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Is testosterone metabolized to estrogen in the forebrain of the Rockland-Swiss mouse?
664
Braht Research, 239 (1982) 664-667 Elsevier Biomedical Press
Is testosterone metabolized to estrogen in the forebrain of the RocklandSwiss mouse?
PETER J. SHERIDAN, STEVEN M. HOWARD and RONALD GANDELMAN Department of Anatomy, The University of Texas, Health Science Center at San Antonio, San Antonio, TX 78284 and (S.M.H. and R.G.) Department of Psychology, Rutgers University, New Brunswick, NJ 08903 (U.S.A.) (Accepted January 28th, 1982) Key words: testosterone metabolism - - estrogen - - Rockland-Swiss mouse - - brain androgen metabolism
The nuclear uptake and retention of [all]testosterone or one of its metabolites was examined in the Rockland-Swiss (R-S) mouse. Castrated male and female mice were injected with 0.2 pg of testosterone [1,2,6,7-aH(N)] per 100 g body weight and killed 1.5 h later. The brains were removed and processed for autoradiography. A nuclear localization of [all]testosterone or one of its metabolites was found in the nucleus (n.) interstitialis striae terminalis and the n. amygdaloideus medialis. This localization is very different from that reported in another strain of mouse after the injection of [aH]dihydrotestosterone. Together with previous reports, these data suggest: (1) that aromatization of T to E2 in the forebrain of the R-S mouse is of minimal consequence; (2) there may be differences in uptake of T or its metabolites in different strains of mice; and (3) different androgens may exhibit uniquz patterns of nuclear uptake and retention. T e s t o s t e r o n e secreted f r o m the testes has long been k n o w n to be essential for the expression o f aggressive b e h a v i o r in male mice 8,8. U n t i l recently it h a d been a s s u m e d that testosterone o r one o f its a n d r o g e n i c m e t a b o l i t e s acted on the b r a i n to m o d u l a t e such behavior. H o w e v e r , it has recently been suggested t h a t the a c t i o n o f circulating testosterone is m e d i a t e d by an i n t r a n e u r o n a l conversion ( a r o m a t i z a t i o n ) o f testosterone to estradiol z,4,5,1°. This suggestion is based on the following facts: (1) estrogen can stimulate intermale aggression in c a s t r a t e d male mice6,U,2°; (2) anti-estrogens can b l o c k testosterone s t i m u l a t e d b e h a v i o r in some strains o f mice4,~,l°; a n d (3) 5a-dihyd r o t e s t o s t e r o n e (a n o n - a r o m a t i z a b l e a n d r o g e n ) does n o t stimulate aggressive b e h a v i o r in some strains o f mice 12. O n the o t h e r hand, a n d r o g e n s themselves can stimulate aggressive b e h a v i o r in at least two strains o f mice. D i h y d r o t e s t o s t e r o n e ( D H T ) has been r e p o r t e d to stimulate i n t e r m a l e aggression in Swiss-Webster a n d R o c k l a n d - S w i s s mice12,16, a n d M E R - 2 5 (an anti-estrogen) does n o t b l o c k the activation o f testos t e r o n e - i n d u c e d aggression in a d u l t R o c k l a n d - S w i s s mice 21. I n the following study we r e p o r t on what we consider to be an u n u s u a l t o p o g r a phical d i s t r i b u t i o n o f nuclear u p t a k e a n d retention o f testosterone or one o f its m e t a b o l i t e s in the f o r e b r a i n o f a strain o f mice in which a r o m a t i z a t i o n does n o t a p p e a r to m e d i a t e a n d r o g e n i n d u c e d aggression. 0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press
665
:.%
.
.
r
. o
G Fig, 1. Autoradiograms of the medialis (b) of castrated male grains can be seen over nuclei stained with hematoxylin and
b
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nucleus (n.) interstitialis striae terminalis (a) and the n. amygdaloideus mice injected with [3H]testosterone. A nuclear concentration of silver of neurons in both areas. Exposure time was 8 months, 4/~m sections eosin.
