyseal and hypothalamic nuclei the former increased faster than the latter. Metabolism o f androgens The amount of radioactivity in these cellular fractions did not permit crystallizaTABLE IV Ratio of peaks of bound to free radioactivity after filtration of cellular fractions (cytosol and nuclear supernatant) on Sephadex G-25. The organs were incubated for 60 min in complete Krebs medium with 3 different concentrations of [SH]testosterone.
Organ
Cellularfraction Ratio of bound to free radioactivity ( [3H]testosterone in ng/ml of medium) 3.4 (8 rats)
10.88 (16 rats)
31.55 (11 rats)
Cytosol Nuclear SN
0.097 1.640
0.131 1.380
0.132 1.650
Hypophysis
Cytosol Nuclear SN
0.009 0.001
0.017 0.031
0.005 0.385
Hypothalamus
Cytosol Nuclear SN
0.001 0.001
0.004 0.033
0.002 0.118
Cerebral cortex
Cytosol Nuclear SN
0.001 0.001
0.002 0.025
-
Prostate
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M. MONBON et al.
tion of isolated steroids. However, the ratio c o u n t s / m i n :/~g of carrier steroid, or the ratio 3H:14C, remained c o n s t a n t in successive chromatographies, once k n o w n interfering steroids were separated from testosterone and D H T . Acetylation in particular did n o t modify the ratio. Therefore the purity of isolated steroids was considered acceptable. Results are shown in Tables V a n d VI. Table V gives the results of a 60-min i n c u b a t i o n . Testosterone, D H T and A4_ a n d r o s t e n e d i o n e were isolated from the cytosol a n d from the whole nuclei (pellet of centrifugation at 800 × g, washed twice). The prostate metabolized testosterone more completely t h a n did the neural tissues, with considerable f o r m a t i o n of D H T . Hyp o t h a l a m u s a n d cortex metabolized only a small part of the testosterone (less than 20 %) a n d little D H T was formed. The pituitary was intermediate between these two groups. F o r m a t i o n of A4-androstenedione was slight in all 4 tissues. Nuclei contained less testosterone a n d more D H T t h a n corresponding cytosols. However, the difference between prostate a n d neural tissues r e m a i n e d large. TABLE V Distribution of radioactivity among isolated androgens of the cytosol and whole nuclei, after cellular fractionation of organs incubated for 60 min in Krebs medium in the presence of 3.4 ng/ml [3H]testosterone. Results expressed as % of extracted radioactivity. Organ
Pituitary Hypothalamus Prostate Cerebral cortex
Cytosol
Nucleus
T
DHT
A4
T
DHT
/[4
63.9 91.0 2.5 77.9
12.1 5.2 46.1 1.8
1.6 0.4 0.1 0.2
42.7 2.0 62.8
27.6 9.0 82.9 6.9
3.7 0.9 0.1 0.5
TABLE VI Distribution of radioactivity between testosterone and dihydrotestosterone isolated from cytosol and nuclear extract of organs incubated in Krebs medium in relation to time, in the presence of tritiated testosterone (3.4 ng/ml). Results expressed as % of extracted radioactivity. Cellular fraction
Organ
Incubation period ( min ) 5 T
Cytosol
10 DHT T
Pituitary 100 Hypothalamus 99.4 1.2 Prostate 21.7 40.5 Cerebralcortex 100 -
Nuclear extract Pituitary 100 Hypothalamus 83.2 < 3 % Prostate 15 82 Cerebral cortex 80.6 -< 3
60
30
DHT T
DHT T
93.7 8.0 50.0 97.0 4.6 8.8 77.8 2.5 95.6 1.5 87.1 -
150 DHT T
DHT
6.0 6 4 . 5 14.8 46.0 31.0 1.6 90.0 6.2 78.0 6.5 33.6 3.7 55.7 - 48.7 1.1 89.0 3.3 90.8 1.4
90 10 76.9 4.9 4 87 78.4 < 3 ~
15 70 3 77
41 18 88 <3~
TESTOSTERONE IN RAT BRAIN TISSUES
147
Table VI shows the results of incubation carried out under the same conditions as before, but for varying lengths of time. No analysis for A4-androstenedione was done since it constituted an insignificant part of the total R.A. Nuclear R.A. was analysed, not as previously in the whole nuclei, but in the soluble nuclear extract obtained as described in the 'cellular fractionation' section. As in the preceding incubation, the prostate metabolized a great deal, the pituitary an appreciable amount, and the hypothalamus and cortex very little testosterone. After 30 min of incubation no further increase of testosterone metabolism in the cytosols was observed in any of the 4 tissues. The amount of DHT obtained increased with time in the pituitary, up to 31 70 after 150 min, but in the prostate the maximum (78 ~) was reached after 10 min of incubation, as in the hypothalamus where the amount reached a low maximum of 6.5 70. Nuclear extract, like whole nuclei, was richer in DHT than was the corresponding cytosol, and metabolism was slower, particularly in the hypothalamus and pituitary. DISCUSSION
This study, which aimed at comparing the behaviour of testosterone in the diencephalon and the prostate on the one hand, and in the diencephalon and the cerebral cortex on the other, permitted us to make several observations. Hormone uptake occurs easily in all the organs studied in vitro but at various rates (Table I). The proportion between rate of uptake and amount of hormone present, together with the observation that rate of uptake is not modified by freezing - - which, however, prevents fixation - - suggests a penetration by diffusion. The ratios (R.A./g of tissue: R.A./ml of medium) we found for the pituitary (6.0), hypothalamus (4.8) and cerebral cortex (5.0) are smaller than those reported by Samperez e t al. la in rats castrated for 40 days. In addition, we found only slight variation within the 4 tissues studied. Thus, under the experimental conditions used, hormone uptake seems rather non-specific to the tissue. In contrast with penetration, cellular binding of the hormone in the different organs studied indicates very clear specificity (Table II), although