Cell Tiss. Res. 183, 379-384 (1977)
Cell and Tissue Research 9 by Springer-Verlag 1977
Cellular and Subcellular Localization of 3H-Diethylstilboestrol in the Central Nervous System* Peter J. Sheridan** *** Department of Anatomy, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA
Summary. The cellular and subcellular localization of radioactivity in the brain of immature female rats was determined by dry-mount autoradiography 2 h after iv injection of 1.0 ~tg of (monethyl-3H) diethylstilboestrol per 100 g body weight. A specific topographic pattern of nuclear concentration of the synthetic oestrogen was obtained similar to that for 3H-oestradiol-17fl in specific neurons of the basal hypothalamus, preoptic region and amygdala. In competition experiments, the nuclear concentration o f radioactivity in all areas studied was inhibited by unlabeled oestradiol, while unlabeled testosterone had no effect. These data suggest that although oestradiol can bind to androgen receptors, the oestrogen receptor itself can account for the localization seen after the injection of 3H-oestradiol. Key words: Brain - Rat - Diethylstilboestrol - Autoradiography.
Introduction Earlier studies (McEwen and Pfaff, 1970; Sheridan et al., 1974) have demonstrated that unlabeled testosterone is capable of inhibiting the uptake and retention of 3Hoestradiol in the preoptic region and basal hypothalamus. The mechanism by which such inhibition occurs is unknown. At least three possibilities immediately present themselves. Testosterone m a y compete with oestradiol for the oestrogen receptor. Testosterone m a y be aromatized to oestradiol and then the converted oestradiol m a y inhibit the binding of 3H-oestradiol to its receptor. Finally, both testosterone and oestradiol m a y be translocated by the androgen receptor. At the m o m e n t it seems highly doubtful that a the doses of androgen used in these studies, the inhibition of oestradiol uptake could be explained by androgen Sendoffprint requests to: Dr. Peter J. Sheridan, Department ofAnatomy, The Universityof TexasHealth
Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78284, USA * This research was supported in part by US PHS Grant No. NS12933 ** NIH Career DevelopmentAwardee No. NS00164 *** The expert technical assistance of Ms. Riki Ison and Ms. Linda Furr is gratefully acknowledged.
380
P.J. Sheridan
c o m p e t i t i o n with the o e s t r o g e n r e c e p t o r (Ruh et al. 1975; R o c h e f o r t a n d Garcia, 1976). O n o t h e r hand, o e s t r a d i o l binds in vitro to b o t h oestrogen a n d a n d r o g e n r e c e p t o r s in the central n e r v o u s system ( K a t o , 1974; Fox, 1975; A t t a r d i et al., 1976; C h a m n e s s et at., 1977). Since oestradiol-17fl binds b o t h o f these receptors (Kd = 1 x 10-1~ for the oestrogen r e c e p t o r a n d K d = 2 • 1 0 - S M for the a n d r o g e n receptor), the nuclear u p t a k e a n d r e t e n t i o n t h a t one sees after the injection o f 3 H - o e s t r a d i o l m a y be the result o f t r a n s l o c a t i o n b y either or b o t h o f these c y t o p l a s m i c proteins. I n contrast, to oestradiol, diethylstilboestrol binds only to the o e s t r o g e n r e c e p t o r in the central n e r v o u s system ( A t t a r d i et al., 1976; C h a m n e s s et al., 1977), a n d thus the nuclear u p t a k e a n d retention seen after the injection o f 3H-diethylstilboestrol will p r o v i d e a m a p o f only the oestrogen receptor. The following s t u d y was c o n d u c t e d to d e t e r m i n e if there are a n y differences between the l o c a l i z a t i o n o f 3H-oestradiol a n d 3H-diethylstilboestrol in the central n e r v o u s system.
