Brain Research, 119 (1977) 375-388
375
© Elsevier/North-HollandBiomedicalPress, Amsterdam- Printed in The Netherlands
NUCLEAR BINDING OF THE OESTROGEN RECEPTOR OF NEONATAL RAT BRAIN AFTER INJECTION OF OESTROGENS AND ANDROGENS; LOCALIZATION AND SEX DIFFERENCES
B. R. WESTLEY and D. F. SALAMAN Department of Anatomy, Medical School, University of Bristol, Bristol BS8 1TD (Great Britain)
(Accepted May 5th, 1976)
SUMMARY We have investigated the nuclear binding of [aH]oestradiol in the neonatal rat brain. The nuclear bound receptor, defined as the diethylstilboestrol (DES) suppressed nuclear bound radioactivity, showed similar sedimentation properties and time course to nuclear bound steroid receptors in other target tissues. After intracerebral injection we found that nuclear binding of [aH]oestradiol was localized to the hypothalamic/ amygdaloid region with relatively little binding in cortical and cerebellar regions. After subcutaneous injection there was less difference between the hypothalamic and cortical regions, though there was still very low binding in the cerebellum. A deficit in nuclear binding of [aH]oestradiol was found in the hypothalamic amygdaloid region of the male compared to the female, which was dependent on the presence of the neonatal testis. We also found that unlabelled testosterone reduced the nuclear binding of [aH]oestradiol when given 3 h before the radioactive steroid, and this reduction was seen predominantly in the hypothalamic/amygdaloid region. The non-aromatizable androgen, 5a-dihydrotestosterone, was without effect on [aH]oestradiol binding. After injection of [all]testosterone, receptor bound radioactivity was found exclusively in nuclei from the hypothalamic/amygdaloidregion, this radioactivity was competed out by DES and testosterone but not by dihydrotestosterone. This study provides evidence that neurally aromatized androgen may bind to an oestrogen receptor in the neonatal hypothalamic/amygdaloid region and effect its translocation into the cell nucleus.
INTRODUCTION Testicular steroids are known to play an essential role in the neonatal rat in determining the sensitivity of the hypothalamo-pituitary axis of the adult to circulat-
376 ing oestrogen and progesterone ,~. We are interested in the mechanism of this permanent differentiation of the brain, and are currently investigating the steroid receptors of the neonatal brain to see whether they have similar characteristics to those of other steroid sensitive tissues. The effects of steroids are known to depend on the presence of receptor proteins which only interact with nuclear chromatin when complexed with the steroid molecule and this interaction is thought to be critical in producing the biological effects of the hormone 16. Early studies suggested the active steroid in the differentiation of the brain was androgen, and testosterone propionate is capable of inducing masculinization when given to neonatal female ratsL More recent evidence has suggested that the androgens are first converted to oestrogens by aromatizing enzymes in the brain and that oestrogen is the biologically active agentlL In support of this mechanism only those androgens which are aromatizable are found to be effective 13 and certain synthetic oestrogens have been shown to be about 1000 times more potent than testosterone propionate 7. From the above it has become apparent that the steroid receptor most likely to be involved in the masculinization would have a high affinity for oestrogens. A large amount of an oestrogen binding protein is indeed present in the neonatal brain, but it differs in many respects from the oestrogen receptor (affinity, intracellular localization, and sedimentation properties)IS, 23. Recently high affinity oestrogen binding activity typical of oestrogen receptors has been demonstrated in cytosol prepared from neonatal brains 1 using methods capable of detecting small amounts of high affinity receptor binding (Ks ~ 10-1° M) in the presence of a large excess of lower affinity bindings. In a subsequent study it was concluded that this putative oestrogen receptor has very similar properties to the previously known oestrogen receptor of the adult female hypothalamusz4. In addition, nuclear uptake of oestrogen and androgen has been demonstrated autoradiographically in the neonatal brain 14,20,2t. In this study we describe the movement of this receptor to the nucleus in vivo in various regions of the brain of both sexes, the sedimentation properties of the steroid receptor complex after extraction from the nucleus and the results of some competition experiments which we believe help to elucidate the role of the oestrogen receptor in the androgen-induced sexual differentiation of the brain. METHODS Animals Male and female 4-day-old Wistar rats (day of birth ~- 0) from our inbred strain were used throughout this study. Neonatal males were castrated within 36 h of birth and used at 4 days of age. Chemicals [2,4,6,7(n)-3H]Oestradiol (specific activity 95 Ci/mrnole), [7(n)-aH]testosterone (specific activity 12.4 Ci/rnmole) and [l,2,4,5,6,7(n)-aH]5a-dihydrotestosterone (specific activity 130 Ci/mmole) were obtained from The Radiochemical Centre,
377 Amersham, Bucks., G.B. The radioactive steroids were stored in benzene-ethanol, 95:5. The solvent was removed under a stream of nitrogen and the steroid then taken up in 0.9 ~ saline. Unlabelled diethylstilboestrol (DES), testosterone and dihydrotestosterone were obtained from Sigma (London) Chemical Co. Ltd., Kingston-UponThames, Surrey, G,B. A stock solution of DES (2 × 10-3 M) in ethanol was diluted directly to the required concentration in buffer. Testosterone and dihydrotestosterone were dissolved in arachis oil (1 mg/ml). All other chemicals were Analar grade and solutions were made up in glass distilled water.
