Brain Research, 514 (1990) 327-333
327
Elsevier
BRES 15388
Immunocytochemical localization of aromatase in the brain Jacques Balthazart 1, Agnes Foidart 1 and Nobuhiro H a r a d a 2 1Laboratory of General and Comparative Biochemistry, University of Lidge, Liege (Belgium) and 2MolecularGenetics, School of Medicine, Fujita-Gakuen Health University, Toyoake, Aichi (Japan) (Accepted 19 September 1989)
Key words: Aromatase immunocytochemistry;Brain aromatase; Preoptic area; Sexually dimorphic nucleus; Japanese quail
An immunocytochemical peroxidase-antiperoxidase procedure using a purified polyclonal antibody raised against human placental aromatase was used to localize aromatase-containing cells in the Japanese quail brain. Immunoreactive cells were found only in the preoptic area and hypothalamus, with a high density of positive cells being present in the sexuallydimorphic medial preoptic nucleus, in the ventromedial nucleus of the hypothalamus and in the infundibulum. The positive material was localized in the perikarya and in adjacent cytoplasmic processes. Aromatase-containingcells were a specificmarker for the sexually dimorphicpreoptic nucleus. Treatment with testosterone produced a 6-fold increase in the aromatase activity of the preoptic area and a 4-fold increase in the number of immunoreactive cells in the medial preoptic nucleus. Thus, the increase in aromatase activity observed after testosterone administration is caused by a change in enzyme concentration.
INTRODUCTION Aromatase was identified in the vertebrate brain more than 15 years ago 21'22. It has since been demonstrated that this enzyme is present in the brain of all vertebrates and plays a critical role in the control of a number of reproductive processes especially in the sexual differentiation and activation of male copulatory behavior TM 12,18,19. The aromatase activity has been determined in microdissected brain nuclei by in vitro radioenzyme assays (measurement of product formation or of the release of tritiated water 26'31) but this leaves unanswered a number of questions concerning the cellular and subcellular distribution of the enzyme such as: is the enzyme present in neurons or in glial cells, is it exclusively microsomal or does it also occur at the synaptic terminals as recently suggested by two independent studies 2s'3s. In addition, the product formation assays cannot reveal what is the mechanism underlying the changes in enzyme activity related to the age, sex or hormonal state of the subjects26'3°'35"36: modification of the number of aromatase-containing cells, of the enzyme concentration within a constant number of cells or modulation of a constant amount of enzyme by specific inhibitors 3°'32"36'37. In the quail and dove, detailed kinetic experiments have shown that the variations in aromatase activity associated with the age and sex of the subjects or with their treatment by testosterone (T) are not related
to a change in the enzyme affinity for the substrate (Kin) suggesting that the modifications of enzyme activity reflect changes in enzyme concentration 3°'32'36"37. However, this type of experiment does not rule out a regulation that would be based on the synthesis/repression of a non-competitive inhibitor (this would also affect the maximum velocity but not the K m of the enzyme). A detection of aromatase by immunocytochemistry (ICC) would provide answers to these questions. Purifications of placental aromatase have been reported and purified antigens have been used to generate polyclonal or monoclonal antibodies 9'14'2°. These were recently used to establish an immunological assay (ELISA) of the aromatase 15 but the sensitivity of the method would have to be increased by several orders of magnitude to be applied on brain samples. Using monoclonal or polyclonal anti-aromatase antibodies, aromatase has been localized in the human ovary and placenta 13'27, but so far this technique has never been applied to the brain of any species. We report here a successful immunocytochemical localization of aromatase in the brain of the Japanese quail. The aromatase ICC demonstrates that this enzyme is a specific marker for the sexually dimorphic T-sensitive nucleus 25"39 of the quail preoptic area (POA). In this nucleus, the induction of aromatase activity which is observed following T-treatment 7,3° is paralleled by an increase in the number of aromatase-positive neurons.
