Brah~ Research, 589 (1992) 327-332 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
327
BRES 18029
Aromatase activity in cultured brain cells: difference between neurons and glia Paola Negri Cesi, Roberto
Cosimo
Melcangi,
Fabio Celotti and Luciano
Martini
Departnlent of Endocrinology. Unh'ersity of Milan, Milan (Italy) (Accepted 31 March 1992)
Key words: Aromatase; Androgen; Neuronal cell; Glial cell: Rat; Culture
At the level of the central nervous systeln (CNS) of several mammalian and non-mammalian species, estrogens may be intracellularly formed from circulating androgens through the action of the aromatase complex. Estrogenic steroids play a crucial role in organizing and directing certain behavioral and neuroendocrine responses both during the fetal/neonatal life and in adulthood. Biochemical and immunocytochemical studies have shown that the aromatase is particularly concentrated in CNS areas involved in the control of reproductive functions, such as the hypoth~lamus, the preoptic area and the limbic system: despite this large body of evidence, the exact cellular localization of this enzymatic complex within the different cell populations of the brain is still uncertain, in the experiments described here, the presence of the aromatase has been evaluated in the two main cellular components of the brain: the neurons and the glia. in these experiments, cultures of neurons obtained from the brains of 14-15-day-old rat embryos, mixed glial cells from l-day-old rats and type 1 astrocytes derived from cultured glial cells, have been utilized. The aromatase has been also evaluated in oligodendrocytes isolated from adult male rat brain by density gradient ultracentrifugation. The arumatase activity has been assayed by an 'in vitro' radiomctric method which quantifies the production of tritiated water from [l~.3H].androstenedione as an index of estrogen formation. The validity of the method has been verified both on the placental microsomes and on rat hypothalamic tissue, in which the actual formation of estrogens has also been measured. Among the different cells tested, only neurons possess the aromatase complex to a significant degree, while mixed glial cell and astrocyte preparations have an extremely low enzymatic activity: oligodendrocytes seem to be completely inactive in this respect. The calculation of the kinetic parameters of the neuronal aromatase has shown that this enzymatic activity is linear with respect to the protein content and the incubation time. The neuronal aromatase follows a Michaelis-Menten model with a Km of 52 nM; this value is analogous to that calculated for other central or peripheral structures in which the enzyme is known tt) be present.
INTRODUCTION The aromatization to estrogens and the process of 5a-reduction are the two major metabolic pathways that allow the transformation of testicular and adrenal androgens into their active metabolites. Both estrogens (estradiol and estrone) and the 5 a - r e d u c e d steroids (5a-androstane-tT/3-ol,3-one: D H T ; 5 a - a n d r o s t a n e 3a,17/3-dioh 3a-diol, etc.) represent the intracellular mediators of the action of testosterone in the different target structures. The process of aromatization is catalyzed by an enzymatic complex formed by a cytochrome-P450 aromatase linked to a N A D P H - r e d u c t a s e responsible for transferring reduced equivalents from N A D P H to aromatase 3~m. T h e h u m a n aromatase has been re-
cently cloned ILtc', and molecular studies have assigned the h u m a n aromatase gene to chromosome 15 H). A large number of studies have demonstrated that the aromatase is expressed in a variety of peripheral tissues (liver, ovary, testis, adipose tissue, breast, etc.), and in the brain of several mammalian and non-mammalian species (see refs. 5, 6, 40 for review). At the level of the central nervous system (CNS), the aromatase has been predominantly found in areas involved in the control of reproductive functions, where estrogenic molecules are required, both prenatally and postnatally, to organize or direct certain behavioral and neuroendocrine responses. For instance, locally formed estrogenic metabolites play a crucial role in the sexual differentiation of the fetal brain towards male patterns of control of gonadotropin secretion and of
Correspondence: P. Negri Cesi, Department of Endocrinology of the University of Milan, Via G. Balzaretti, 9, 20133, Milano, Italy.
328 sexual behavior ~4, as well as in the control of male sexual behavior ~s and of gonadotropin secretion 25 once sexual maturity is achieved. Estrogens have also been shown to be crucial for facilitating synaptogenesis and other early events in the ontogenesis of the CNS 24. The distribution of the aromatase activity has been extensively studied in different areas of the CNS in several animal species including rats 3°, birds 35, hamsters :'6 and non-human primates 3°'3s. The data obtained utilizing both macro- and micro-dissection 27 techniques have shown that the aromatase (as measured by the conversion of androstenedione into estrone) is localized especially within discrete brain areas like the hypothalamus, the preoptic area and the limbic system; little or no activity is evident in other brain regions or in the anterior pituitary 2,~..~5..~7.3s.These findings have been recently confirmed also by immunocytochemical studies performed both in rats a3 and in birds 1'2 using specific polyclonal antibodies. In particular, Sanghera and coworkers 33 have found an intense aromatase immunoreactivity in the rostral region of the hypothalamus, at the level of the olfactory tract and in the anterior cortical amygdaloid nuclei. The medial basal hypothalamus showed, on the contrary, little labeling, except for the ventral region of the arcuate nucleus. This discrete localization of the aromatase system within specific brain regions contrasts with the more diffuse distribution of the 5a-reductase (see ref. 9 for details). Previous work of this laboratory -''~ has analyzed the cellular distribution of the 5a-reductase activity using two types of techniques, i.e. the isolation by density gradient ultracentrifugation of neurons, astrocytes and oligodendroeytes from the adult male rat brain, and the culture of neurons and glial cells, respectively, from the fetal and neonatal brain. The results have shown that the 5a-reductase is present in all types of brain cells. However, neurons express an activity which is higher than that of the different types of glial cells studied; among these, astrocytes seem to possess an enzymatic activity higher than that of oligodendrocytes 23. It is still an open question whether the aromatase is localized in neurons, in glial cells or in both. Indirect evidence provided by Canik and coworkers TM seems to suggest that the aromatization of androgens occurs mainly in neuronal cells; however, a direct proof of this is still lacking. The present work was undertaken in order to directly analyze the distribution of the aromatase complex in neurons and in the glia. In the present experiments cultures of neurons obtained from the brain of 14-15-day.old rat embryos, mixed glial cells obtained
from the brains of l-day-old rats, and type 1 astrocytes purified from glial cells have been utilized. Due to the technical difficulties of growing oligodendrocytes in culture, these cells were isolated from the brain of adult male rats by density gradient ultracentrifugation. The kinetic parameters of the aromatase were determined in those cells where the enzyme was found to be present. MATERIALS AND METHODS
Animals For the experiments on isolated oligodendrocytes, adult normal male Sprague-Dawley rats (crl: CD~BR, Charles River, Calco, Italy) were used. The animals were maintained in animal quarters with controlled temperature and humidity. The light schedule was 14 h light and 10 h dark (lights on at 06.30 h). Animals were fed a standard pellet diet and water was provided ad libitum. For the cell culture experiments, neurons were obtained from the brains of 14-15-day-old rat embryos and mixed glial cells from the brain~ of l-day-old rats, whose mothers had been maintained in the conditions mentioned above.