Nuclear uptake of radioactivity Animals were injected intracerebrally, using the method of Barnea and Lindner 3, with [3H]oestradiol (2.5 #Ci, 26.3 pmol) in saline (10 #1), with or without DES (526 pmol). For subcutaneous injection, animals were injected with 20/~Ci of radioactive steroid (211 pmol [3H]oestradiol, 154 pmol [3H]dihydrotestosterone, 1613 pmol [3H]testosterone) in saline (50/tl). [3H]Oestradiol was injected 1 h, and [3HItestosterone and [SH]dihydrotestosterone 2 h prior to killing. DES (4200 pmol) in saline (50 #1) was injected subcutaneously in competition experiments simultaneously with the radioactive steroid. Testosterone and dihydrotestosterone were used in competition experiments at a dose of 50 #g (174 nmol) per animal and were given 3 h before the radioactive steroid. Radioactivity was measured in the initial homogenate and in purified nuclear fractions prepared using the method of Plapinger and McEwen 17. In preliminary time course experiments nuclei were prepared from the whole cerebrum, i.e. all brain tissue anterior to the mammillary bodies. In all subsequent experiments the brain was divided into 4 parts, 'ANT CORT' including all brain anterior to the optic chiasm, 'CEREB' including all brain posterior to the mammillary bodies, 'SUP CORT' consisting of the dorsal half of the remaining cerebral tissue and 'HYP' the ventral half containing the hypothalamus and amygdalae. In localization experiments, where total levels of radioactivity in nuclei were measured, nuclei were prepared from pooled tissues from 4 animals. In experiments where radioactivity was extracted from nuclei for analysis on sucrose gradients, nuclei were prepared from pooled tissues from 8 animals.
Extraction of nuclear radioactivity Nuclei were extracted using KC1 (0.3 ml of 0.4 M, pH 8.) for 1 h at 0 °C and were sonicated in ice at times 0 and 30 min for 5 sec with a Dawe soniprobe, with microtip, set at 30 W. The extracts were then centrifuged for 30 min at 2° C and 100,000 × g in an MSE 10 × 10 ml angle rotor. The supernatant (200 #1) was layered onto 4.5 ml 7-30~ sucrose gradients containing 0.4 M KCI and 10 mM sodium phosphate (pH 7.3). Sucrose gradients were centrifuged for 16 h at 2 °C and 300,000 × g in an MSE 6 × 5 ml swing-out rotor using bovine serum albumin (BSA, 4.6S) on a parallel gradient as a sedimentation marker.
Measurement of radioactivity Samples to be counted were collected in polypropylene vials and extracted over-
378
night with 10 ml of scintillation fluid (PPO, 4 g/l, POPOP, 0.1 g/l in toluene) before counting with an efficiency of approximately 50 ~ in a Beckmann model LS100 liquid scintillation counter. D N A was measured in the nuclear preparations by the method of Burton 6. Statistical analysis was by Student's t-test. RESULTS
To determine the optimum time after injection for the study of nuclear binding, we initially looked at the nuclear accumulation of radioactivity in the whole cerebrum of neonatal rats at various times after intracerebral injection of radioactive oestradiol. a JNITIAL HOMOGENATF
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Fig. 1. Time course of whole tissue and nuclear accumulation of [aH]oestradiol in cerebrum of (a) female and (b) male neonatal rats. Animals were injected intracerebrally with 26.3 pmol (2.5 #Ci) [aH]oestradiol (11), or 26.3 pmol (2.5 #Ci) [aH]oestradiol together with 526 pmol DES (A). Radioactivity was measured in the initial homogenate of cerebral tissue and in the purified nuclear fraction prepared as in the Methods. ( 0 ) represents the DES-suppressed radioactivity in the nuclear fraction. Bars indicate S.E.M. limits where greater than the size of the symbol.