Correspondence: J. Balthazart, Universit6 de Liege, Biochimie G6n6rale et Compar6e, (Bat. L1), 17 place Delcour, B-4020 Liege, Belgium. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
328 T h i s d e m o n s t r a t e s f o r t h e first t i m e t h a t t h e i n d u c t i o n o f aromatase
activity corresponds
to a n i n c r e a s e in t h e
a c t u a l c o n c e n t r a t i o n o f t h e e n z y m e in t h e P O A . MATERIALS AND METHODS
Animals Male japanese quail (Coturnix coturnix japonica) were obtained from a local breeder at the age of 3 weeks. Throughout their life at the breeding colony and in the laboratory, birds were exposed to a photoperiod of 16 h of light and 8 h of dark per day. From hatching to the start of the experiments, birds were raised in groups of both sexes. During the experiments, they were isolated in individual cages. Six of the birds were kept intact and studied by immunocytochemistry after they reached sexual maturity (7 weeks posthatch). Thirty other males were castrated at the age of 4 weeks under total anesthesia (Hypnodil, Janssen Pharmaceutica, Beerse, Belgium; 15 mg/kg i.m.) using procedures previously described 29. At the age of 7 weeks, some of the castrates (n = 19) were treated with testosterone (2 x 20 mm silastic implants) as previously described 29. Control castrated birds (n = 11) received empty silastic implants. Birds were killed 5 days after the implantation of the silastic capsules. Brains were immediately dissected and frozen on powdered dry ice. They were stored at -70 °C until used for immunocytoehemistry or radioenzyme assays.
Immunocytochemistry A standard peroxidase-antiperoxidase procedure using diaminobenzidine as the chromogen was used throughout. Brains were cut in 20 p m coronal sections on a cryostat. The sections were fixed for 2-3 h in 4% paraformaidehyde in PBST (0.01 M phosphate buffer, 0.125 M NaCI, 0.1% Triton X-100) at room temperature. Endogenous peroxidase was blocked by immersing the sections in a solution of 0.6% hydrogen peroxide in methanol for 20 rain. Sections were incubated overnight at 4 °C with the primary antibody diluted at 1/1000 in PBST. We used a polyclonal antibody raised in rabbit against human placental aromatase and purified by ammonium sulfate fractionation and affinity chromatography with antigenconjugated Sepharose 4B (ref. 14). This antibody appears to be monospecific as determined in classical biochemical and immunological tests TM. On the next day, sections were processed according to the peroxidase-antiperoxidase (PAP) technique. The goat anti-rabbit (dilution 1/60 for 30 rain) and PAP complex (1/300 for 30 min) were both diluted in PBST. Extensive rinses in PBST were made between each step. The peroxidase was finally revealed by immersing slides for 6 min in a solution of diaminobenzidine (DAB; 20 mg in 50 ml PBST containing 20 pl hydrogen peroxide at 30%). Controls included the omission of the primary, secondary or tertiary antibody and the use of a preimmune rabbit serum in place of the primary antibody. In addition, primary anti-aromatase antibody preabsorbed with an excess of purified human placental aromatase was used in one experiment. The specific preparation used in the present study showed about 90% purity judging from SDS-polyacrylamide gel electrophoresis (see ref. 14 for the preparation and characteristics of this preparation). To study the neuroanatomical distribution of the aromatase in intact sexually mature males (n = 6), 20/~m frozen sections were collected every 100 p m in the POA and every 300/~m in the rest of the hypothalamus. Alternate sections were also saved and stained with Toluidine blue to confirm neuroanatomical localizations. The neuroanatomical nomenclature used in this paper is based on previously published work on the quail and chicken brain s'16'17'39. The brain of two sexually mature male Wistar rats were also studied in an attempt to localize the aromatase by ICC in this species. Sections coming from areas known to contain the highest levels of aromatase activity, the preoptic area, the bed nucleus of the stria terminalis and the amygdala 26 were stained by the same immunocytochemical procedure.
In the castration and T-replacement experiment, for each bird, 8 consecutive sections in the POA located 100 p m apart were stained by ICC. Castrates (n = 5) and T-treated birds (n = 5) were processed in matched pairs to avoid confounding experimental effects with procedural differences. The immunocytochemical staining was performed as described above. In each bird, the numbers of labelled cells were counted in 4 sections located in the medial portion of POM in the rostral-caudal axis. The area of the POM in the adjacent Nissl-stained sections was measured with the help of a graphic tablet connected to a microcomputer 25'39.