Cell preparations Isolation of oligodendrocytes by uitracentrifugation. The isolation of these cells was performed as previously described 23, using the whole brain (without the cerebellum) as the starting material. At the end of purification, the oligodendrocyte fraction was washed and centrifuged twice with the aromatase assay buffer (see below) at 1,000 × g for 10 rain, resuspended in the same buffer and used for the aromatase assay. The purity of the oligodendrocyte preparations was assessed by light and electron microscopy as previously described 23.
Caltured cells Neuronal ctdtures, Neuronal cell cultures were performed as previ. ously described -',~, using as starting material the cerebral region of 14-15-day.old rat fetuses. The cultured neurons were used for the aromatase assay on the 6th day in culture (d.i.v.). The Petri dishes were washed 3 times with Krebs-Ringer buffer, and the cell layer was scraped, collected, washed and centrifuged twice with the aromatase assay buffer at 1,000× g for 10 rain, resuspended in the same buffer and used for the aromatase assay. Mixed glial cell cultures. These cultures were performed as previously described 23 starting from the hemispheres of I-day-old rats and were used at the 14-16 d.i.v. Mixed glial cells were processed in the same way as neurons before the aromatase assay. Type i astrocyte cultures, Mixed glial primary cultures were obtained as described by Melcangi et al. 2~, with the exception that the cells were initially plated at high density (20× I(P cells) in 75 cm 2 flasks according to the method of McCarthy and DeVellis21. The mixed glial cells were cultured for two weeks and the bedlayer, which consists of type I astrocytes, was obtained by shaking the cultures. The astrocytes were processed in the same way as neurons before the aromatase assay. The purity of the neuronal cultures was evaluated by immunofluorescence, utilizing monoclonal antibodies against the 160 K d neurofilament neuropeptide and the microtubule-associated protein (MAP 2)q. The purity of neuronal cultures was > 90%. in the case of mixed glial cells and type I astrocytes an antibody against glial fibrillary acidic protein (GFAP) was utilized`j. When immunostained with this antibody, the glial cell cultures consisted of more than 90% of GFAP-positive cells (i.e., astrocytes).
Aromatase assay The aromatase activity was estimated from the synthesis of 3H 20 from [l/~-3H]-androstenedione (NEN, Research Product, DuPont, Boston, MA, S.A. 27.5 Ci/mmol), utilizing a procedure previously
329 described for the rat 2s and hamster brain 2('. Briefly, either isolated or cultured cells were resuspended in a small volume of 10 mM phosphate buffer (pH 7.4:i:0.1) containing 100 mM KCI, 10 mM dithiothreitol and 1 mM EDTA and sonicated by means of a Branson sonifier cell disruptor BI5 (10 s, 30% duty cycle). The volume was then adjusted in order to have an appropriate protein concentration (see the relative tables and figures) in 100 /zl of phosphate buffer. The reaction was started by adding 100/tl of a preincubated generating system (5 mM glucose-6P, disodium salt, Boehringer, Mannheim; I mM NADP +, disodium salt, Boehringer, Mannheim; 2 U / m l glucose-6P-dehydrogenase from yeast grade 1, Boehringer, Mannheim, in phosphate buffer) and an adequate concentration of [l/3-3H]-androstenedione to 100/zl of cell suspensions. In the determination of the kinetic parameters, pooled samples from 20-30 Petri dishes were used. The samples were then incubated at 37°C in a Dubnoff metabolic bath. The incubation conditions are detailed in each table and figure. The reaction was stopped with 10% lrichloroacetic acid containing 20 mg charcoal/ml, the generated tritiated water was purified by column chromatography utilizing two different mesh of Dowex 50W-X4 ion exchange resin (Bio-Rad Laboratories, Richmond, CA). The eluted tritiated water (2.5 ml) was added to 10 ml of the scintillation cocktail (picoaqua, Packard, Canberra Co.). The samples were counted in a Packard 1600 CA liquid scintillation spectrometer. Quench corrected dpm of the isotope were obtained by a calibration standard curve. Corrections for recoveries and blanks were made as previously described 2('. The aromatase activity was expressed as fmol of tritiated water/rag of proteins. In order to confirm the validity of the tritiated water assay as a measure of estrogen synthesis, either human term placental microsprees prepared as described by Santner et al. 34, or adult male rat hypothalamic homogenates were tested in an independent set of experiments. The tritiated water generated from [I/3-'~H]-andros tenedione (NEN, Research Product, DuPont, Boston, MA, S.A. 27.5 Ci/mmol) and the actual formation of estrogens from [I,2,6,7-N•~H]-androstenedione (Amersham, UK, S.A. 80 Ci/mmol) were assayed in parallel utilizing the same conditions in terms of substrate concentration, cofactor availability and incubation times, except that for the actual evaluation of estrogen formation, the reaction was stopped by deep freezing the samples. The labelled estrogenic products were isolated and quantified as follows. Known amounts of carbon-labelled estrone and estradiol were added to each sample in order to calculate the recoveries and the samples were then extracted 3 times with 5.5 ml of diethylether. The extracts were evaporated to dryness under a stream of nitrogen, redissolved in 1(10 /zl of ethanol containing cold estrone, estradiol and androstenedione and applied on silica gel chromatography plates (60 F25o, Merk, F.R.G.). The samples were eluted 3 times in diethylether/n-hexane (3:2 by vol.) at 4°C. The radioactivity corresponding to authentic estrone and estradiol were separately scraped off, re-extracted with chloroform/methanol (95:5 by vol.), subjected to overnight acetylation and chromatographed on silica gel chromatography plates using toluene:ethylacetate (8:2 by vol.) as solvent system. The areas corresponding to the appropriate acetates were scraped off and counted in a liquid scintillation counter. The data were expressed as fmol (hypothalamus) or nmol (placental microsomes) of estrone plus estradiol/mg of proteins, in all experiments, protein concentrations were determined in an aliquot of each sample by the Bradford method 4.