we only studied the whole protein fraction and did not try, in this study, to isolate the specific molecule. In the prostate, despite relatively low uptake, binding is considerable and rapid, reaching its maximum after 30 min in cytosol and nuclear supernatant. At the same time the cytosolic metabolism of testosterone is intense (Table VI). The principal metabolite is DHT, which accounts for almost 80 ~ of the radioactivity after 10 min. The subsequent lower rate can probably be attributed to slower formation of other metabolites, since our results are expressed as percentages. These other metabolites constitute a smaller percentage of the nuclear extract where DHT remains the major metabolite: 88 70 in 150 min. Analysis of whole nuclei confirms these results (Table V), which are in agreement with the results from other laboratories~,3,7, 23. The rate of hormone binding by cytosolic macromolecules of the pituitary is much slower than for the prostate: even in the 150-min incubation, fixation is not maximal. At the same time, DHT accumulates in cytosol and nuclei, suggesting either
148
M. MONBONet al.
slow D H T formation or an insignificant subsequent metabolism. The percentage of A4-androstenedione, even if greater than in the other organs studied, remains very low (Table V). The rate of hormone binding in hypothalamic cytosol is indistinguishable from that in the cortex. There is a distinctly smaller accumulation of D H T than in the pituitary, particularly in the cytosol. On the other hand, hypothalamic nuclei form D H T more effectively after a longer period of time (18 ~ after 150 min) than do cortical nuclei. An investigation of binding in relation to concentration of hormone in solution (Table III) shows that the physiological amount used was non-saturating; even a concentration about 10 times greater (31.6 ng/ml) did not saturate the cytosolic macromolecules binding testosterone or its metabolites. The determination of the binding capacity would need a competitive study. A relatively small quantity of radioactivity is bound within the nuclear fraction; the percentage of total nuclear radioactivity remaining in the pellet after extraction is approximately 50 for all tissues studied. Mainwaring et al. 8 have studied this unextractable radioactivity in the prostate: it corresponds to hormonal fixation by nuclear chromatin. In the different organs (Table IV), the ratio of bound to free radioactivity is greater in the nuclei than in the cytosol, which suggests greater specificity of fixation, mainly in the prostate. In this organ the ratio of bound to free radioactivity seems relatively independent of hormonal concentration. In contrast, it increases sharply with hormonal concentration in nuclear supernatants of the hypothalamus and especially the pituitary. This indicates a displacement of the equilibrium in favour of bound radioactivity. This displacement suggests modifications of the 'receptor' activity as hormonal concentration increases. If this phenomenon is effective in vivo, it could play a role in the feedback regulatory mechanisms characteristic of these tissues. In all 4 tissues studied, the nucleus is richer in D H T than is the cytosol. This may equally well suggest either formation in situ, or preferential transport of D H T formed elsewhere. It appears that in the prostate 5a-reductase exists in all subcellular fractions 2°. In neural tissues this enzyme seems to be mainly present in microsomes, and in relatively larger quantity in the hypothalamus 16. Testosterone and D H T represent a large proportion of the radioactivity in nuclear extract. However, other metabolites exist in this fraction as in the whole nuclei, since the sum of the two steroids does not account for 100K of total radioactivity. Most of these other metabolites are more polar than testosterone, and more abundant in the neural tissues than in the prostate. Investigating the same neural tissues, Jouan et al. 4 found that after incubation the main metabolite bound to cytosolic proteins was a slightly polar compound. Little D H T was formed - - never more than 10700. In contrast with the high affinity of brain for oestradiol, the low affinity of these tissues for testosterone makes difficulties in attempts to demonstrate specific protein 'receptors' at physiological concentration. Moreover, in the entire hypothalamus only certain groups of neurones form the releasing factor for gonadotrophins; hence it is logical to assume that these cells are very active in D H T formation and in androgen
TESTOSTERONE IN RAT BRAIN TISSUES
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binding, while the other neurones behave like those of e.g. the frontal cortex. Then, comparing our results with the study in vivo of Stern and Eisenfeld22, it seems difficult to extrapolate results obtained in vitro to the situation in vivo. However, the presence at a relatively high proportion of metabolites other than DHT in neural tissues, and the possible presence of an aromatizing system of androgens in the diencephalonn, 12, may be responsible for qualitative differences found between 'receptors' of the central nervous system and the prostate. In addition, since we used 3-month-old adult rats only, this study is limited to a static image of what is in fact an extremely dynamic system of regulation. Research into the evolution of androgenic receptors in the course of sexual maturation would define more clearly the overall function of this regulatory system.
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