Radioenzyme assay Aromatase activity was measured in homogenates prepared from pooled preoptic areas-hypothalami (POA-HYP) using an in vitro radioenzyme assay previously described for the quail brain 31'33. Briefly, the brain samples were homogenized at a concentration of 40 mg fresh weight/ml STMM buffer (0.25 M sucrose, 10 mM Tris-HC1, pH 7.4 at 20 °C, 5 mM MgCI2, 1 mM fl-mercaptoethanol) and the homogenates were immediately snap-frozen in an acetonedry ice bath. For the assay, 100-200/A of these homogenates were incubated in the presence of [1,2-a-3H]testosterone (Amersham, U.K.; specific activity = 54.1 Ci/mmol) at a final concentration of 10-100 nM depending on the experiment. At the start of the incubation, STMM buffer containing N A D P H 2 (final concentration = 1.2 mM) was added to the tubes and these were then maintained at 41 °C for 15 rain under constant agitation. The radioactive metabolites produced were then extracted by diethylether, estrogens and androgens were separated by phenolic partition and both fractions were purified by thin layer chromatography on silicagel plates 33. Three metabolites of T were quantified: estradiol (E2), 5ct-dihydrotestosterone (5a-DHT), and 5fl-dihydrotestosterone (5flDHT). This assay has been fully validated for the quail brain 31"33 and the identity and purity of the metabolites after chromatography have been confirmed by recrystallizations to constant specific activity or constant isotopic ratio 33. The protein content of the homogenates was determined by the method of Bradford 1° and the amounts of metabolites produced were expressed in fmol/mg protein/h. In one experiment, 200 pl aliquots of the homogenates prepared with the brains of castrated males (n = 6) or of T-treated males (n = 6) were incubated with increasing concentrations of radioactive testosterone (in the range 10-100 nM) in order to determine the effects of T on the maximum velocity of the aromatase. All assays were performed in duplicate. The production of E2 only was measured in this case. In the other experiment, I00 pl aliquots of the homogenates prepared with the brains of T-treated birds (n = 9) were incubated with radioactive testosterone (20 nM) and 50pl of anti-aromatase antibody at various dilutions (1/10-1/100 000 thus corresponding to amounts of pure antibody between 5 and 0.0005 #1) to determine whether the antibody prepared against human placental aromatase inhibited the quail brain aromatase. The production of E 2 as well as 5a-DHT and 5fl-DHT was quantified to evaluate the specificity of the inhibition. In this experiment, assays were performed in duplicate except for the control condition (no antibody) which was measured in quadruplicate. In a parallel and similar experiment, the anti-aromatase antibody was replaced by rabbit preimmune serum to test further the specificity of the inhibition of enzymatic activity produced by the antibody. RESULTS Radioenzyme
assay of quail hypothalamic
homoge-
n a t e s in t h e p r e s e n c e o f a n t i b o d y d i r e c t e d a g a i n s t h u m a n a r o m a t a s e c o n f i r m e d t h a t this a n t i b o d y w a s specifically i n t e r a c t i n g w i t h t h e q u a i l e n z y m e (Fig. 1). The
aromatase
activity
was
inhibited
in
a
dose-
dependent manner by the antibody (one way ANOYA: /75,13 = 77.67, P < 0.0001) a n d at t h e h i g h e s t c o n c e n -
329
Fig. 1. Inhibition of quail aromatase activity by a polyclonal anti-aromatase antibody. Aliquots of preoptic-hypothalamichomogenates (100/tl) were incubated with radioactive T (20 riM) and 50 /~l of anti-aromatase antibody at various dilutions (1/10-1/100 000 corresponding to 5-0.0005/~1 antibody). Concentration of the pure antibody was calculated to be I mg/ml by the method of Bradford TM. The activity of the 3 T-metabolizing enzymes has been expressed in percents of the non-inhibited controls to permit direct comparison (aromatase = 327 fmol/mg protein/h, 5a-reductase = 602 fmol/mg protein/h and 5fl-reductase = 6510 fmol/mg protein/h). Asterisks indicate significant difference by comparison with the non-inhibited control (P < 0.05 by the Fisher protected least-significant difference test following a significant ANOVA).
A O~ 100.
Q
.~:A~r-.
..,,S~-"~---°'--.