Materials and Methods Twenty-five-day-old female rats (n = 5 per group) were injected iv with 1.0 gg of (monoethyl-aH) diethylstilboestrol (62.4 Ci/mmol) per 100 g body wt dissolved in 25 % ethanol saline only, or in 25 % ethanol saline containing 15 gg per 100 g body wt of either unlabeled oestradiol-17fl or testosterone. The unlabeled steroids were administered for the purpose of demonstrating the specificity and saturabilityof the nuclear uptake and retention of 3H-diethylstilboestrol. In order to minimize the potential competition due to the aromatization of testosterone to oestradiol, the unlabeled steroids were administered simultaneously with the labeled oestrogen and in much smaller quantities than those used in past studies (McEwen and Pfaff, 1970; Sheridan et al., 1974). The animals were killed 2 h later, and the brains were removed, sliced, and mounted on tissue holders. The mounted tissue was then frozen in liquified propane and stored in liquid nitrogen until processing. Four micron frozen sections were cut in a cyrostat and mounted for autoradiography on emulsioncoated slides according to the dry- nount procedure (Stumpf, 1971). The slides were exposed at - 15~ for 8 weeks, then photographically processed and stained with methylgreen-pyronin. Grain counts were made to determine the effects of the unlabeled steroids on the nuclear uptake and retention of the 3H-diethylstilboestrol. The number of grains in twenty-five randomly selected labeled cell nuclei from three regions of the hypothalamus (Nucleus (n.) preopticus medialis, n. interstitialis striae terminalis and n. arcuatus hypothalami) were counted. Cells were considered labeled if the number of grains exceeded twice the silver grain counts in adjacent extracellular spaces and cytoplasm. The grain counts were averaged for each area for each animal before the backgrounds were subtracted. Background was estimated by averaging the number of grains in equivalent areas of extracellular space.
Results In a u t o r a d i o g r a m s o f the b r a i n s f r o m a n i m a l s injected with 3H-diethylstilboestrol only, r a d i o a c t i v i t y was f o u n d c o n c e n t r a t e d with v a r y i n g intensity o f nuclei o f n e u r o n s o f the following areas (Figs. 1 a, d, 2): Nucleus (n.) preopticus, p a r s s u p r a c h i a s m a t i c a ; n. p r e o p t i c u s medialis; n. p r e o p t i c u s lateralis; n. periventricularis; n. interstitialis striae terminalis; n. septi lateralis; n. paraventricularis, p a r s m a g n o c e l l u l a r i s ; n. a r c u a t u s h y p o t h a l a m i ; n. v e n t r o m e d i a l i s h y p o t h a l a m i , p a r s v e n t r o l a t e r a l i s ; n. p r e m a m m i l l a r i s ventralis; n. medialis a m y g d e a l a e ; n. corticalis a m y g d a l a e ; n. basalis a m y g d a l a e p a r s medialis.
Diethylstilboestrol Localization in the Brain
381
Fig. 1 a-f. Autoradiograms of the medial nuclei of the preoptic region (a-e) and the amygdala (d-f) of the immature female rat 2 h after the injection o f 3H-diethylstilboestrol. Injection of 3H-diethylstilboestrol alone produced a nuclear concentration of radioactivity in certain neurons, as seen in the medial preoptic nucleus (a) and amygdala (d). Simultaneous treatment with a 15 fold greater dose of unlabeled oestradiol completely inhibited the nuclear concentration o f radioactivity in all areas studied (b and e). On the other hand, a simultaneous treatment with a 15 fold greater dose of unlabeled testosterone had little or no effect on nuclear uptake and retention (c and O
382
P.J. Sheridan
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Fig. 2. A topographic distribution of diethylstilboestrol binding neurons in the female rat brain is schematically represented in frontal cross sections of the preoptic, central hypothalamus and central amygdala. Abbreviations for the areas with dots, representing accumulations of radioactively labeled neurons: aco n. amygdaloideus cortcalis; a m n. medialis amygdalae; ar n. arcuatus hypothalami; hpv n. periventricularis hypothalami; h v m n . ventromedialis hypothalami; p o l n. preopticus lateralis; p o m n. preopticus medialis;pv n. premammillaris ventralis; sln. septi lateralis; st n. interstitialis striae terminalis (for other abbreviations see K6nig and Klippel, 1963)
T h e s i m u l t a n e o u s a d m i n i s t r a t i o n o f u n l a b e l e d oestradiol-17fl c o m p l e t e l y i n h i b i t e d the n u c l e a r c o n c e n t r a t i o n o f r a d i o a c t i v i t y in all areas studies (Fig. 1 b, e). O n the o t h e r h a n d , g r a i n c o u n t s i n three areas i n d i c a t e d t h a t the s i m u l t a n e o u s a d m i n i s t r a t i o n o f t e s t o s t e r o n e was w i t h o u t a n y a p p a r e n t effect (Fig. 1 c, f, T a b l e 1).