Adult Rockland-Swiss (R-S) mice (12 males and 12 females) were castrated 1 week prior to the experiment. On the day of the experiment, each animal was injected intravenously with 0.2 /~g [l,2,6,7-3H(N)]testosterone (New England Nuclear, 98.8 Ci/mmol) per 100 g body weight. Two males and two females also received 20/~g/100 g body weight of radioinert testosterone to serve as controls. The animals were killed 1.5 h after the injection of [3H]testosterone. The brains of the animals were removed, sliced and mounted on tissue holders. The mounted forebrains were then frozen in liquified propane and stored in nitrogen until processing. Four #m frozen sections were cut in a wide-range cryostat (Harris, North Billerica, MA) and mounted for autoradiography on emulsion-coated slides according to the thaw-mount procedure9. 23. After 4 or 8 months of exposure time at --15 °C, the slides were photographically developed (D-19 for 45 s), fixed (5 min) and stained with hematoxylin and eosin. In autoradiograms from animals injected with [3H]testosterone, nuclear concentration of silver grains appeared in only two areas of the forebrain: the nucleus (n.) interstitialis striae terminalis (Fig. 1) and the posterior portion of the n. amygdaloideus medialis (Fig. 1). The cells in the n. interstitialis striae terminalis which concentrated the steroid formed a 'tube' which began anteriorly near the lateral ventricles and ran both ventrally and medially in more posterior sections. No differences were found between males and females. Simultaneous injection of unlabeled testosterone inhibited nuclear uptake and retention of the [3H]steroid (not shown). The topographical distribution reported here after the injection of [3H]testosterone in the mouse is quite different from that reported after the injection of [3H]dihydrotestosterone ([aH]DHT)16. The localization seen after the injection of [3H]DHT in the mouse was quite extensive in the hypothalamus, amygdala and hippocampus. If both testosterone (T) and DHT bind to the same receptors as is generally assumed1, 7 one would expect a similar pattern of localization. The localization of
666 D H T and T observed in the rat is very similar if the tritium associated with testosterone is not put in the 1fl and 2fl positions exclusively (the tritium is lost in these positions if testosterone is converted to estradiol) 15. A number of explanations could account for the differences in localization of the labeled neurons in these two studies. First, the strain of mouse used in the present study (Rockland-Swiss) is different from the strain used in the D H T study (normal male littermates of Tfm/y carrying tabby). Perhaps there are differences in the number and location of androgen receptors in these strains. A second possibility is that testosterone is metabolized to another steroid before it reaches the androgen receptor. Testosterone has been reported to be metabolized to D H T la,19,22, estradio114 and other polar metabolites in the hypothalamus of rats. Metabolism of testosterone to estradiol is considered unlikely for the following reasons. Most of the tritium would remain with the steroid if it were metabolized to estradiol, and nuclear uptake and retention of [3H]estradiol would result in a more widespread distribution of labeled neurons. Moreover MER-25, an anti-estrogen, does not block testosterone-induced aggressive behavior in this strain of mice 2°. In the rat, aromatization of testosterone to estradiol does not occur in either the medial nucleus of the amygdala or the bed nucleus of the interstitialis striae terminalis is. However, the possibility of conversion of testosterone to an inactive metabolite cannot be ruled out at this time. Diferences due to dose of steroid used or exposure time cannot be ruled out, although at this time we consider them unlikely. At the moment, it is attractive to postulate that the differences in neuronal labeling seen in this strain of mouse represent a unique distribution pattern and/or a different receptor than that found in the other strains of mice or in rats and that these differences are related to the differences in the behavioral effects of androgen in this and other strains of mice and rats. I f we are correct, these data also suggest that little or no aromatization of testosterone to estradiol occurs in the forebrain of this strain of mouse, and provide additional evidence that aromatization is not involved in androgen-induced aggression in the Rockland-Swiss mouse 21. Additional experiments are needed in both this strain and in the Tfm/y carrier strain of mice with D H T and testosterone, respectively, to definitely establish the strain differences and/or the differences found with the different androgens in nuclear uptake and retention. This work was supported by USPHS Research Career Development Award K04 NS00164, U S P H S Grants R01NSI2933 and MH-28660 and N S F Grant BNS-07347.