"~:'~,~"
~'~,,
....cJ5~-DHT "\ 5/3-DHT
80. 1>
327
Elsevier
BRES 15388
Immunocytochemical localization of aromatase in the brain Jacques Balthazart 1, Agnes Foidart 1 and Nobuhiro H a r a d a 2 1Laboratory of General and Comparative Biochemistry, University of Lidge, Liege (Belgium) and 2MolecularGenetics, School of Medicine, Fujita-Gakuen Health University, Toyoake, Aichi (Japan) (Accepted 19 September 1989)
Key words: Aromatase immunocytochemistry;Brain aromatase; Preoptic area; Sexually dimorphic nucleus; Japanese quail
An immunocytochemical peroxidase-antiperoxidase procedure using a purified polyclonal antibody raised against human placental aromatase was used to localize aromatase-containing cells in the Japanese quail brain. Immunoreactive cells were found only in the preoptic area and hypothalamus, with a high density of positive cells being present in the sexuallydimorphic medial preoptic nucleus, in the ventromedial nucleus of the hypothalamus and in the infundibulum. The positive material was localized in the perikarya and in adjacent cytoplasmic processes. Aromatase-containingcells were a specificmarker for the sexually dimorphicpreoptic nucleus. Treatment with testosterone produced a 6-fold increase in the aromatase activity of the preoptic area and a 4-fold increase in the number of immunoreactive cells in the medial preoptic nucleus. Thus, the increase in aromatase activity observed after testosterone administration is caused by a change in enzyme concentration.
INTRODUCTION Aromatase was identified in the vertebrate brain more than 15 years ago 21'22. It has since been demonstrated that this enzyme is present in the brain of all vertebrates and plays a critical role in the control of a number of reproductive processes especially in the sexual differentiation and activation of male copulatory behavior TM 12,18,19. The aromatase activity has been determined in microdissected brain nuclei by in vitro radioenzyme assays (measurement of product formation or of the release of tritiated water 26'31) but this leaves unanswered a number of questions concerning the cellular and subcellular distribution of the enzyme such as: is the enzyme present in neurons or in glial cells, is it exclusively microsomal or does it also occur at the synaptic terminals as recently suggested by two independent studies 2s'3s. In addition, the product formation assays cannot reveal what is the mechanism underlying the changes in enzyme activity related to the age, sex or hormonal state of the subjects26'3°'35"36: modification of the number of aromatase-containing cells, of the enzyme concentration within a constant number of cells or modulation of a constant amount of enzyme by specific inhibitors 3°'32"36'37. In the quail and dove, detailed kinetic experiments have shown that the variations in aromatase activity associated with the age and sex of the subjects or with their treatment by testosterone (T) are not related
to a change in the enzyme affinity for the substrate (Kin) suggesting that the modifications of enzyme activity reflect changes in enzyme concentration 3°'32'36"37. However, this type of experiment does not rule out a regulation that would be based on the synthesis/repression of a non-competitive inhibitor (this would also affect the maximum velocity but not the K m of the enzyme). A detection of aromatase by immunocytochemistry (ICC) would provide answers to these questions. Purifications of placental aromatase have been reported and purified antigens have been used to generate polyclonal or monoclonal antibodies 9'14'2°. These were recently used to establish an immunological assay (ELISA) of the aromatase 15 but the sensitivity of the method would have to be increased by several orders of magnitude to be applied on brain samples. Using monoclonal or polyclonal anti-aromatase antibodies, aromatase has been localized in the human ovary and placenta 13'27, but so far this technique has never been applied to the brain of any species. We report here a successful immunocytochemical localization of aromatase in the brain of the Japanese quail. The aromatase ICC demonstrates that this enzyme is a specific marker for the sexually dimorphic T-sensitive nucleus 25"39 of the quail preoptic area (POA). In this nucleus, the induction of aromatase activity which is observed following T-treatment 7,3° is paralleled by an increase in the number of aromatase-positive neurons.