379 Fig. 1 shows the level of radioactivity in the initial homogenates and purified nuclear fractions after intracerebral injection of [aH]oestradiol alone or in the presence of a 20-fold molar excess of non-radioactive DES. The concentration of radioactivity in the homogenates declined from the earliest time of measurement (15 min) and there were no differences between the plus and minus DES injected groups. However in the purified nuclear fractions, DES-suppressible radioactivity was detected which was maximal 0.5-1 h after injection and declined thereafter in both males and females. Fig. 2 shows the sedimentation profiles of 0.4 M KCI extracts of purified nuclei 1 h after injection. All of the DES-suppressed radioactivity sedimented at 4.5S, a value typical of nuclear bound steroid receptors on sucrose gradients containing 0.4 M KCI. The DES-suppressed radioactivity on sucrose gradients, expressed per unit of DNA extracted, was higher in each case in females than in males. The values were, female 0.53 ~z 0.036 disint./min/#g DNA and male 0.34 ± 0.030 disint./min//~g DNA (P < 0.02). We then looked at the localization of nuclear bound radioactivity, using the dissection described in the Methods, 1 h after the intracerebral injection of [3H]oestradiol plus and minus DES. Although the levels of radioactivity in the homogenates were similar in each area, the majority of the nuclear bound, DES-suppressed radioactivity was found in the 'HYP' region (,,~5 times more than in the other regions). There was also more DES-suppressed radioactivity (P < 0.02) in the female than in the male 'HYP' (Fig. 3a and b) but there were no differences in the other 3 regions. To test whether this difference was due to the neonatal testis, groups of males were castrated within 36 h of birth. In these animals DES-suppressed radioactivity in the 'HYP' region was equivalent to that seen in normal females and significantly greater (P ~ 0.02) than in normal and sham-operated males (Fig. 3c and d). The cytoplasmic oestradiol receptor of the neonatal brain is found in equal concentration throughout the forebrain of both male and femaleI and we therefore
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375
© Elsevier/North-HollandBiomedicalPress, Amsterdam- Printed in The Netherlands
NUCLEAR BINDING OF THE OESTROGEN RECEPTOR OF NEONATAL RAT BRAIN AFTER INJECTION OF OESTROGENS AND ANDROGENS; LOCALIZATION AND SEX DIFFERENCES
B. R. WESTLEY and D. F. SALAMAN Department of Anatomy, Medical School, University of Bristol, Bristol BS8 1TD (Great Britain)
(Accepted May 5th, 1976)
SUMMARY We have investigated the nuclear binding of [aH]oestradiol in the neonatal rat brain. The nuclear bound receptor, defined as the diethylstilboestrol (DES) suppressed nuclear bound radioactivity, showed similar sedimentation properties and time course to nuclear bound steroid receptors in other target tissues. After intracerebral injection we found that nuclear binding of [aH]oestradiol was localized to the hypothalamic/ amygdaloid region with relatively little binding in cortical and cerebellar regions. After subcutaneous injection there was less difference between the hypothalamic and cortical regions, though there was still very low binding in the cerebellum. A deficit in nuclear binding of [aH]oestradiol was found in the hypothalamic amygdaloid region of the male compared to the female, which was dependent on the presence of the neonatal testis. We also found that unlabelled testosterone reduced the nuclear binding of [aH]oestradiol when given 3 h before the radioactive steroid, and this reduction was seen predominantly in the hypothalamic/amygdaloid region. The non-aromatizable androgen, 5a-dihydrotestosterone, was without effect on [aH]oestradiol binding. After injection of [all]testosterone, receptor bound radioactivity was found exclusively in nuclei from the hypothalamic/amygdaloidregion, this radioactivity was competed out by DES and testosterone but not by dihydrotestosterone. This study provides evidence that neurally aromatized androgen may bind to an oestrogen receptor in the neonatal hypothalamic/amygdaloid region and effect its translocation into the cell nucleus.