Diethylstilboestrol Localization in the Brain
383
Table 1. Effect of simultaneous treatment with unlabeled steroids on the nuclear uptake and retention of 3H-diethylstilboestrol Treatment
3H-DES O N L Y 3H-DES + Estradiol 3H-DES + Testosterone
Average n u m b e r of silver grains + standard error Nucleus preopticus
Nucleus interstitialis
Nucleus arcuatus
medialis
Striae terminalis
hypothalami
22.1 ---0.8 0 23.7 + 0.9
15.1 -+0.6 0 14.6 + 0.7
15.3---0.3 0 16.2 + 0.5
Discussion
A specific topographic pattern of nuclear concentration of radioactivity was obtained in specific neurons of the basal hypothalamus, preoptic region and amygdala. Since the localization is inhibited by the simultaneous treatment with oestradiol, but not by testosterone, the data indicate that the radioactivity is still associated with the diethylstilboestrol or a biologically active metabolite, and that the binding is of limited capacity. This pattern of localization is similar to that seen after the injection of 3H-oestradiol (Stumpf, 1970; Sheridan et al., 1974). Since the nuclear uptake and retention of diethylstilboestrol is due only to the estrogen receptor, it appears that for the doses of 3H-oestradiol that have been used in past autoradiographic studies (Stumpf, 1970; Sheridan, 1974) one need not invoke the androgen receptor for localization that has been reported. In previous studies (McEwen and Pfaff, 1970; Sheridan et al., 1974) it has been reported that unlabeled testosterone when given in large quantities prior to 3H-oestradiol will inhibit nuclear uptake and retention of 3H-oestradiol in many areas of the brain. Since the oestrogen is binding only to oestrogen receptors at this dosage, and since testosterone does not compete for the oestrogen receptors except at extremely high doses (Ruh et al., 1975; Rochefort et al., 1976), it would seem that the only explanation for the observed inhibition of nuclear uptake and retention of 3Hoestradiol by testosterone is the conversion of unlabeled testosterone to oestradiol. Once converted, the unlabeled oestradiol could compete for bindings sites with the labeled estradiol. Support for this hypothesis comes from a number of studies. It has been demonstrated that androgens can be converted to oestrogens in the hypothalamus and limbic system (Naftolin et al., 1972; Weisz and Gibbs, 1974; Reddy et al., 1974), and recently it has been reported that 3H-oestradiol has been found in purified nuclear preparations from the hypothalamus and amygdala of the rat after the injection of 3H-testosterone (Lieberburg and McEwen, 1975, 1976). All of the above studies are consistent with the idea that the observed inhibition of nuclear uptake and retention of oestradiol by testosterone was mediated by the conversion of testosterone to oestradiol.