1 Attardi, B. and Ohno, S., Androgen and estrogen receptors in the developing mouse brain, Endocrinology, 99 (1976) 1279-1290. 2 Beatty, W. W., Gonadal hormones and sex differences in nonreproductive behavior in rodents: organizational and activational influences, Horrn. Behav., 12 (1979) 112-163. 3 Beeman, E. A., The effect of male hormone on aggressive behavior in mice, PhysioL Zool., 20 (1947) 373-405. 4 Bowden, N. J. and Brain, P. D., Blockade of testosterone-maintained intermate fighting in albino laboratory mice by an aromatization inhibitor, Physiol. Behav., 20 (1978) 543-546.
667 5 Clark, C. R. and Nowell, N. W., The effect of the anti-estrogen CI-628 on androgen-induced aggressive behavior in castrated male mice, Horm. Behav., 12 (1979) 205-211. 6 Edwards, D. A. and Burge, K. G., Estrogenic arousal of aggressive behavior and masculine sexual behavior in male and female mice, Horm. Behav., 2 (1971) 239 245. 7 Fox, T. O., Androgen- and estrogen-binding macromolecules in developing mouse brain: biochemical and genetic evidence, Proc. nat. Acad. Sci. U.S.A., 72 (1975) 4303-4307. 8 Gandelman, R., Gonadal hormones and the induction of intraspecific fighting in mice, Neurosci. Biobehav. Rev., 4 (1980) 133-140. 9 lson, E. J. and Sheridan, P. J., Autoradiography of diffusible substances - - a practical approach, Amer. J. Med. Tech., 47 (1981) 38-42. 10 Luttge, W. G., Anti-estrogen inhibition of testosterone-stimulated aggression in mice, Experientia, 35 (1979) 273. 11 Luttge, W. G. and Hall, N. R., Androgen-induced agonistic behavior in castrate male SwissWebster mice: comparison of four naturally occurring androgens, Behav. Biol., 8 (1973) 725 732. 12 Luttge, W. G., Hall, N. R. and Wallis, C. J., Studies on the neuroendocrine, somatic and behavioral effectiveness of testosterone and its 5a-reduced metabolites in Swiss-Webster mice, Physiol. Behav., 13 (1974) 553-561. 13 Marsa, R., Stupnicka, E., Kniewald, Z. and Martini, L., The transformation of testosterone into dihydrotestosterone by the brain and the anterior pituitary, J. Steroid Biochem., 3 (1972) 383-389. 14 Naftolin, F., Ryan, K., Davies, I., Reddy, V., FIores, F., Petro, Z., Kuhn, M., White, R. J., Takaoka, Y. and Wolin, L., The formation of estrogens by central neuroendocrine tissues, Rec. Progr. Horm. Res., 31 (1975) 295-315. 15 Sar, M. and Stumpf, W. E., Distribution of androgen target cells in rat forebrain and pituitary after [3H]dihydrotestosterone administration, J. Steroid. Biochem., 8 (1977) 1131-1135. 16 Schechter, D., Howard, S. M. and Gandelman, R., Dihydrotestosterone promotes fighting behavior of female mice, Horm. Behav., 15 (1981) 233-237. 17 Sheridan, P. J., Localization of androgen and estrogen concentrating neurons in the diencephalon and telencephalon of the mouse, Endocrinology, 103 (1978) 1328-1334. 18 Sheridan, P. J., The nucleus interstitialis striae terminalis and the medial nucleus of the amygdala, prime targets for androgen in the rat forebrain, Endocrinology, 104 (1979) 130-136. 19 Sholiton, L. J., Jones, C. E. and Werk, E. E., The uptake and metabolism of [1,2-3H]testosterone by the brains of functionally hepatectomized and totally eviscerated male rats, Steroids, 20 (1972) 399-415. 20 Simon, N. G. and Gandelman, R., The aggression-promoting and aggression-eliciting properties of estrogen in male mice, Physiol. Behav., 21 (1978) 161-164. 21 Simon, N. G., Gandelman, R. and Howard, S. M., MER-25 does not inhibit the activation of aggression by testosterone in adult Rockland-Swiss Mice, Psychonettroendocrinology, 6 (1981) 131-137. 22 Stern, J. M. and Eisenfeld, A. J., Distribution and metabolism of [3H]testosterone in castrated male rats; effects of cyproterone, progesterone and unlabeled testosterone, Endocrinology, 88 (1971) 1117-1125. 23 Stumpf, W. E., Autoradiographic techniques for localization of hormones and drugs at the cellular and subcellular level, Acta Endocr. Kbh., Suppl. 153 (1971) 205-221.