Statistics Where necessary, the statistical analysis of the data was performed by a one-way analysis of variance; to determine the levels of significance of the responses, the t-values were compared with the values of Dunnett's tables for multiple comparison 12. The regression curves were evaluated according to the linear regression method, using a computer statistical package; the K m and Vma,, values were calculated using both the Lineweaver-Burke transformation and the Macintosh version of a specific non-linear least-squares curve fitting program (ENZYME i,)).
RESULTS
The results obtained in the validation of the tritiated water assay are shown in Table 1. It is evident that the aromatase activity present in the placental microsomes is greater than that present in the rat hypothalamus by about 5 orders of magnitude, independently of whether this activity is evaluated with the tritiated water assay (substrate = [lfl-3H]-androstene dione) or with the actual measurement of the formation of estrone plus estradiol (substrate = [1,2,6,7-N3H]-androstenedione); however, both in the case of an extremely high aromatase concentration (placental microsomes) or of a low enzymatic activity (hypothalamic homogenates), no significant differences exist between the formation of tritiated water and the actual production of estrogens. On the basis of these data, the tritiated water assay appears to be a sensitive and reliable method for the measurement of the aromatase activity. It must be pointed out that, in the present study, placental microsomes have been shown to form both estrone and estradiol, while the hypothalamus forms almost exclusively estrone. This last finding is in agreement with a previous report by Roselli and coworkers 31. It seems then that the tritiated water method provides a reliable evaluation of the aromatase activity, even when the ratio of the end products of such a process (estrone and estradiol) are different. Table II shows the aromatase activity present in cultured neurons, mixed gliai cells and type 1 astrocytes obtained from the fetal or neonatal brain as well as in oligodendrocytes isolated from the adult rat brain. It is apparent that only neurons are able to aromatize androgens with considerable yields; the capability of the glial cells and of type 1 astrocytes to form estrogens appears to be minimal and closely related to the sensiTABLE ! Formation of tritiated water or estrogens as products of the aromatase actit'ity in human term plao, nta microsomes and adult malt, rat hypothalamic homogenat,,s
Placental microsomes (0.017 mg of proteins) or hypothalamic homogenate (0.93 mg of proteins) were incubated for I h at 37°C with the same concentration (about 0.5 /.tM) of either [l/3-'~H]-andros tenedione (tritiated water production) or [I,2,6,7-N-3H]-andros tenedione (estrogen formation). Data are expressed as mean +S.D. In parentheses, the number of samples assayed. Products
Placental microsomes (nmol/mg protein) Hypothalamus (fmol/mg protein)
Tritiated water
Estrone + estradiol
3.71 + 0.32 (5)
2.96 + 0.53 (5)
27.14 + 4.53 (5)
19.41 + 2.95 (5)
330 40
TABLE !1 Aromatase actil'ity in cuhured neurons, mL~ed glial ('ells and type ! astrocytes and in isolated oligodendrocyws
in parentheses the number of determinations each consisting in a pool of two or more Petri dishes (about 0.1 mg of proteins/sample). The samples were incubated for 1 h at 37°C in the presence of [lfl--~H]-androstenedione (0.7/zM). Data are expressed as mean+ S.D.n.d. ffi not detectable.
I Km=52+20nM Vmax = 25.94 + 9.49 fmol3H20 1 mg protein I h
35 p.
.E 30
"~ 25 n
m
~' 2o ~ 0.10
_~ lO
Aromatase actii'ity fmol "~H,0 / mg protein
~e
0.05
5
Cultured Neurons
18.06 + 9.28 (19) !.01 + 1.75 (3) 1.01 + i.15 (7)
Mixed glial cells Type 1 astrocytes Isolated Oligodendrocytes
.;o -;o
0
0.0
o ,o
14S]
3'o " 4'o " s'o
i
i
l
l
0.2
0.4
0.6
0.8
,ml
.0
Androstenedione Odd)
Fig. 2. Michaelis-Menten constant (K m) and maximum velocity (Vm~x) of neuronal aromatase activity. Samples of pooled Petri dishes (about 0.1 mg of proteins) were incubated for 1 h at 37°C in the presence of [l/]-3H]-androstenedione (0.02-0.4 /~M). Inset: Lineweaver-Burke transformation of the data. V = fmol ~ H 2 0 / m g protein/h; [S] = [1 fl- 3H]-androstenedione concentrations (~t M). The data in the figure are derived from two independent experiments. K m and Vm,,x consequently represent the mean +S.D. of these two experiments. Moreover, curves perfectly superimposable to the one presented may be derived using the data of each experiment sepa rately.
n.d.