INTRODUCTION Testicular steroids are known to play an essential role in the neonatal rat in determining the sensitivity of the hypothalamo-pituitary axis of the adult to circulat-
376 ing oestrogen and progesterone ,~. We are interested in the mechanism of this permanent differentiation of the brain, and are currently investigating the steroid receptors of the neonatal brain to see whether they have similar characteristics to those of other steroid sensitive tissues. The effects of steroids are known to depend on the presence of receptor proteins which only interact with nuclear chromatin when complexed with the steroid molecule and this interaction is thought to be critical in producing the biological effects of the hormone 16. Early studies suggested the active steroid in the differentiation of the brain was androgen, and testosterone propionate is capable of inducing masculinization when given to neonatal female ratsL More recent evidence has suggested that the androgens are first converted to oestrogens by aromatizing enzymes in the brain and that oestrogen is the biologically active agentlL In support of this mechanism only those androgens which are aromatizable are found to be effective 13 and certain synthetic oestrogens have been shown to be about 1000 times more potent than testosterone propionate 7. From the above it has become apparent that the steroid receptor most likely to be involved in the masculinization would have a high affinity for oestrogens. A large amount of an oestrogen binding protein is indeed present in the neonatal brain, but it differs in many respects from the oestrogen receptor (affinity, intracellular localization, and sedimentation properties)IS, 23. Recently high affinity oestrogen binding activity typical of oestrogen receptors has been demonstrated in cytosol prepared from neonatal brains 1 using methods capable of detecting small amounts of high affinity receptor binding (Ks ~ 10-1° M) in the presence of a large excess of lower affinity bindings. In a subsequent study it was concluded that this putative oestrogen receptor has very similar properties to the previously known oestrogen receptor of the adult female hypothalamusz4. In addition, nuclear uptake of oestrogen and androgen has been demonstrated autoradiographically in the neonatal brain 14,20,2t. In this study we describe the movement of this receptor to the nucleus in vivo in various regions of the brain of both sexes, the sedimentation properties of the steroid receptor complex after extraction from the nucleus and the results of some competition experiments which we believe help to elucidate the role of the oestrogen receptor in the androgen-induced sexual differentiation of the brain. METHODS Animals Male and female 4-day-old Wistar rats (day of birth ~- 0) from our inbred strain were used throughout this study. Neonatal males were castrated within 36 h of birth and used at 4 days of age. Chemicals [2,4,6,7(n)-3H]Oestradiol (specific activity 95 Ci/mrnole), [7(n)-aH]testosterone (specific activity 12.4 Ci/rnmole) and [l,2,4,5,6,7(n)-aH]5a-dihydrotestosterone (specific activity 130 Ci/mmole) were obtained from The Radiochemical Centre,
377 Amersham, Bucks., G.B. The radioactive steroids were stored in benzene-ethanol, 95:5. The solvent was removed under a stream of nitrogen and the steroid then taken up in 0.9 ~ saline. Unlabelled diethylstilboestrol (DES), testosterone and dihydrotestosterone were obtained from Sigma (London) Chemical Co. Ltd., Kingston-UponThames, Surrey, G,B. A stock solution of DES (2 × 10-3 M) in ethanol was diluted directly to the required concentration in buffer. Testosterone and dihydrotestosterone were dissolved in arachis oil (1 mg/ml). All other chemicals were Analar grade and solutions were made up in glass distilled water.
Nuclear uptake of radioactivity Animals were injected intracerebrally, using the method of Barnea and Lindner 3, with [3H]oestradiol (2.5 #Ci, 26.3 pmol) in saline (10 #1), with or without DES (526 pmol). For subcutaneous injection, animals were injected with 20/~Ci of radioactive steroid (211 pmol [3H]oestradiol, 154 pmol [3H]dihydrotestosterone, 1613 pmol [3H]testosterone) in saline (50/tl). [3H]Oestradiol was injected 1 h, and [3HItestosterone and [SH]dihydrotestosterone 2 h prior to killing. DES (4200 pmol) in saline (50 #1) was injected subcutaneously in competition experiments simultaneously with the radioactive steroid. Testosterone and dihydrotestosterone were used in competition experiments at a dose of 50 #g (174 nmol) per animal and were given 3 h before the radioactive steroid. Radioactivity was measured in the initial homogenate and in purified nuclear fractions prepared using the method of Plapinger and McEwen 17. In preliminary time course experiments nuclei were prepared from the whole cerebrum, i.e. all brain tissue anterior to the mammillary bodies. In all subsequent experiments the brain was divided into 4 parts, 'ANT CORT' including all brain anterior to the optic chiasm, 'CEREB' including all brain posterior to the mammillary bodies, 'SUP CORT' consisting of the dorsal half of the remaining cerebral tissue and 'HYP' the ventral half containing the hypothalamus and amygdalae. In localization experiments, where total levels of radioactivity in nuclei were measured, nuclei were prepared from pooled tissues from 4 animals. In experiments where radioactivity was extracted from nuclei for analysis on sucrose gradients, nuclei were prepared from pooled tissues from 8 animals.