384
P.J. Sheridan
References Attardi, B., Geller, L.N., Ohno, S.: Androgen and estrogen receptors in the brain cytosol from male, female, and testicular feminized ( T f m / y ) mice. Endocrinology 98, 864-874 (1976) Chamness, G.C., King, T.W., Sheridan, PJ.: Estrogen-binding androgen receptors in the rat brain. The 59th Annual Endocrine Society Meeting, Chicago, Abstract (1977) Fox, T.O.: Androgen- and estrogen binding macromolecules in developing mouse brain: Biochemical and genetic evidence. Proc. nat. Acad. Sci. (Wash.) 72, 4303-4307 (1975) Kato, J.: Receptor proteins for androgen and estrogen in the hypothalamo-hypophyseal unit, BrainEndocrine Interaction, Symposium II, Tokyo, Abstract (1974) Kfnig, J.F.R., Klippel, R.A.: The rat brain. Baltimore: Williams and Wilkins 1963 Lieberburg, I., McEwen, B.S.: Estradiol-17/~: A metabolite of testosterone recovered in cell nuclei from limbic areas of adult male rat brains. Brain Res. 91, 171-174, (1975) Lieberburg, I., McEwen, B.S.: Brain cell nuclear retention of testosterone metabolites, 5-~ dihydrotestosterone and estradiol-17/3, in adult rats. Endocrinology 100, 588-597 (1977) McEwerh B.S., Pfaff, D.W.: Factors influencing sex hormone uptake by rat brain regions. I. Effects of neonatal treatment, hypophysectomy, and competing steroid on estradiol uptake. Brain Res. 21, 1 16 (1970) Naflolin, F., Ryan, K.J., Petro, Z.: Aromatization of androstenedione by the anterior hypothalamus of adult male and female rats. Endocrinology 90, 295-298 (1972) Reddy, V.V.R., Naftolin, F., Ryan, K.J.: Conversion of androstenedione to estrone by neural tissues from fetal and neonatal rats. Endocrinology 94, 117-121 (1974) Rochefort, H., Garcia, M.: Androgen on the estrogen receptor I. Binding and in vivo nuclear translocation. Steroids 28, 549-560 (1976) Ruh, T.S., Wassilak, S.G., Ruh, M.F.: Androgen-induced nuclear accumulation of the estrogen receptor. Steroids 25, 257-273 (1975) Sheridan, P.J., Sar, M., Stumpf, W.E.: Autoradiographic localization of 3H-estradiol or its metabolites in the central nervous system of the developing rat. Endocrinology 94, 1386-1390 (1974) Stumpf, W.E.: Autoradiographic techniques for the localization of hormones and drugs at the cellular and subcellular level. Acta endocr. (Suppl.) 153, 205-221 (1971) Stumpf, W.E.: Estrogen-neurons and estrogen-neuron systems in the periventricular brain. Amer. J. Anat. 129, 207-217 (1970) Weisz, J., Gibbs, C.: Conversion of testosterone and androstenedione to estrogen in vitro by the brain of female rats. Endocrinology 94, 616-620 (1974)
Accepted May 26, 1977
Cell and Tissue Research 9 by Springer-Verlag 1977
Cellular and Subcellular Localization of 3H-Diethylstilboestrol in the Central Nervous System* Peter J. Sheridan** *** Department of Anatomy, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA
Summary. The cellular and subcellular localization of radioactivity in the brain of immature female rats was determined by dry-mount autoradiography 2 h after iv injection of 1.0 ~tg of (monethyl-3H) diethylstilboestrol per 100 g body weight. A specific topographic pattern of nuclear concentration of the synthetic oestrogen was obtained similar to that for 3H-oestradiol-17fl in specific neurons of the basal hypothalamus, preoptic region and amygdala. In competition experiments, the nuclear concentration o f radioactivity in all areas studied was inhibited by unlabeled oestradiol, while unlabeled testosterone had no effect. These data suggest that although oestradiol can bind to androgen receptors, the oestrogen receptor itself can account for the localization seen after the injection of 3H-oestradiol. Key words: Brain - Rat - Diethylstilboestrol - Autoradiography.
Introduction Earlier studies (McEwen and Pfaff, 1970; Sheridan et al., 1974) have demonstrated that unlabeled testosterone is capable of inhibiting the uptake and retention of 3Hoestradiol in the preoptic region and basal hypothalamus. The mechanism by which such inhibition occurs is unknown. At least three possibilities immediately present themselves. Testosterone m a y compete with oestradiol for the oestrogen receptor. Testosterone m a y be aromatized to oestradiol and then the converted oestradiol m a y inhibit the binding of 3H-oestradiol to its receptor. Finally, both testosterone and oestradiol m a y be translocated by the androgen receptor. At the m o m e n t it seems highly doubtful that a the doses of androgen used in these studies, the inhibition of oestradiol uptake could be explained by androgen Sendoffprint requests to: Dr. Peter J. Sheridan, Department ofAnatomy, The Universityof TexasHealth
Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78284, USA * This research was supported in part by US PHS Grant No. NS12933 ** NIH Career DevelopmentAwardee No. NS00164 *** The expert technical assistance of Ms. Riki Ison and Ms. Linda Furr is gratefully acknowledged.