A) 40
tivity of the method. Moreover, oligodendrocytes are completely inactive in this respect. The kinetic parameters of the neuronal aromatase are shown in Figs. 1 and 2. The production of tritiated water is linear with increasing amounts of protein concentration up to 0.4 mg (R 2 - 0.99, Fig. IA) and is also directly correlated with the time of incubation up to 3 h (R 2 - 0.98, Fig. I B). In the experimental conditions adopted, the neuronal enzyme exhibits an apparent Michaelis-Menten constant (Kin) of 52 + 20 nM, with a maximum rate of aromatase activity (Vm,x) calculated to be 25.94 + 9.49 fmol/mg of proteins/h (Fig.
3O
I0
I
I
0,1
I
0,2 0,3 mg protein
I
I
0,4
05
B)
2). 40 35 30 25 20 15 10 5 ~" 0 0
DISCUSSION
RLo.976 i.
30
i
60
I
!
90 120 minutes
!
!
150
180
Fig. I. Linear regression analysis of the neuronal aromatase activity in function of protein content (A) and incubation time (B). Incubation conditions: A: samples from pooled Petri dishes (0.05-0.4 mg protein) were incubated for I h at 37°C in the presence of [I/3-3H]androstenedione ((I.5/zM). B: samples from pooled Petri dishes (0.18 mg protein) were incubated for different times at 37°C in presence of [ 1,8-3H]-androstenedione (0.6 p.M).
The results obtained in the experiments described here show that among the different cell populations tested only neurons seem to possess in a significant degree the enzyme involved in the aromatization of androgens. Moreover, the data also indicate that, in the present experimental conditions, the kinetics of the neuronal enzyme are linear with respect to the protein concentration and the incubation time; the formation of estrogens follows the Michaelis-Menten model. To the authors' knowledge, this paper provides the first direct evidence that, in the brain, the aromatase activity is practically localized only in neurons; in fact, the low levels of tritiated water recovered during the
331 incubation of androstenedione with mixed glial cells and with type 1 astrocytes are at the limit of the sensitivity of the method, and do not allow one to derive firm conclusions on the real presence of the enzyme in these cells. Moreover, oligodendrocytes seem to be completely devoid of aromatase activity. It must be pointed out that the total inactivity of oligodendrocytes might be due to the fact that these were obtained from adult rather than fetal-neonatal animals. Previous work had suggested that in the brain the aromatase activity decreases from embryonic life to adulthood ~3"2°. Moreover, oligodendrocytes were purified from the whole brain and not specifically from regions known to possess a rather elevated aromatase activity (e.g., amygdala, preoptic region, etc.). The present results agree with previous data by Canik and coworkers TM who used, however, a totally indirect approach. These authors described a 6-fold increase in the aromatase activity in primary cultures of fetal rat hypothalamic cells in which the neuronal component had been enriched by the addition to the medium of cytosine arabinoside, a factor which inhibits the proliferation of non-neuronal cells s. An analogous conclusion was reached by the same authors 7 on the basis of a reverse experiment; a dose-dependent decrease of the aromatase activity was found in hypothalamic cell cultures in which the neuronal component had been diminished by the addition to the cultures of kainic acid, a neurotoxic agent. As previously mentioned, prevalence of the aromatase within neurons has also been shown by immunocytochemical studies performed both in rats "~:~and in birds ~a. The aromatase present in neurons possesses a Michaelis-Menten constant (Km 52 4- 20 nM) which is not significantly different from that calculated for the human purified enzyme 'v, and for the aromatase present in brain homogenates of different animal species 26"28'32'36. This indicates that the culture conditions adopted do not modify the kinetic characteristics of the aromatase. Moreover, preliminary results obtained in our laboratory (data not shown) indicate that a known aromatase inhibitor (4-hydroxy-androstenedione), when added to the neuronal cultures at the beginning of the aromatase assay, practically abolishes the formation of tritiated water. The amounts of tritiated water formed by neurons (as evaluated by the Vmax) appear to be lower than those described for the aromatase present in embryonic or adult male rat hypothalamic fragments ~3 or in particular nuclei of the adult male rat brain 2~. Therefore it might be hypothesized that the aromatase is not equally expressed in all neuronal population, but might be more concentrated in neurons localized in particu-
lar regions of the CNS. In this respect, Sanghera and coworkers aa have recently shown that a polyclonal antibody raised against a synthetic peptide corresponding to a 20 aminoacid segment common to the rat, human and chicken aromatase, is able to immunostain with decreasing intensities the amygdaloid structures, the supraoptic nucleus, the paraventricular and arcuate nuclei as well as the hippocampus. Neurons in the bed nucleus of the stria terminalis, the medial basal hypothalamus and the preoptic area show little aromatase immunoreactivity. On the contrary, high yields of immunoreactivity were detected in specific brain regions not previously recognized to contain the enzyme (i.e., the reticular thalamic nucleus, the olfactory tract and the piriform cortex) a3. Comparing the data of the present paper with those obtained in this laboratory on the 5a-reductase activity 23, it appears that the two main androgen metabolizing pathways coexist only in neurons, while the glial component of the brain apparently utilizes androgens only via the 5a-reductase pathway. It is interesting that androgen and estrogen receptors are present in neurons 3'22. The intracellularly formed estrogens and 5areduced androgens could either exert separate actions in the neurons, or synergize with each other in the regulation of various neuroendocrine functions. In this context, it may be underlined that, in the rat, estradiol administration to castrated animals is able to increase androgen receptor levels in specific brain areas, such as the medial preoptic nucleus and the medial amygdala ~5, in which the aromatase has been shown to be present 2~. On the other hand, the aromatase activity of the rat brain appears to be under androgenic control probably through a receptor-mediated mechanism 3°'3~. It is then possible to postulate that DHT formed via the 5a-reductase pathway might increase the intraneutonal formation of estrogens.