Extraction of nuclear radioactivity Nuclei were extracted using KC1 (0.3 ml of 0.4 M, pH 8.) for 1 h at 0 °C and were sonicated in ice at times 0 and 30 min for 5 sec with a Dawe soniprobe, with microtip, set at 30 W. The extracts were then centrifuged for 30 min at 2° C and 100,000 × g in an MSE 10 × 10 ml angle rotor. The supernatant (200 #1) was layered onto 4.5 ml 7-30~ sucrose gradients containing 0.4 M KCI and 10 mM sodium phosphate (pH 7.3). Sucrose gradients were centrifuged for 16 h at 2 °C and 300,000 × g in an MSE 6 × 5 ml swing-out rotor using bovine serum albumin (BSA, 4.6S) on a parallel gradient as a sedimentation marker.
Measurement of radioactivity Samples to be counted were collected in polypropylene vials and extracted over-
378
night with 10 ml of scintillation fluid (PPO, 4 g/l, POPOP, 0.1 g/l in toluene) before counting with an efficiency of approximately 50 ~ in a Beckmann model LS100 liquid scintillation counter. D N A was measured in the nuclear preparations by the method of Burton 6. Statistical analysis was by Student's t-test. RESULTS
To determine the optimum time after injection for the study of nuclear binding, we initially looked at the nuclear accumulation of radioactivity in the whole cerebrum of neonatal rats at various times after intracerebral injection of radioactive oestradiol. a JNITIAL HOMOGENATF
E
NUCLEAR PELLET DIFFERENCE
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.
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.
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I ~ - ~ . . . . i e~
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Fig. 1. Time course of whole tissue and nuclear accumulation of [aH]oestradiol in cerebrum of (a) female and (b) male neonatal rats. Animals were injected intracerebrally with 26.3 pmol (2.5 #Ci) [aH]oestradiol (11), or 26.3 pmol (2.5 #Ci) [aH]oestradiol together with 526 pmol DES (A). Radioactivity was measured in the initial homogenate of cerebral tissue and in the purified nuclear fraction prepared as in the Methods. ( 0 ) represents the DES-suppressed radioactivity in the nuclear fraction. Bars indicate S.E.M. limits where greater than the size of the symbol.
379 Fig. 1 shows the level of radioactivity in the initial homogenates and purified nuclear fractions after intracerebral injection of [aH]oestradiol alone or in the presence of a 20-fold molar excess of non-radioactive DES. The concentration of radioactivity in the homogenates declined from the earliest time of measurement (15 min) and there were no differences between the plus and minus DES injected groups. However in the purified nuclear fractions, DES-suppressible radioactivity was detected which was maximal 0.5-1 h after injection and declined thereafter in both males and females. Fig. 2 shows the sedimentation profiles of 0.4 M KCI extracts of purified nuclei 1 h after injection. All of the DES-suppressed radioactivity sedimented at 4.5S, a value typical of nuclear bound steroid receptors on sucrose gradients containing 0.4 M KCI. The DES-suppressed radioactivity on sucrose gradients, expressed per unit of DNA extracted, was higher in each case in females than in males. The values were, female 0.53 ~z 0.036 disint./min/#g DNA and male 0.34 ± 0.030 disint./min//~g DNA (P < 0.02). We then looked at the localization of nuclear bound radioactivity, using the dissection described in the Methods, 1 h after the intracerebral injection of [3H]oestradiol plus and minus DES. Although the levels of radioactivity in the homogenates were similar in each area, the majority of the nuclear bound, DES-suppressed radioactivity was found in the 'HYP' region (,,~5 times more than in the other regions). There was also more DES-suppressed radioactivity (P < 0.02) in the female than in the male 'HYP' (Fig. 3a and b) but there were no differences in the other 3 regions. To test whether this difference was due to the neonatal testis, groups of males were castrated within 36 h of birth. In these animals DES-suppressed radioactivity in the 'HYP' region was equivalent to that seen in normal females and significantly greater (P ~ 0.02) than in normal and sham-operated males (Fig. 3c and d). The cytoplasmic oestradiol receptor of the neonatal brain is found in equal concentration throughout the forebrain of both male and femaleI and we therefore
,•1001
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