Braht Research, 239 (1982) 664-667 Elsevier Biomedical Press
Is testosterone metabolized to estrogen in the forebrain of the RocklandSwiss mouse?
PETER J. SHERIDAN, STEVEN M. HOWARD and RONALD GANDELMAN Department of Anatomy, The University of Texas, Health Science Center at San Antonio, San Antonio, TX 78284 and (S.M.H. and R.G.) Department of Psychology, Rutgers University, New Brunswick, NJ 08903 (U.S.A.) (Accepted January 28th, 1982) Key words: testosterone metabolism - - estrogen - - Rockland-Swiss mouse - - brain androgen metabolism
The nuclear uptake and retention of [all]testosterone or one of its metabolites was examined in the Rockland-Swiss (R-S) mouse. Castrated male and female mice were injected with 0.2 pg of testosterone [1,2,6,7-aH(N)] per 100 g body weight and killed 1.5 h later. The brains were removed and processed for autoradiography. A nuclear localization of [all]testosterone or one of its metabolites was found in the nucleus (n.) interstitialis striae terminalis and the n. amygdaloideus medialis. This localization is very different from that reported in another strain of mouse after the injection of [aH]dihydrotestosterone. Together with previous reports, these data suggest: (1) that aromatization of T to E2 in the forebrain of the R-S mouse is of minimal consequence; (2) there may be differences in uptake of T or its metabolites in different strains of mice; and (3) different androgens may exhibit uniquz patterns of nuclear uptake and retention. T e s t o s t e r o n e secreted f r o m the testes has long been k n o w n to be essential for the expression o f aggressive b e h a v i o r in male mice 8,8. U n t i l recently it h a d been a s s u m e d that testosterone o r one o f its a n d r o g e n i c m e t a b o l i t e s acted on the b r a i n to m o d u l a t e such behavior. H o w e v e r , it has recently been suggested t h a t the a c t i o n o f circulating testosterone is m e d i a t e d by an i n t r a n e u r o n a l conversion ( a r o m a t i z a t i o n ) o f testosterone to estradiol z,4,5,1°. This suggestion is based on the following facts: (1) estrogen can stimulate intermale aggression in c a s t r a t e d male mice6,U,2°; (2) anti-estrogens can b l o c k testosterone s t i m u l a t e d b e h a v i o r in some strains o f mice4,~,l°; a n d (3) 5a-dihyd r o t e s t o s t e r o n e (a n o n - a r o m a t i z a b l e a n d r o g e n ) does n o t stimulate aggressive b e h a v i o r in some strains o f mice 12. O n the o t h e r hand, a n d r o g e n s themselves can stimulate aggressive b e h a v i o r in at least two strains o f mice. D i h y d r o t e s t o s t e r o n e ( D H T ) has been r e p o r t e d to stimulate i n t e r m a l e aggression in Swiss-Webster a n d R o c k l a n d - S w i s s mice12,16, a n d M E R - 2 5 (an anti-estrogen) does n o t b l o c k the activation o f testos t e r o n e - i n d u c e d aggression in a d u l t R o c k l a n d - S w i s s mice 21. I n the following study we r e p o r t on what we consider to be an u n u s u a l t o p o g r a phical d i s t r i b u t i o n o f nuclear u p t a k e a n d retention o f testosterone or one o f its m e t a b o l i t e s in the f o r e b r a i n o f a strain o f mice in which a r o m a t i z a t i o n does n o t a p p e a r to m e d i a t e a n d r o g e n i n d u c e d aggression. 0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press
665
:.%
.
.
r
. o
G Fig, 1. Autoradiograms of the medialis (b) of castrated male grains can be seen over nuclei stained with hematoxylin and
b
-
nucleus (n.) interstitialis striae terminalis (a) and the n. amygdaloideus mice injected with [3H]testosterone. A nuclear concentration of silver of neurons in both areas. Exposure time was 8 months, 4/~m sections eosin.