Acknowledgemous. Thanks are due to Dr. R. Maggi, Department of Endocrinology, University of Milan, for providing the Macintosh version of the 'ENZYME' program. The experimentsdescribed here were supported by grants of the Consiglio Nazionale delle Ricerche through the following target projects: 'Biotechnologyand Bioinstrumentation' N. 91.0192PF20, 'Factors of disease' N. 91.002t)7PF4, 'Aging' N. 91.00419PF40 and through the bilateral project ItalyYugoslavia N. 90.01445.04. Such support is gratefullyacknowledged. REFERENCES I Balthazart, J., Foidart, A. and Harada, N., immunocytochemical localization of aromatase in the brain, Brain Res., 514 (1990) 327-333. 2 Balthazart,J., Foidart, A., Surlemont, C., Vockel,A. and Harada, N., Distribution of aromatase in the brain of the Japanese quail, Ring dove, and Zebra finch: an immunocytochemical study, J. Comp. Neuroi., 301 (1990) 276-288.
332 3 Blaustein, J.D. and Turcotte, J.C., Estrogen receptor-immunostaining of neuronal cytoplasmic processes as well as nuclei in the guinea pig brain, Brab~ Res., 495 (19891 75-82. 4 Bradford, M.M., A rapid and sensitive method for the quantitalion of microgram quantities of protein utilizing the principle of protein-dye binding, Anal, Biochem., 72 (19761 248-254. 5 Callard, G.V., Petro. Z. and Ryan, K.J.. Phylogenetical distribution of aromatase and other androgen-converting enzymes in the central nervous system, Endocrinology, 103 (19781 2283-2290. 6 Callard, G.V., Petro, Z. and Ryan, K.J., Conversion of androgens to estrogens and other steroids in the vertebrate brain, Am. Zool., 18 (19781 511-523. 7 Canik, J.A., Vaccaro, D.E., Livingston, E.M., Leeman, S.E., Ryan, K.J. and Fox, T.O., Localization of aromatase and 5a-reductase to neuronal and non-neuronal cells in the fetal rat hypothalamus, Brain Res., 372 (1986) 277-282. 8 Canik, J.A., Vaccaro. D.E., Ryan, K.J. and Leeman, S.E., The aromatization of androgens in primary cultures of fetal rat hypothalamus, Endocrinology, 100 (19771 250-253. 9 Celotti, F., Melcangi, R.C. and Martini, L., The 5alpha-reductase in the brain: molecular aspects and relation to brain function, in L. Martini and W.F. Ganong (Eds.), Frontiers in Neuroendocrino/ogy. Vol. 13, Raven, New York, 1992, pp. 163-215. l0 Chen, S., Beaman, M.J., Sparkes, R.S., Zollman, S., Kiisak, I., Mohandaa, T., Hall. P.F. and Shively, J.E., Human aromatase: eDNA cloning, Southern blot analysis, and assignment of the gene to chromosome 15, DNA, 7 (19881 27-38. Ii Corbin, C.J., Graham-l.x~rence. S., McPhaul, M., Mason, J.I., Mendelson, C.R. and Simpson, E.R., Isolation of a full-length eDNA insert encoding human aromatase system cytochrome P450 and its expression in non steroidogenic cells, Proc. Nat/. Acad. Sci. USA, 85 (1988) 8948-8952. 12 Dunnett, C.W., A multiple comparison procedure for comparing several treatments with a control, J. Am. Stat. Assoc., 50 (19551 1096-1121. 13 George, F W. ,'rod Ojeda, S.R., Changes in aromatase activity in the rat brain during embryonic, neonatal, and infantile development, l':mh)crino/ox~.', I I I (1982) 522-529. 14 Goy, R.W. and McEwen, B.S.. Se~'ual Diffi,rentiation of the Brain, MIT, Cambridge, 1980. 15 tlanda. R.J., Rosdli, C.E., |torton, L. and Resko, J.A., The quantitative distribution of cytosolie androgen receptors in nil. crodisseeted areas of the male rat brain: effects of estrogen treatments, l"ndocrinologv, 121 (1987) 233-240. 16 llarada N., Cloning of a complete eDNA encoding human aromatase: immunocytoehemieal identification and sequence analysis, Biochem. Biophys. Res. Commun., 156 (1988)725-732. 17 Kellis, J.T and Vickery, L.E.. Purification and characterization of human placental aromatase cytochrome P-450, J. Biol. Chem,, 262 (1987) 4413-4420. 18 Larsson, K., Features of the neuroendocrine regulation of masculine sexual behavior. In C. Beyer (Ed.), Endocrine Control of Seruai Behavior, Raven, New York, 1979, pp. 77-163, 19 Lutz, R.A, Bull, C. and Rodbard, D., Computer analysis of enzyme-substrate-inhibitor kinetic data with automatic model selection using IBM-PC compatible microcomputers, Enzyme, 36 (1986) 197-206. 20 MacLusky, N.J., Philip, A., Hulburt, C. and Naflolin, F,, Estrogen formation in the developing rat brain: sex difference in aromatase activity during early post-natal life, Psyconeuroemh)crinolog~.,, 10 (1985) 355-361. 21 McCarthy, K.D. and DeVellis, J., Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue, J. Cell. Bit)L, 85 (1980) 890-902. 22 McEwen, B.S., Bigeon, A., Davis, P.G., Krey, L.C., Luine, V,N., McGinnis, M.Y., Paden, C.M., Parsons, B. and Rainbow, C.M., Steroid hormones: humoral signals which alter brain cell properties and function, Rec. Prot,,. thmn. Res., 38 (1982)41-92,