Correspondence: J. Balthazart, Universit6 de Liege, Biochimie G6n6rale et Compar6e, (Bat. L1), 17 place Delcour, B-4020 Liege, Belgium. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
328 T h i s d e m o n s t r a t e s f o r t h e first t i m e t h a t t h e i n d u c t i o n o f aromatase
activity corresponds
to a n i n c r e a s e in t h e
a c t u a l c o n c e n t r a t i o n o f t h e e n z y m e in t h e P O A . MATERIALS AND METHODS
Animals Male japanese quail (Coturnix coturnix japonica) were obtained from a local breeder at the age of 3 weeks. Throughout their life at the breeding colony and in the laboratory, birds were exposed to a photoperiod of 16 h of light and 8 h of dark per day. From hatching to the start of the experiments, birds were raised in groups of both sexes. During the experiments, they were isolated in individual cages. Six of the birds were kept intact and studied by immunocytochemistry after they reached sexual maturity (7 weeks posthatch). Thirty other males were castrated at the age of 4 weeks under total anesthesia (Hypnodil, Janssen Pharmaceutica, Beerse, Belgium; 15 mg/kg i.m.) using procedures previously described 29. At the age of 7 weeks, some of the castrates (n = 19) were treated with testosterone (2 x 20 mm silastic implants) as previously described 29. Control castrated birds (n = 11) received empty silastic implants. Birds were killed 5 days after the implantation of the silastic capsules. Brains were immediately dissected and frozen on powdered dry ice. They were stored at -70 °C until used for immunocytoehemistry or radioenzyme assays.
Immunocytochemistry A standard peroxidase-antiperoxidase procedure using diaminobenzidine as the chromogen was used throughout. Brains were cut in 20 p m coronal sections on a cryostat. The sections were fixed for 2-3 h in 4% paraformaidehyde in PBST (0.01 M phosphate buffer, 0.125 M NaCI, 0.1% Triton X-100) at room temperature. Endogenous peroxidase was blocked by immersing the sections in a solution of 0.6% hydrogen peroxide in methanol for 20 rain. Sections were incubated overnight at 4 °C with the primary antibody diluted at 1/1000 in PBST. We used a polyclonal antibody raised in rabbit against human placental aromatase and purified by ammonium sulfate fractionation and affinity chromatography with antigenconjugated Sepharose 4B (ref. 14). This antibody appears to be monospecific as determined in classical biochemical and immunological tests TM. On the next day, sections were processed according to the peroxidase-antiperoxidase (PAP) technique. The goat anti-rabbit (dilution 1/60 for 30 rain) and PAP complex (1/300 for 30 min) were both diluted in PBST. Extensive rinses in PBST were made between each step. The peroxidase was finally revealed by immersing slides for 6 min in a solution of diaminobenzidine (DAB; 20 mg in 50 ml PBST containing 20 pl hydrogen peroxide at 30%). Controls included the omission of the primary, secondary or tertiary antibody and the use of a preimmune rabbit serum in place of the primary antibody. In addition, primary anti-aromatase antibody preabsorbed with an excess of purified human placental aromatase was used in one experiment. The specific preparation used in the present study showed about 90% purity judging from SDS-polyacrylamide gel electrophoresis (see ref. 14 for the preparation and characteristics of this preparation). To study the neuroanatomical distribution of the aromatase in intact sexually mature males (n = 6), 20/~m frozen sections were collected every 100 p m in the POA and every 300/~m in the rest of the hypothalamus. Alternate sections were also saved and stained with Toluidine blue to confirm neuroanatomical localizations. The neuroanatomical nomenclature used in this paper is based on previously published work on the quail and chicken brain s'16'17'39. The brain of two sexually mature male Wistar rats were also studied in an attempt to localize the aromatase by ICC in this species. Sections coming from areas known to contain the highest levels of aromatase activity, the preoptic area, the bed nucleus of the stria terminalis and the amygdala 26 were stained by the same immunocytochemical procedure.
In the castration and T-replacement experiment, for each bird, 8 consecutive sections in the POA located 100 p m apart were stained by ICC. Castrates (n = 5) and T-treated birds (n = 5) were processed in matched pairs to avoid confounding experimental effects with procedural differences. The immunocytochemical staining was performed as described above. In each bird, the numbers of labelled cells were counted in 4 sections located in the medial portion of POM in the rostral-caudal axis. The area of the POM in the adjacent Nissl-stained sections was measured with the help of a graphic tablet connected to a microcomputer 25'39.