380
P.J. Sheridan
c o m p e t i t i o n with the o e s t r o g e n r e c e p t o r (Ruh et al. 1975; R o c h e f o r t a n d Garcia, 1976). O n o t h e r hand, o e s t r a d i o l binds in vitro to b o t h oestrogen a n d a n d r o g e n r e c e p t o r s in the central n e r v o u s system ( K a t o , 1974; Fox, 1975; A t t a r d i et al., 1976; C h a m n e s s et at., 1977). Since oestradiol-17fl binds b o t h o f these receptors (Kd = 1 x 10-1~ for the oestrogen r e c e p t o r a n d K d = 2 • 1 0 - S M for the a n d r o g e n receptor), the nuclear u p t a k e a n d r e t e n t i o n t h a t one sees after the injection o f 3 H - o e s t r a d i o l m a y be the result o f t r a n s l o c a t i o n b y either or b o t h o f these c y t o p l a s m i c proteins. I n contrast, to oestradiol, diethylstilboestrol binds only to the o e s t r o g e n r e c e p t o r in the central n e r v o u s system ( A t t a r d i et al., 1976; C h a m n e s s et al., 1977), a n d thus the nuclear u p t a k e a n d retention seen after the injection o f 3H-diethylstilboestrol will p r o v i d e a m a p o f only the oestrogen receptor. The following s t u d y was c o n d u c t e d to d e t e r m i n e if there are a n y differences between the l o c a l i z a t i o n o f 3H-oestradiol a n d 3H-diethylstilboestrol in the central n e r v o u s system.
Materials and Methods Twenty-five-day-old female rats (n = 5 per group) were injected iv with 1.0 gg of (monoethyl-aH) diethylstilboestrol (62.4 Ci/mmol) per 100 g body wt dissolved in 25 % ethanol saline only, or in 25 % ethanol saline containing 15 gg per 100 g body wt of either unlabeled oestradiol-17fl or testosterone. The unlabeled steroids were administered for the purpose of demonstrating the specificity and saturabilityof the nuclear uptake and retention of 3H-diethylstilboestrol. In order to minimize the potential competition due to the aromatization of testosterone to oestradiol, the unlabeled steroids were administered simultaneously with the labeled oestrogen and in much smaller quantities than those used in past studies (McEwen and Pfaff, 1970; Sheridan et al., 1974). The animals were killed 2 h later, and the brains were removed, sliced, and mounted on tissue holders. The mounted tissue was then frozen in liquified propane and stored in liquid nitrogen until processing. Four micron frozen sections were cut in a cyrostat and mounted for autoradiography on emulsioncoated slides according to the dry- nount procedure (Stumpf, 1971). The slides were exposed at - 15~ for 8 weeks, then photographically processed and stained with methylgreen-pyronin. Grain counts were made to determine the effects of the unlabeled steroids on the nuclear uptake and retention of the 3H-diethylstilboestrol. The number of grains in twenty-five randomly selected labeled cell nuclei from three regions of the hypothalamus (Nucleus (n.) preopticus medialis, n. interstitialis striae terminalis and n. arcuatus hypothalami) were counted. Cells were considered labeled if the number of grains exceeded twice the silver grain counts in adjacent extracellular spaces and cytoplasm. The grain counts were averaged for each area for each animal before the backgrounds were subtracted. Background was estimated by averaging the number of grains in equivalent areas of extracellular space.
Results In a u t o r a d i o g r a m s o f the b r a i n s f r o m a n i m a l s injected with 3H-diethylstilboestrol only, r a d i o a c t i v i t y was f o u n d c o n c e n t r a t e d with v a r y i n g intensity o f nuclei o f n e u r o n s o f the following areas (Figs. 1 a, d, 2): Nucleus (n.) preopticus, p a r s s u p r a c h i a s m a t i c a ; n. p r e o p t i c u s medialis; n. p r e o p t i c u s lateralis; n. periventricularis; n. interstitialis striae terminalis; n. septi lateralis; n. paraventricularis, p a r s m a g n o c e l l u l a r i s ; n. a r c u a t u s h y p o t h a l a m i ; n. v e n t r o m e d i a l i s h y p o t h a l a m i , p a r s v e n t r o l a t e r a l i s ; n. p r e m a m m i l l a r i s ventralis; n. medialis a m y g d e a l a e ; n. corticalis a m y g d a l a e ; n. basalis a m y g d a l a e p a r s medialis.
