Prychoneuuroendocrinology, 1977, Vol. 2. pp. 173-178. Pergamon Press. Printed in Great Britain

NUCLEAR LOCALIZATION OF TESTOSTERONE, DIHYDROTESTOSTERONE AND ESTRADIOL-178 IN BASAL RAT BRAIN DONALD L. REZEK* Department

of Psychobiology,

University of California, Irvine, CA 92717, U.S.A. SUMMARY

(1) One-half hour after the intravenous injection of [3H]testosterone, basal brain cell nuclei isolated from castrated male rats contained more radioactivity with the thin layer chromatography mobility of estradiol-178 and dihydrotestosterone (DHT) than nuclei from female rats. (2) The sex differences in the subcellular localization of [‘HJDHT and [3H]estradiol-17f3 were not reflected in the ratio of the nuclear to whole homogenate radioactivity. (3) In the basal brain, estradiol-17/l was the only metabolite examined that showed more DPM/mg protein in the nuclear fraction than in the whole homogenate. (4) In the basal brain and pituitary, unlabeled DHT was more effective than testosterone in blocking the nuclear localization of [“HItestosterone and 13H]DHT. (5) Unlabeled estradiol-17/I only blocked the nuclear concentration of [3H]estradiol-17,9 and not [3H]testosterone or r3H]DHT. Key Words--testosterone cell nuclei.

metabolism;

hypothalamic

androgen metabolites;

estradiol-17,9 in

INTRODUCTION

of testosterone into active metabolites is considered important in the hormone’s mechanism of action in target tissues. Two possible active metabolites, dihydrotestosterone (DHT) and estradiol-17fi, are known to be produced from testosterone by neural tissue. Behavioral studies indicate that these two metabolites may act synergistically in support of male sexual behavior when administered to the male rat (Baum & Vreeburg, 1973; Larsson, Sodersten & Beyer, 1973). Since current models of steroid hormone action emphasize the role of the hormone within cell nuclei, one would predict that the active metabolites should be concentrated in cell nuclei within target regions of the brain. In a recent study Lieberburg & McEwen (197%) examined the amount of estradiol-1715 found in brain cell nuclei. They found after the administration of [3H]testosterone that [3H]estradiol-17/3 was concentrated in cell nuclei of hypothalamic and limbic brain regions. The present study was conducted to determine whether DHT and estradiol-17/3 are concentrated in the rat’s hypothalamus and ventral midbrain, brain regions responsible for the regulation of androgen-dependent sexual behavior. Perinatal hormonal secretion influences the sensitivity of target tissues to androgens in later life. Adult female rats are less likely to display male behavior following an equal dose of testosterone than adult maIe rats which have been affected by their early endogenous

THE CONVERSION

* Present address: Albert Einstein College of Medicine, Department Avenue, Bronx. NY 10461, U.S.A. 173 P.N.E.C. 2/2--e