Adult Rockland-Swiss (R-S) mice (12 males and 12 females) were castrated 1 week prior to the experiment. On the day of the experiment, each animal was injected intravenously with 0.2 /~g [l,2,6,7-3H(N)]testosterone (New England Nuclear, 98.8 Ci/mmol) per 100 g body weight. Two males and two females also received 20/~g/100 g body weight of radioinert testosterone to serve as controls. The animals were killed 1.5 h after the injection of [3H]testosterone. The brains of the animals were removed, sliced and mounted on tissue holders. The mounted forebrains were then frozen in liquified propane and stored in nitrogen until processing. Four #m frozen sections were cut in a wide-range cryostat (Harris, North Billerica, MA) and mounted for autoradiography on emulsion-coated slides according to the thaw-mount procedure9. 23. After 4 or 8 months of exposure time at --15 °C, the slides were photographically developed (D-19 for 45 s), fixed (5 min) and stained with hematoxylin and eosin. In autoradiograms from animals injected with [3H]testosterone, nuclear concentration of silver grains appeared in only two areas of the forebrain: the nucleus (n.) interstitialis striae terminalis (Fig. 1) and the posterior portion of the n. amygdaloideus medialis (Fig. 1). The cells in the n. interstitialis striae terminalis which concentrated the steroid formed a 'tube' which began anteriorly near the lateral ventricles and ran both ventrally and medially in more posterior sections. No differences were found between males and females. Simultaneous injection of unlabeled testosterone inhibited nuclear uptake and retention of the [3H]steroid (not shown). The topographical distribution reported here after the injection of [3H]testosterone in the mouse is quite different from that reported after the injection of [3H]dihydrotestosterone ([aH]DHT)16. The localization seen after the injection of [3H]DHT in the mouse was quite extensive in the hypothalamus, amygdala and hippocampus. If both testosterone (T) and DHT bind to the same receptors as is generally assumed1, 7 one would expect a similar pattern of localization. The localization of
666 D H T and T observed in the rat is very similar if the tritium associated with testosterone is not put in the 1fl and 2fl positions exclusively (the tritium is lost in these positions if testosterone is converted to estradiol) 15. A number of explanations could account for the differences in localization of the labeled neurons in these two studies. First, the strain of mouse used in the present study (Rockland-Swiss) is different from the strain used in the D H T study (normal male littermates of Tfm/y carrying tabby). Perhaps there are differences in the number and location of androgen receptors in these strains. A second possibility is that testosterone is metabolized to another steroid before it reaches the androgen receptor. Testosterone has been reported to be metabolized to D H T la,19,22, estradio114 and other polar metabolites in the hypothalamus of rats. Metabolism of testosterone to estradiol is considered unlikely for the following reasons. Most of the tritium would remain with the steroid if it were metabolized to estradiol, and nuclear uptake and retention of [3H]estradiol would result in a more widespread distribution of labeled neurons. Moreover MER-25, an anti-estrogen, does not block testosterone-induced aggressive behavior in this strain of mice 2°. In the rat, aromatization of testosterone to estradiol does not occur in either the medial nucleus of the amygdala or the bed nucleus of the interstitialis striae terminalis is. However, the possibility of conversion of testosterone to an inactive metabolite cannot be ruled out at this time. Diferences due to dose of steroid used or exposure time cannot be ruled out, although at this time we consider them unlikely. At the moment, it is attractive to postulate that the differences in neuronal labeling seen in this strain of mouse represent a unique distribution pattern and/or a different receptor than that found in the other strains of mice or in rats and that these differences are related to the differences in the behavioral effects of androgen in this and other strains of mice and rats. I f we are correct, these data also suggest that little or no aromatization of testosterone to estradiol occurs in the forebrain of this strain of mouse, and provide additional evidence that aromatization is not involved in androgen-induced aggression in the Rockland-Swiss mouse 21. Additional experiments are needed in both this strain and in the Tfm/y carrier strain of mice with D H T and testosterone, respectively, to definitely establish the strain differences and/or the differences found with the different androgens in nuclear uptake and retention. This work was supported by USPHS Research Career Development Award K04 NS00164, U S P H S Grants R01NSI2933 and MH-28660 and N S F Grant BNS-07347.
1 Attardi, B. and Ohno, S., Androgen and estrogen receptors in the developing mouse brain, Endocrinology, 99 (1976) 1279-1290. 2 Beatty, W. W., Gonadal hormones and sex differences in nonreproductive behavior in rodents: organizational and activational influences, Horrn. Behav., 12 (1979) 112-163. 3 Beeman, E. A., The effect of male hormone on aggressive behavior in mice, PhysioL Zool., 20 (1947) 373-405. 4 Bowden, N. J. and Brain, P. D., Blockade of testosterone-maintained intermate fighting in albino laboratory mice by an aromatization inhibitor, Physiol. Behav., 20 (1978) 543-546.