Radioenzyme assay Aromatase activity was measured in homogenates prepared from pooled preoptic areas-hypothalami (POA-HYP) using an in vitro radioenzyme assay previously described for the quail brain 31'33. Briefly, the brain samples were homogenized at a concentration of 40 mg fresh weight/ml STMM buffer (0.25 M sucrose, 10 mM Tris-HC1, pH 7.4 at 20 °C, 5 mM MgCI2, 1 mM fl-mercaptoethanol) and the homogenates were immediately snap-frozen in an acetonedry ice bath. For the assay, 100-200/A of these homogenates were incubated in the presence of [1,2-a-3H]testosterone (Amersham, U.K.; specific activity = 54.1 Ci/mmol) at a final concentration of 10-100 nM depending on the experiment. At the start of the incubation, STMM buffer containing N A D P H 2 (final concentration = 1.2 mM) was added to the tubes and these were then maintained at 41 °C for 15 rain under constant agitation. The radioactive metabolites produced were then extracted by diethylether, estrogens and androgens were separated by phenolic partition and both fractions were purified by thin layer chromatography on silicagel plates 33. Three metabolites of T were quantified: estradiol (E2), 5ct-dihydrotestosterone (5a-DHT), and 5fl-dihydrotestosterone (5flDHT). This assay has been fully validated for the quail brain 31"33 and the identity and purity of the metabolites after chromatography have been confirmed by recrystallizations to constant specific activity or constant isotopic ratio 33. The protein content of the homogenates was determined by the method of Bradford 1° and the amounts of metabolites produced were expressed in fmol/mg protein/h. In one experiment, 200 pl aliquots of the homogenates prepared with the brains of castrated males (n = 6) or of T-treated males (n = 6) were incubated with increasing concentrations of radioactive testosterone (in the range 10-100 nM) in order to determine the effects of T on the maximum velocity of the aromatase. All assays were performed in duplicate. The production of E2 only was measured in this case. In the other experiment, I00 pl aliquots of the homogenates prepared with the brains of T-treated birds (n = 9) were incubated with radioactive testosterone (20 nM) and 50pl of anti-aromatase antibody at various dilutions (1/10-1/100 000 thus corresponding to amounts of pure antibody between 5 and 0.0005 #1) to determine whether the antibody prepared against human placental aromatase inhibited the quail brain aromatase. The production of E 2 as well as 5a-DHT and 5fl-DHT was quantified to evaluate the specificity of the inhibition. In this experiment, assays were performed in duplicate except for the control condition (no antibody) which was measured in quadruplicate. In a parallel and similar experiment, the anti-aromatase antibody was replaced by rabbit preimmune serum to test further the specificity of the inhibition of enzymatic activity produced by the antibody. RESULTS Radioenzyme
assay of quail hypothalamic
homoge-
n a t e s in t h e p r e s e n c e o f a n t i b o d y d i r e c t e d a g a i n s t h u m a n a r o m a t a s e c o n f i r m e d t h a t this a n t i b o d y w a s specifically i n t e r a c t i n g w i t h t h e q u a i l e n z y m e (Fig. 1). The
aromatase
activity
was
inhibited
in
a
dose-
dependent manner by the antibody (one way ANOYA: /75,13 = 77.67, P < 0.0001) a n d at t h e h i g h e s t c o n c e n -
329
Fig. 1. Inhibition of quail aromatase activity by a polyclonal anti-aromatase antibody. Aliquots of preoptic-hypothalamichomogenates (100/tl) were incubated with radioactive T (20 riM) and 50 /~l of anti-aromatase antibody at various dilutions (1/10-1/100 000 corresponding to 5-0.0005/~1 antibody). Concentration of the pure antibody was calculated to be I mg/ml by the method of Bradford TM. The activity of the 3 T-metabolizing enzymes has been expressed in percents of the non-inhibited controls to permit direct comparison (aromatase = 327 fmol/mg protein/h, 5a-reductase = 602 fmol/mg protein/h and 5fl-reductase = 6510 fmol/mg protein/h). Asterisks indicate significant difference by comparison with the non-inhibited control (P < 0.05 by the Fisher protected least-significant difference test following a significant ANOVA).
A O~ 100.
Q
.~:A~r-.
..,,S~-"~---°'--.
"~:'~,~"
~'~,,
....cJ5~-DHT "\ 5/3-DHT
80. 1>