23 Melcangi, R.C., Celotti, F., Ballabio, M., Castano, P., Massarelli, R., Poletti, A. and Martini, L., 5a-Reductase activity in isolated and cultured neuronal and glial cells of the rat, Brab~ Res., 516 (1990) 229-236. 24 Naftolin, F., MacLusky, N.J., Leranth, C.Z., Sakamoto, H.S. and Garcia-Segura, L.M., The cellular effects of estrogens on neuroendocrine tissues, J. StcroM Biochem., 30 (19881 195-207. 25 Negri Cesi, P., Celotti, F. and Martini, L., Androgen metabolism in the brain: role in sexual differentiation and in the control of gonado!ropin secretion, Monogr. Neural Sci., 12 (19861 7-16. 26 Negri Cesi, P., Celotti, F. and Martini. L., Androgen metabolism in the male hamster: 2. Aromatization of androstenedione in the hypothalamus and in the cerebral cortex; kinetic parameters and effect of exposure to different photoperiods, J. Steroid Biochem., 32 (19891 65-70. 27 Palkovitz, M., Isolated removal of hypothalamic or other brain nuclei of the rat, Braht Res., 59 (19731 449-450. 28 Roselli, C.E., Ellinwood, W.E. and Resko, J.A., Regulation of brain aromatase activity in rats, Endocrinology, 114 (19841 192200. 29 Roselli, C.E., Horton, L.E. and Resko, J.A., Distribution and regulation of aromatase activity in the rat hy0othalamus and limbic system, Endocrinology, ! 17 (1985) 2471-2477. 30 Roselli, C.E. and Resko, J.A., The distribution and regulation of aromatase activity in the central nervous system, Steroids, 50 (1987) 495-508. 31 Roselli, C.E., Salisbury, R.L. and Resko, J.A., Genetic evidence fur an androgen-dependent and independent control of aromatase activity in the rat brain, Endocrinology, 121 (1987) 2205221(}. 32 Roselli, C.E., Stadelman, H., Horton, L.E.' and Resko, J.A., Regulation of androgen metabolism and luteinizing hormone-releasing hormone content in discrete hypothalamic and limbic areas of male Rhesus macaques, Endocrinology, 120 (19871 97106. 33 Sanghera, M.K., Simpson, E.R., McPhaul, M.J., Kozlowski, G., Conley, A.J. and Lephart E.D., lmmunocytochemical distribution of ammatase cytochrome P-450 in the rat brain using peptidegenerated polyclonal antibodies, Endocrinology, 129 (1991)28342844. 34 Santncr, S.I., Rosen, H., Osawa, Y, and Santen, R,J., Additive effects of aminoglutethimide, testonolacton¢, and 4-hydroxyandrostenedione as inhibitors of aromatasc, J. Steroid Biochem., 2(1 (19841 1239-1242. 35 Schumaker, M. and Balthazart, J., Neuroanatomieal distribution of testosterone metabolizing enzymes in the Japanese quail, Braba Res., 422 (1987) 137-148. 36 Schumaker, M., Contenti, E. and Balthazart, J., Partial characterization of testosterone-metabolizing enzymes in the quail brain, Brain Res., 305 ( 19841 5 !-59. 37 Selmanoff, M.K., Bmdkin, L.D., Weiner, R.I. and Siiteri, P,K., Aromatization and 5a-reduction of androgens in discrete hypothalamic and limbic regions of the male and female rat, Endocrinok~gy, 101 (1977) 841-848. 38 Sholl, S.A. and Kim, K.L., Aromatase, 5a-reductase and androgen receptor level in the fetal monkey brain during early development, Neuroendocrinology, 52 (19901 94-98. 39 Simmons, D.L., Lalley, P.A. and Kasper, C.B., Chromosomal assignments of genes coding for components of the mixed-function oxidasc system in mice, J. Biol. Chem., 260 (19851 515-521. 40 Simpson, E.R., Merril, J.C., Hollub, A.J., Graham-Lorence, S. and Mendelson, C.R., Regulation of estrogen biosynthesis by human adipose cells, Endocr. Rec., 10 (19891 136-148. 41 Thompson Jr,, E.A. and Siiteri, P.K., Utilization of oxygen and reduced nicotinamide dinucleotide phosphate by human placental microsomes during aromatization of androstenedione, J. Biol. Chem., 249 (197415364-5372.
327
BRES 18029
Aromatase activity in cultured brain cells: difference between neurons and glia Paola Negri Cesi, Roberto
Cosimo
Melcangi,
Fabio Celotti and Luciano
Martini
Departnlent of Endocrinology. Unh'ersity of Milan, Milan (Italy) (Accepted 31 March 1992)
Key words: Aromatase; Androgen; Neuronal cell; Glial cell: Rat; Culture
At the level of the central nervous systeln (CNS) of several mammalian and non-mammalian species, estrogens may be intracellularly formed from circulating androgens through the action of the aromatase complex. Estrogenic steroids play a crucial role in organizing and directing certain behavioral and neuroendocrine responses both during the fetal/neonatal life and in adulthood. Biochemical and immunocytochemical studies have shown that the aromatase is particularly concentrated in CNS areas involved in the control of reproductive functions, such as the hypoth~lamus, the preoptic area and the limbic system: despite this large body of evidence, the exact cellular localization of this enzymatic complex within the different cell populations of the brain is still uncertain, in the experiments described here, the presence of the aromatase has been evaluated in the two main cellular components of the brain: the neurons and the glia. in these experiments, cultures of neurons obtained from the brains of 14-15-day-old rat embryos, mixed glial cells from l-day-old rats and type 1 astrocytes derived from cultured glial cells, have been utilized. The aromatase has been also evaluated in oligodendrocytes isolated from adult male rat brain by density gradient ultracentrifugation. The arumatase activity has been assayed by an 'in vitro' radiomctric method which quantifies the production of tritiated water from [l~.3H].androstenedione as an index of estrogen formation. The validity of the method has been verified both on the placental microsomes and on rat hypothalamic tissue, in which the actual formation of estrogens has also been measured. Among the different cells tested, only neurons possess the aromatase complex to a significant degree, while mixed glial cell and astrocyte preparations have an extremely low enzymatic activity: oligodendrocytes seem to be completely inactive in this respect. The calculation of the kinetic parameters of the neuronal aromatase has shown that this enzymatic activity is linear with respect to the protein content and the incubation time. The neuronal aromatase follows a Michaelis-Menten model with a Km of 52 nM; this value is analogous to that calculated for other central or peripheral structures in which the enzyme is known tt) be present.