Diethylstilboestrol Localization in the Brain
381
Fig. 1 a-f. Autoradiograms of the medial nuclei of the preoptic region (a-e) and the amygdala (d-f) of the immature female rat 2 h after the injection o f 3H-diethylstilboestrol. Injection of 3H-diethylstilboestrol alone produced a nuclear concentration of radioactivity in certain neurons, as seen in the medial preoptic nucleus (a) and amygdala (d). Simultaneous treatment with a 15 fold greater dose of unlabeled oestradiol completely inhibited the nuclear concentration o f radioactivity in all areas studied (b and e). On the other hand, a simultaneous treatment with a 15 fold greater dose of unlabeled testosterone had little or no effect on nuclear uptake and retention (c and O
382
P.J. Sheridan
cp
I~ II
/S
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' %).
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! • : - ,~• i"--"
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.
oj
, 9 .m . - , .'* * - . .b, ;t "....:/.- .... ..,,.. . . . ~ o . . . . ~.:.., _.,
..
.:....:,~f
,
Fig. 2. A topographic distribution of diethylstilboestrol binding neurons in the female rat brain is schematically represented in frontal cross sections of the preoptic, central hypothalamus and central amygdala. Abbreviations for the areas with dots, representing accumulations of radioactively labeled neurons: aco n. amygdaloideus cortcalis; a m n. medialis amygdalae; ar n. arcuatus hypothalami; hpv n. periventricularis hypothalami; h v m n . ventromedialis hypothalami; p o l n. preopticus lateralis; p o m n. preopticus medialis;pv n. premammillaris ventralis; sln. septi lateralis; st n. interstitialis striae terminalis (for other abbreviations see K6nig and Klippel, 1963)
T h e s i m u l t a n e o u s a d m i n i s t r a t i o n o f u n l a b e l e d oestradiol-17fl c o m p l e t e l y i n h i b i t e d the n u c l e a r c o n c e n t r a t i o n o f r a d i o a c t i v i t y in all areas studies (Fig. 1 b, e). O n the o t h e r h a n d , g r a i n c o u n t s i n three areas i n d i c a t e d t h a t the s i m u l t a n e o u s a d m i n i s t r a t i o n o f t e s t o s t e r o n e was w i t h o u t a n y a p p a r e n t effect (Fig. 1 c, f, T a b l e 1).
Diethylstilboestrol Localization in the Brain
383
Table 1. Effect of simultaneous treatment with unlabeled steroids on the nuclear uptake and retention of 3H-diethylstilboestrol Treatment
3H-DES O N L Y 3H-DES + Estradiol 3H-DES + Testosterone
Average n u m b e r of silver grains + standard error Nucleus preopticus
Nucleus interstitialis
Nucleus arcuatus
medialis
Striae terminalis
hypothalami
22.1 ---0.8 0 23.7 + 0.9
15.1 -+0.6 0 14.6 + 0.7
15.3---0.3 0 16.2 + 0.5
Discussion
A specific topographic pattern of nuclear concentration of radioactivity was obtained in specific neurons of the basal hypothalamus, preoptic region and amygdala. Since the localization is inhibited by the simultaneous treatment with oestradiol, but not by testosterone, the data indicate that the radioactivity is still associated with the diethylstilboestrol or a biologically active metabolite, and that the binding is of limited capacity. This pattern of localization is similar to that seen after the injection of 3H-oestradiol (Stumpf, 1970; Sheridan et al., 1974). Since the nuclear uptake and retention of diethylstilboestrol is due only to the estrogen receptor, it appears that for the doses of 3H-oestradiol that have been used in past autoradiographic studies (Stumpf, 1970; Sheridan, 1974) one need not invoke the androgen receptor for localization that has been reported. In previous studies (McEwen and Pfaff, 1970; Sheridan et al., 1974) it has been reported that unlabeled testosterone when given in large quantities prior to 3H-oestradiol will inhibit nuclear uptake and retention of 3H-oestradiol in many areas of the brain. Since the oestrogen is binding only to oestrogen receptors at this dosage, and since testosterone does not compete for the oestrogen receptors except at extremely high doses (Ruh et al., 1975; Rochefort et al., 1976), it would seem that the only explanation for the observed inhibition of nuclear uptake and retention of 3Hoestradiol by testosterone is the conversion of unlabeled testosterone to oestradiol. Once converted, the unlabeled oestradiol could compete for bindings sites with the labeled estradiol. Support for this hypothesis comes from a number of studies. It has been demonstrated that androgens can be converted to oestrogens in the hypothalamus and limbic system (Naftolin et al., 1972; Weisz and Gibbs, 1974; Reddy et al., 1974), and recently it has been reported that 3H-oestradiol has been found in purified nuclear preparations from the hypothalamus and amygdala of the rat after the injection of 3H-testosterone (Lieberburg and McEwen, 1975, 1976). All of the above studies are consistent with the idea that the observed inhibition of nuclear uptake and retention of oestradiol by testosterone was mediated by the conversion of testosterone to oestradiol.