of Neurology,

1300 Morris Park

174

DONALD L. REZEK

h o r m o n e levels. W h i c h c e l l u l a r p r o c e s s e s are a l t e r e d is n o t well u n d e r s t o o d . O n e possibility is a c h a n g e in the w a y t a r g e t n e u r a l tissues p r o d u c e o r t r a n s p o r t a c t i v e m e t a b o l i t e s w i t h i n t h e cell. I n o r d e r to test this h y p o t h e s i s , the ability o f m a l e a n d f e m a l e r a t b r a i n cells to c o n c e n t r a t e t e s t o s t e r o n e , D H T a n d estradiol-17/3 w i t h i n cell nuclei was c o m p a r e d . In vitro studies h a v e s h o w n t h a t m a l e a n d f e m a l e rat b r a i n tissue p r o d u c e d i f f e r e n t a m o u n t s o f t h e s e m e t a b o l i t e s ( N a f t o l i n , R y a n & P e t r o , 1972; D e n e f , M a g n u s & M c E w e n , 1973) s u g g e s t i n g t h a t s i m i l a r effects w o u l d be p r e s e n t in civo. I n the s e c o n d p a r t o f t h e p r e s e n t i n v e s t i g a t i o n , t h e a b i l i t y o f u n l a b e l e d steroids to r e d u c e t h e n u c l e a r c o n c e n t r a t i o n o f t h e r a d i o a c t i v i t y w i t h the t h i n layer c h r o m a t o g r a p h y ( T L C ) c h a r a c t e r i s t i c s o f [ 3 H ] t e s t o s t e r o n e , [ 3 H ] D H T , a n d [3H]estradiol-17fl was e x a m i n e d . METHODS Nine albino male rats (Simonsen Laboratories, Gilroy, Cal.), nine female rats of the same age as the males (61 days), and nine female rats with the same weight as the males (276 g) were used in the first portion of the study. All animals were gonadectomized 2 days before the experiment. Each animal was injected with 70/~Ci(0.24 p.g)/100 g body weight of [1,2,6,7-3H]testosterone (specific activity 85 Ci/mmol, New England Nuclear) in a 1 0 ~ ethanol solution via the jugular vein 30 min before the animal was killed and samples removed. A basal brain sample that included the preoptic area posterior through the ventral midbrain, bordered laterally by the hypothalamic sulci and extending 2.5 mm dorsally was excised. A portion of gray matter of the sensorimotor cortex from the dorsal surface of the brain opposite the basal brain sample was used as a cortical sample. Whole pituitary samples were also taken. After the corresponding samples from three animals were combined and weighed, enriched nuclear fractions were prepared using the method of McEwen & Zigmond (1972). Homogenization was performed in a pH 6.5 1 mM KH2PO4 buffer including 3 mM MgCI2 and 0.25Yo Triton X-100 in 0.32 M sucrose. After centrifugation at 850 g for l0 rain the pellet was washed once in the same buffer without Triton X-100 and spun at the same speed. For the final centrifugation the sample was resuspended in the same buffer (without Triton X-100) in 2 M sucrose and spun at 63,000 g for 45 rain. The nuclear pellets were resuspended in 2.0 ml 0.01 M citric acid and extracted 4 times with 5.0 ml of toluene. Unlabeled testosterone, DHT, and estradiol-17/3 were added in /~g quantities as carriers. Several other androgens included in a standard carrier mixture were also added and are listed elsewhere (Rezek & Whalen, 1975). Samples of the whole homogenate were diluted with water and the carrier steroids plus 200 CPM each of [~4C]-labeled testosterone and DHT (both 56.1 mCi/mmol, New England Nuclear) as recovery standards were added prior to the sample being extracted three times with 3.0 ml dichloromethane. Protein determinations were performed on samples taken prior to the extraction using the Miller (1959) modification of the Lowry, Rosebrough, Farr & Randall (1951) method. The organic extracts were dried under nitrogen and applied to precoated silica gel TLC plates (EM brand, silica gel 60). The toluene extracts from the nuclear samples were developed in a system of benzenemethanol (98:2), followed by a second run in benzene-ethyl acetate (60:40), and a third in a system of chloroform-benzene-ethyl acetate-methanol (65:20:13: 2). The whole homogenate extracts were first run for 5 cm in a solvent system of benzene-methanol (95: 5) and then developed twice for a full 16 cm in the third solvent system used for the nuclear extracts. The plates were allowed to air dry between each run. This procedure yielded similar mobilities for the androgens from both the nuclear and whole homogenate samples but caused the estradiol-17fl to move with DHT instead of testosterone for the nuclear extracts. The DHT-estradiol-17fl zone from the nuclear extracts and the testosterone-estradiol-17fl and D H T zones from the whole homogenate extracts were scraped and the steroids eluted. The eluates were acetylated overnight (in 0.15 ml acetic anhydride and 0.25 ml pyridine) and then replated and run twice in a system of benzene-cyclohexane-ethyl acetate (71:23: 6). Final zones with the mobilities of the authentic unlabeled steroids were scraped into scintillation vials. After the addition of 1.0 ml methanol followed after several hours with 10 ml of toluene based scintillation fluid, the radioactivity was determined using a Beckman 150 liquid scintillation counter. Based on [14C] recovery standards remaining in the samples being studied (for D H T and testosterone) or in similarly treated parallel samples (for estradiol-17fl); the recoveries were 70 for DHT, 80 ~ for testosterone, and 77 ~ for estradiol-17fl. Testosterone acetate and DHT acetate from the whole homogenate samples were corrected for loss during recovery using the [~4Cl recovery standards and all CPM converted to DPM as described in Rezek & Whalen (1975). To determine the specificity of the binding of the steroids to the nuclear fraction, six males were injected

ANDROGEN METABOLITESIN BASAL BRAIN CELL NUCLEI

175

intravenously with 200/~g of testosterone, DHT, or estradiol-17fl 30 min before the animals were injected with 200 ~Ci of radiolabeled testosterone. The same regions used for the first part of the study were taken. However, the regions from only two instead of three animals were pooled. The rest of the procedure was the same as in the first part of the study. RESULTS Figure 1 shows that for nuclei no sex difference was present in the amount of radioactivity recovered from the testosterone zones from the TLC plates. However, the figure illustrates that the males had more radioactivity with the TLC mobility of D H T acetate and estradiol17/3 diacetate present than did either group of females. Using a one way analysis of variance

6°°f

Nuclei

-t--~+-S.E.M.