667 5 Clark, C. R. and Nowell, N. W., The effect of the anti-estrogen CI-628 on androgen-induced aggressive behavior in castrated male mice, Horm. Behav., 12 (1979) 205-211. 6 Edwards, D. A. and Burge, K. G., Estrogenic arousal of aggressive behavior and masculine sexual behavior in male and female mice, Horm. Behav., 2 (1971) 239 245. 7 Fox, T. O., Androgen- and estrogen-binding macromolecules in developing mouse brain: biochemical and genetic evidence, Proc. nat. Acad. Sci. U.S.A., 72 (1975) 4303-4307. 8 Gandelman, R., Gonadal hormones and the induction of intraspecific fighting in mice, Neurosci. Biobehav. Rev., 4 (1980) 133-140. 9 lson, E. J. and Sheridan, P. J., Autoradiography of diffusible substances - - a practical approach, Amer. J. Med. Tech., 47 (1981) 38-42. 10 Luttge, W. G., Anti-estrogen inhibition of testosterone-stimulated aggression in mice, Experientia, 35 (1979) 273. 11 Luttge, W. G. and Hall, N. R., Androgen-induced agonistic behavior in castrate male SwissWebster mice: comparison of four naturally occurring androgens, Behav. Biol., 8 (1973) 725 732. 12 Luttge, W. G., Hall, N. R. and Wallis, C. J., Studies on the neuroendocrine, somatic and behavioral effectiveness of testosterone and its 5a-reduced metabolites in Swiss-Webster mice, Physiol. Behav., 13 (1974) 553-561. 13 Marsa, R., Stupnicka, E., Kniewald, Z. and Martini, L., The transformation of testosterone into dihydrotestosterone by the brain and the anterior pituitary, J. Steroid Biochem., 3 (1972) 383-389. 14 Naftolin, F., Ryan, K., Davies, I., Reddy, V., FIores, F., Petro, Z., Kuhn, M., White, R. J., Takaoka, Y. and Wolin, L., The formation of estrogens by central neuroendocrine tissues, Rec. Progr. Horm. Res., 31 (1975) 295-315. 15 Sar, M. and Stumpf, W. E., Distribution of androgen target cells in rat forebrain and pituitary after [3H]dihydrotestosterone administration, J. Steroid. Biochem., 8 (1977) 1131-1135. 16 Schechter, D., Howard, S. M. and Gandelman, R., Dihydrotestosterone promotes fighting behavior of female mice, Horm. Behav., 15 (1981) 233-237. 17 Sheridan, P. J., Localization of androgen and estrogen concentrating neurons in the diencephalon and telencephalon of the mouse, Endocrinology, 103 (1978) 1328-1334. 18 Sheridan, P. J., The nucleus interstitialis striae terminalis and the medial nucleus of the amygdala, prime targets for androgen in the rat forebrain, Endocrinology, 104 (1979) 130-136. 19 Sholiton, L. J., Jones, C. E. and Werk, E. E., The uptake and metabolism of [1,2-3H]testosterone by the brains of functionally hepatectomized and totally eviscerated male rats, Steroids, 20 (1972) 399-415. 20 Simon, N. G. and Gandelman, R., The aggression-promoting and aggression-eliciting properties of estrogen in male mice, Physiol. Behav., 21 (1978) 161-164. 21 Simon, N. G., Gandelman, R. and Howard, S. M., MER-25 does not inhibit the activation of aggression by testosterone in adult Rockland-Swiss Mice, Psychonettroendocrinology, 6 (1981) 131-137. 22 Stern, J. M. and Eisenfeld, A. J., Distribution and metabolism of [3H]testosterone in castrated male rats; effects of cyproterone, progesterone and unlabeled testosterone, Endocrinology, 88 (1971) 1117-1125. 23 Stumpf, W. E., Autoradiographic techniques for localization of hormones and drugs at the cellular and subcellular level, Acta Endocr. Kbh., Suppl. 153 (1971) 205-221.