384
P.J. Sheridan
References Attardi, B., Geller, L.N., Ohno, S.: Androgen and estrogen receptors in the brain cytosol from male, female, and testicular feminized ( T f m / y ) mice. Endocrinology 98, 864-874 (1976) Chamness, G.C., King, T.W., Sheridan, PJ.: Estrogen-binding androgen receptors in the rat brain. The 59th Annual Endocrine Society Meeting, Chicago, Abstract (1977) Fox, T.O.: Androgen- and estrogen binding macromolecules in developing mouse brain: Biochemical and genetic evidence. Proc. nat. Acad. Sci. (Wash.) 72, 4303-4307 (1975) Kato, J.: Receptor proteins for androgen and estrogen in the hypothalamo-hypophyseal unit, BrainEndocrine Interaction, Symposium II, Tokyo, Abstract (1974) Kfnig, J.F.R., Klippel, R.A.: The rat brain. Baltimore: Williams and Wilkins 1963 Lieberburg, I., McEwen, B.S.: Estradiol-17/~: A metabolite of testosterone recovered in cell nuclei from limbic areas of adult male rat brains. Brain Res. 91, 171-174, (1975) Lieberburg, I., McEwen, B.S.: Brain cell nuclear retention of testosterone metabolites, 5-~ dihydrotestosterone and estradiol-17/3, in adult rats. Endocrinology 100, 588-597 (1977) McEwerh B.S., Pfaff, D.W.: Factors influencing sex hormone uptake by rat brain regions. I. Effects of neonatal treatment, hypophysectomy, and competing steroid on estradiol uptake. Brain Res. 21, 1 16 (1970) Naflolin, F., Ryan, K.J., Petro, Z.: Aromatization of androstenedione by the anterior hypothalamus of adult male and female rats. Endocrinology 90, 295-298 (1972) Reddy, V.V.R., Naftolin, F., Ryan, K.J.: Conversion of androstenedione to estrone by neural tissues from fetal and neonatal rats. Endocrinology 94, 117-121 (1974) Rochefort, H., Garcia, M.: Androgen on the estrogen receptor I. Binding and in vivo nuclear translocation. Steroids 28, 549-560 (1976) Ruh, T.S., Wassilak, S.G., Ruh, M.F.: Androgen-induced nuclear accumulation of the estrogen receptor. Steroids 25, 257-273 (1975) Sheridan, P.J., Sar, M., Stumpf, W.E.: Autoradiographic localization of 3H-estradiol or its metabolites in the central nervous system of the developing rat. Endocrinology 94, 1386-1390 (1974) Stumpf, W.E.: Autoradiographic techniques for the localization of hormones and drugs at the cellular and subcellular level. Acta endocr. (Suppl.) 153, 205-221 (1971) Stumpf, W.E.: Estrogen-neurons and estrogen-neuron systems in the periventricular brain. Amer. J. Anat. 129, 207-217 (1970) Weisz, J., Gibbs, C.: Conversion of testosterone and androstenedione to estrogen in vitro by the brain of female rats. Endocrinology 94, 616-620 (1974)
Accepted May 26, 1977