40O .E

200

N

Whole "~-

~000

a

500

N

Group

M F I F2 TC DC EC

TLC zone

Te~os'l'erone

homogenates

nnBOn M FI F2 TC DC EC DI4T

M FI F2 TC DC EC Estrodiol

FIG. 1. Basal brain androgen metabolites. The amounts of radioactivity in the testosterone (acetate), DHT (acetate) and estradiol-17fl (diacetate) TLC zones from the basal brain samples following [3H]testosterone administration are presented. The groups M, F1, and F2 represent adult male rats, female rats of the same weight as the males and those of the same age, respectively. Groups TC, DC, and EC represent males pretreated with unlabeled testosterone, DHT, or estradiol-17fl 30 rain before [3H]testosterone administration. for D H T acetate, F ( 2 , 6 ) = 9.14, p < 0.05; and for estradiol-17fl diacetate, F ( 2 , 6 ) = 7.95, p < 0.05. The Newman-Keuls ranking procedure with qo.os(r,6) demonstrated that the amount of radioactivity was greater in the males than in the females for both compounds. A similar pattern was seen in the whole homogenate samples. Figure 1 and Table I indicate that D H T acetate and estradiol-17fl diacetate appeared to show sex differences similar to those seen in the nuclear fraction. The difference was significant for D H T a c e t a t e (F(2,6) = 6.95, p < 0.05) but not for estradiol-17fl diacetate. As with the nuclear samples, the whole homogenate samples showed no difference between males and females in the amount of testosterone acetate present. The ratio of metabolite in the nuclear fraction to that in the whole homogenate in the basal brain samples did not display sex differences for any of the three steroids. This can be seen in Table II. These ratios also indicate that estradiol-17fi was the only one of the compounds that concentrated in the brain nuclei to a greater extent than in the whole homogenate samples. The cortex generally contained less metabolite than the basal brain. This can be most clearly seen in the ratio of radioactivity for each metabolite found in the basal brain nuclei

DONALD L. REZEK

176

TABLE I. DISTRIBUTION OF RADIOACTIVITY(DPM/mg PROTEIN) IN MALEAND FEMALE NUCLEI AND WHOLEHOMOGENATES Nuclei Females--l*

Females--2

543 4- 60 206 4- 32 2180 4- 360

419 -4- 89 246 4- 11 3920 4- 1180

478 q- 20 326 4- 40 5220 4- 300

939 4- 114 705 4- 84 1850 4- 180

954 4- 231 1027 4- 225 2270 4- 326

636 4- 43 503 4- 24 2480 4- 240

96 4- 7 49 4- 5 310 4- 50

47 4- 5 11 ± 6 270 4- 82

70 ± 10 62 4- 13 452 4- 93

369 4- 39 254 q- 31 280 -4- 33

209 4- 45 216 ± 51 166 4- 33

168 4- 36 165 4- 33 255 4- 35

373 4- 45 69 4- 2 110 4- 30

152 4- 22 40 -4- 20 58 4- 14

216 4- 49 137 4- 291" 150 4- 23

25 4- 5 9 q- 1 110 4- 10

13 4- 2 14 4- 10 57 4- 32

Males Testosterone BBr Cx Pit DHT BBr Cx Pit Estradiol BBr Cx Pit

Whole homogenates Males Females--I Females--2

13 4- 2 7 4- 2 51 4- 7

Values represent mean 4- S.E.M. for the basal brain (BBr), cortex (Cx), and pituitary (Pit) samples. * Females--I = females that equal males' weights; Females--2 = females that equal males' ages. t Some values are somewhat higher than would be expected from work indicating no estradiol is produced in the cortex and pituitary or bound in the cortex. TABLE II. RATIOS OF METABOLITESIN THE NUCLEARFRACTIONTO THOSEIN THE WHOLE HOMOGENATEOF THE BASALBRAIN SAMPLES