INTRODUCTION The aromatization to estrogens and the process of 5a-reduction are the two major metabolic pathways that allow the transformation of testicular and adrenal androgens into their active metabolites. Both estrogens (estradiol and estrone) and the 5 a - r e d u c e d steroids (5a-androstane-tT/3-ol,3-one: D H T ; 5 a - a n d r o s t a n e 3a,17/3-dioh 3a-diol, etc.) represent the intracellular mediators of the action of testosterone in the different target structures. The process of aromatization is catalyzed by an enzymatic complex formed by a cytochrome-P450 aromatase linked to a N A D P H - r e d u c t a s e responsible for transferring reduced equivalents from N A D P H to aromatase 3~m. T h e h u m a n aromatase has been re-
cently cloned ILtc', and molecular studies have assigned the h u m a n aromatase gene to chromosome 15 H). A large number of studies have demonstrated that the aromatase is expressed in a variety of peripheral tissues (liver, ovary, testis, adipose tissue, breast, etc.), and in the brain of several mammalian and non-mammalian species (see refs. 5, 6, 40 for review). At the level of the central nervous system (CNS), the aromatase has been predominantly found in areas involved in the control of reproductive functions, where estrogenic molecules are required, both prenatally and postnatally, to organize or direct certain behavioral and neuroendocrine responses. For instance, locally formed estrogenic metabolites play a crucial role in the sexual differentiation of the fetal brain towards male patterns of control of gonadotropin secretion and of
Correspondence: P. Negri Cesi, Department of Endocrinology of the University of Milan, Via G. Balzaretti, 9, 20133, Milano, Italy.
328 sexual behavior ~4, as well as in the control of male sexual behavior ~s and of gonadotropin secretion 25 once sexual maturity is achieved. Estrogens have also been shown to be crucial for facilitating synaptogenesis and other early events in the ontogenesis of the CNS 24. The distribution of the aromatase activity has been extensively studied in different areas of the CNS in several animal species including rats 3°, birds 35, hamsters :'6 and non-human primates 3°'3s. The data obtained utilizing both macro- and micro-dissection 27 techniques have shown that the aromatase (as measured by the conversion of androstenedione into estrone) is localized especially within discrete brain areas like the hypothalamus, the preoptic area and the limbic system; little or no activity is evident in other brain regions or in the anterior pituitary 2,~..~5..~7.3s.These findings have been recently confirmed also by immunocytochemical studies performed both in rats a3 and in birds 1'2 using specific polyclonal antibodies. In particular, Sanghera and coworkers 33 have found an intense aromatase immunoreactivity in the rostral region of the hypothalamus, at the level of the olfactory tract and in the anterior cortical amygdaloid nuclei. The medial basal hypothalamus showed, on the contrary, little labeling, except for the ventral region of the arcuate nucleus. This discrete localization of the aromatase system within specific brain regions contrasts with the more diffuse distribution of the 5a-reductase (see ref. 9 for details). Previous work of this laboratory -''~ has analyzed the cellular distribution of the 5a-reductase activity using two types of techniques, i.e. the isolation by density gradient ultracentrifugation of neurons, astrocytes and oligodendroeytes from the adult male rat brain, and the culture of neurons and glial cells, respectively, from the fetal and neonatal brain. The results have shown that the 5a-reductase is present in all types of brain cells. However, neurons express an activity which is higher than that of the different types of glial cells studied; among these, astrocytes seem to possess an enzymatic activity higher than that of oligodendrocytes 23. It is still an open question whether the aromatase is localized in neurons, in glial cells or in both. Indirect evidence provided by Canik and coworkers TM seems to suggest that the aromatization of androgens occurs mainly in neuronal cells; however, a direct proof of this is still lacking. The present work was undertaken in order to directly analyze the distribution of the aromatase complex in neurons and in the glia. In the present experiments cultures of neurons obtained from the brain of 14-15-day.old rat embryos, mixed glial cells obtained
from the brains of l-day-old rats, and type 1 astrocytes purified from glial cells have been utilized. Due to the technical difficulties of growing oligodendrocytes in culture, these cells were isolated from the brain of adult male rats by density gradient ultracentrifugation. The kinetic parameters of the aromatase were determined in those cells where the enzyme was found to be present. MATERIALS AND METHODS
Animals For the experiments on isolated oligodendrocytes, adult normal male Sprague-Dawley rats (crl: CD~BR, Charles River, Calco, Italy) were used. The animals were maintained in animal quarters with controlled temperature and humidity. The light schedule was 14 h light and 10 h dark (lights on at 06.30 h). Animals were fed a standard pellet diet and water was provided ad libitum. For the cell culture experiments, neurons were obtained from the brains of 14-15-day-old rat embryos and mixed glial cells from the brain~ of l-day-old rats, whose mothers had been maintained in the conditions mentioned above.
Cell preparations Isolation of oligodendrocytes by uitracentrifugation. The isolation of these cells was performed as previously described 23, using the whole brain (without the cerebellum) as the starting material. At the end of purification, the oligodendrocyte fraction was washed and centrifuged twice with the aromatase assay buffer (see below) at 1,000 × g for 10 rain, resuspended in the same buffer and used for the aromatase assay. The purity of the oligodendrocyte preparations was assessed by light and electron microscopy as previously described 23.