Males Females*

Testosterone

DHT

Estradiol-17/3

0.58 4- 0.05 0.60 4- 0.08

0.26 4- 0.01 0.29 4- 0.03

15.89 4- 2.66 15.30 -t- 3.50

* Female groups 1 and 2 are combined for the ratios. TABLE III. NUCLEARBASALBRAINTO CEREBRALCORTEXRATIOS

Males Females

Testosterone

DHT

Estradiol-17/3

2.65 4- 0.15 1.62 4- 0.22

1.98 4- 0.18 1.85 4- 0.42

5.39 4- 0.46 5.55 4- 3.50

to that found in the cortex nuclei (see Table III). Only testosterone showed a consistently greater basal brain to cortex ratio in males when compared to females. Very little difference was present in the basal brain to cortex ratios in the whole homogenate samples. Unlike the brain, in the pituitary more radioactivity representing testosterone and DHT was found in the nuclei than in the whole homogenate (Table I). The effect was greater in females than in males. In males the ratios were 1.21 4- 0.26 for testosterone and 1.13 ~ 0.20 for DHT. For the females the ratios were 1.99 ± 0.31 and 1.80 :~ 0.31, respectively. Differences were found in the way the three unlabeled steroids blocked the uptake of the radioactivity in the second part of the study. This is illustrated in Fig. 1. Testosterone was

ANDROGEN METABOLITESIN BASALBRAIN CELL NUCLEI

177

most effective in inhibiting the concentration of radioactivity in the nuclear fraction since it decreased the amount of radioactivity in the TLC zones corresponding to all three hormones studied. DHT was effective in blocking the radioactivity from the nuclear testosterone and DHT acetate zones. In the basal brain and pituitary DHT was more effective in inhibiting the binding of testosterone than was unlabeled testosterone. Analyses of variance demonstrated that the differences were significant; F~3,8) : 37.4, p < 0.001 for the basal brain and F~3,8) : 18.4, p < 0.001 for the pituitary. The Newman-Keuls procedure showed that the males that had not been injected with unlabeled hormone had more radioactivity in the testosterone zone than did the animals pretreated with 200/zg of the same hormone which in turn had more than those pretreated with 200/zg of DHT. Estradiol-17/3 was effective only in blocking radioactivity with the TLC mobility of estradiol-17/3 diacetate from the nuclear extracts of the basal brain samples. These effects can be seen in Fig. 1. Unlike the nuclear samples, in the whole homogenates pretreatment with testosterone or DHT seemed to increase rather than decrease the amount of these compounds present. However, the effect was not significant. The levels of these compounds were also higher in the systemic blood from animals for which plasma samples were obtained. The radioactivity with the mobility of estradiol-17/3 diacetate in the whole homogenate paralleled that seen in the nuclear fraction but at a much lower level. DISCUSSION One half hour following the injection of radiolabeled testosterone male rat basal brain cell nuclei contain higher levels of DHT and estradiol-17/3 than do female nuclei. This finding is consistent with earlier findings (Naftolin et al., 1972; Denef et al., 1973) which showed that neural tissue from male and female rats differ in the amount of reduced and aromatized products they produce from androgen. Although there were differences in the amount of DHT and estradiol-1713 in the nuclei, there were no sex differences in the ratio of the amount of metabolite in the nuclei to that in the whole homogenate. This implies the difference between males and females is not due to a greater ability of male basal brain cells to concentrate the steroid in the cell nuclei. If male and female rats differ in their response to androgens in the display of male sexual behavior as is generally believed, the phenomenon may be mediated by differences in the way the animals metabolize the hormone and not in the manner in which the metabolite is taken up into the nucleus. Since the completion of this study Lieberburg & McEwen (1975b) have reported finding no major sex differences in the amount of DHT and estradiol-17/3 in brain cell nuclei of rats. Earlier Lieberburg & McEwen (1975a) reported finding less estradiol-17/3 in cell nuclei from the cortex and pituitary than was found in the present study. Because they injected the [all]testosterone into gonadectomized and adrenalectomized animals and killed the animals 2 hr later, the procedural dissimilarities may explain the different results. The different results emphasize the limitations of the method of using pulse injections of radioactive tracers. Factors such as the length of time between the injection and the killing of the animal sometimes may influence the interpretation of the findings. The ability of the unlabeled hormones to block the localization of testosterone and its metabolites in the basal brain cell nuclei demonstrated a selective and saturable uptake system. The presence of higher levels of radioactive testosterone and DHT in the whole

178

DONALD L. REZEK

h o m o g e n a t e samples f r o m a n i m a l s p r e t r e a t e d with u n l a b e l e d testosterone a n d D H T at first a p p e a r s to c o n t r a d i c t this idea. The finding is n o t the same as t h a t o f Stern & Eisenfeld (1971) o r Naess & A t t r a m a d a l (1974), w h o f o u n d a significant i n h i b i t i o n at the whole tissue level. I n those experiments the c o m p e t i n g u n l a b e l e d h o r m o n e was injected m u c h closer to the time o f the tracer h o r m o n e injection. In the present study the higher levels in the b a s a l b r a i n were paralleled in the cortical samples a n d also in the systemic b l o o d . T h u s it is likely t h a t the higher levels in the whole h o m o g e n a t e were due to a slower clearance o f the m e t a b o l i t e s f r o m the b l o o d . T h e ability o f estradiol-17fl to be c o n c e n t r a t e d in the nuclei o f the h y p o t h a l a m i c cells a n d the specificity o f the u n l a b e l e d estradiol-17fl in b l o c k i n g its u p t a k e indicate that the m e t a b o l i s m o f testosterone to estradiol-17fl m a y be functionally significant. T h e a p p a r e n t s u p e r i o r i t y o f D H T in i n h i b i t i n g the u p t a k e o f testosterone into nuclei m a y indicate t h a t u n d e r p h y s i o l o g i c a l c o n d i t i o n s D H T is able to c o m p e t e m o r e successfully t h a n testosterone for r e c e p t o r sites in the nucleus. These factors i m p l y t h a t estradiol-17/3 a n d D H T are p r o b a b l e active m e t a b o l i t e s o f testosterone on the nuclear level in the brain. This research was supported by grant HD-00893 to Richard E. Whalen from the National Institute of Child Health and Human Development. The author is grateful for Dr. Whalen's comments and suggestions on preliminary drafts of this paper. This paper is based on a portion of the research done by the author in partial fulfillment of the requirements for the Ph.D. degree at UC-Irvine. REFERENCES ]BAUM,M. J. & VREEBURG,J. T. M. (1973) Copulation in castrated male rats following combined treatment with estradiol and dihydrotestosterone. Science 182, 283-285. DENEF, C., MAGNUS, C. & MCEWEN, B. S. (1973) Sex differences and hormonal control of testosterone metabolism in rat pituitary and brain. J. Endocr. 59, 605-621. LARSSON,K., S()DERSTEN,P. & BEYER, C. (1973) Induction of male sexual behavior by oestradiol benzoate in combination with dihydrotestosterone. J. Endocr. 57, 563-564. LIEBERBURG, I. & McEWEN, B. S. (1975a) Estradiol-17fl: a metabolite of testosterone recovered in cell nuclei from limbic areas of adult male rat brains. Brain Res. 91, 171-174. LIEBERBURG,I. & MCEWEN,B. S. (1975b) Estradiol-17fl and 5~-dihydrotestosterone: testosterone metabolites recovered in cell nuclei from adult rat brains. Neurosci. Abst. 1,455. LOWRY, O. H., ROSEBROUGH,N. J., FARR,A. L. & RANDALL,R. J. (1951) Protein measurement with Folin phenol reagent. J. biol. Chem. 193, 265-275. MCEWEN, B. S. & ZIGMOND,R. E. (1972) Isolation of brain cell nuclei. In Research Methods in Neurochemistry, N. Marks and R. Rodnight (Eds.), pp. 139-161. Plenum, New York. MILLER,G. L. (1959) Protein determination for large numbers of samples. Analyt. Chem. 31, 954. NAESS, O. & ATI'RAMADAL,A. (1974) Uptake and binding of androgens by the anterior pituitary gland, hypothalamus, preoptic area, and brain cortex of rats. Acta endocr., Copenh. 76, 417-430. NAFTOLIN, F., RYAN, K. J. & PETRO, Z. (1972) Aromatization of androstenedione by the anterior hypothalamus of adult male and female rats. Endocrinology 90, 295-298. REZEK, D. L. & WHALEN,R. E. (1975) Localization of intravenously administered [all]testosterone and its metabolites in the brain of the male rat: the absence of a major effect related to the time of day of the injection. 3. Steroid Biochem. 6, 1193-1199. STERN, J. M. & EISENFELD,A. J. (1971) Distribution and metabolism of all-testosterone in castrated male rats: effects of cyperterone, progesterone, and unlabeled testosterone. Endocrinology 88, 1117-1125.