Caltured cells Neuronal ctdtures, Neuronal cell cultures were performed as previ. ously described -',~, using as starting material the cerebral region of 14-15-day.old rat fetuses. The cultured neurons were used for the aromatase assay on the 6th day in culture (d.i.v.). The Petri dishes were washed 3 times with Krebs-Ringer buffer, and the cell layer was scraped, collected, washed and centrifuged twice with the aromatase assay buffer at 1,000× g for 10 rain, resuspended in the same buffer and used for the aromatase assay. Mixed glial cell cultures. These cultures were performed as previously described 23 starting from the hemispheres of I-day-old rats and were used at the 14-16 d.i.v. Mixed glial cells were processed in the same way as neurons before the aromatase assay. Type i astrocyte cultures, Mixed glial primary cultures were obtained as described by Melcangi et al. 2~, with the exception that the cells were initially plated at high density (20× I(P cells) in 75 cm 2 flasks according to the method of McCarthy and DeVellis21. The mixed glial cells were cultured for two weeks and the bedlayer, which consists of type I astrocytes, was obtained by shaking the cultures. The astrocytes were processed in the same way as neurons before the aromatase assay. The purity of the neuronal cultures was evaluated by immunofluorescence, utilizing monoclonal antibodies against the 160 K d neurofilament neuropeptide and the microtubule-associated protein (MAP 2)q. The purity of neuronal cultures was > 90%. in the case of mixed glial cells and type I astrocytes an antibody against glial fibrillary acidic protein (GFAP) was utilized`j. When immunostained with this antibody, the glial cell cultures consisted of more than 90% of GFAP-positive cells (i.e., astrocytes).
Aromatase assay The aromatase activity was estimated from the synthesis of 3H 20 from [l/~-3H]-androstenedione (NEN, Research Product, DuPont, Boston, MA, S.A. 27.5 Ci/mmol), utilizing a procedure previously
329 described for the rat 2s and hamster brain 2('. Briefly, either isolated or cultured cells were resuspended in a small volume of 10 mM phosphate buffer (pH 7.4:i:0.1) containing 100 mM KCI, 10 mM dithiothreitol and 1 mM EDTA and sonicated by means of a Branson sonifier cell disruptor BI5 (10 s, 30% duty cycle). The volume was then adjusted in order to have an appropriate protein concentration (see the relative tables and figures) in 100 /zl of phosphate buffer. The reaction was started by adding 100/tl of a preincubated generating system (5 mM glucose-6P, disodium salt, Boehringer, Mannheim; I mM NADP +, disodium salt, Boehringer, Mannheim; 2 U / m l glucose-6P-dehydrogenase from yeast grade 1, Boehringer, Mannheim, in phosphate buffer) and an adequate concentration of [l/3-3H]-androstenedione to 100/zl of cell suspensions. In the determination of the kinetic parameters, pooled samples from 20-30 Petri dishes were used. The samples were then incubated at 37°C in a Dubnoff metabolic bath. The incubation conditions are detailed in each table and figure. The reaction was stopped with 10% lrichloroacetic acid containing 20 mg charcoal/ml, the generated tritiated water was purified by column chromatography utilizing two different mesh of Dowex 50W-X4 ion exchange resin (Bio-Rad Laboratories, Richmond, CA). The eluted tritiated water (2.5 ml) was added to 10 ml of the scintillation cocktail (picoaqua, Packard, Canberra Co.). The samples were counted in a Packard 1600 CA liquid scintillation spectrometer. Quench corrected dpm of the isotope were obtained by a calibration standard curve. Corrections for recoveries and blanks were made as previously described 2('. The aromatase activity was expressed as fmol of tritiated water/rag of proteins. In order to confirm the validity of the tritiated water assay as a measure of estrogen synthesis, either human term placental microsprees prepared as described by Santner et al. 34, or adult male rat hypothalamic homogenates were tested in an independent set of experiments. The tritiated water generated from [I/3-'~H]-andros tenedione (NEN, Research Product, DuPont, Boston, MA, S.A. 27.5 Ci/mmol) and the actual formation of estrogens from [I,2,6,7-N•~H]-androstenedione (Amersham, UK, S.A. 80 Ci/mmol) were assayed in parallel utilizing the same conditions in terms of substrate concentration, cofactor availability and incubation times, except that for the actual evaluation of estrogen formation, the reaction was stopped by deep freezing the samples. The labelled estrogenic products were isolated and quantified as follows. Known amounts of carbon-labelled estrone and estradiol were added to each sample in order to calculate the recoveries and the samples were then extracted 3 times with 5.5 ml of diethylether. The extracts were evaporated to dryness under a stream of nitrogen, redissolved in 1(10 /zl of ethanol containing cold estrone, estradiol and androstenedione and applied on silica gel chromatography plates (60 F25o, Merk, F.R.G.). The samples were eluted 3 times in diethylether/n-hexane (3:2 by vol.) at 4°C. The radioactivity corresponding to authentic estrone and estradiol were separately scraped off, re-extracted with chloroform/methanol (95:5 by vol.), subjected to overnight acetylation and chromatographed on silica gel chromatography plates using toluene:ethylacetate (8:2 by vol.) as solvent system. The areas corresponding to the appropriate acetates were scraped off and counted in a liquid scintillation counter. The data were expressed as fmol (hypothalamus) or nmol (placental microsomes) of estrone plus estradiol/mg of proteins, in all experiments, protein concentrations were determined in an aliquot of each sample by the Bradford method 4.
Statistics Where necessary, the statistical analysis of the data was performed by a one-way analysis of variance; to determine the levels of significance of the responses, the t-values were compared with the values of Dunnett's tables for multiple comparison 12. The regression curves were evaluated according to the linear regression method, using a computer statistical package; the K m and Vma,, values were calculated using both the Lineweaver-Burke transformation and the Macintosh version of a specific non-linear least-squares curve fitting program (ENZYME i,)).
RESULTS
The results obtained in the validation of the tritiated water assay are shown in Table 1. It is evident that the aromatase activity present in the placental microsomes is greater than that present in the rat hypothalamus by about 5 orders of magnitude, independently of whether this activity is evaluated with the tritiated water assay (substrate = [lfl-3H]-androstene dione) or with the actual measurement of the formation of estrone plus estradiol (substrate = [1,2,6,7-N3H]-androstenedione); however, both in the case of an extremely high aromatase concentration (placental microsomes) or of a low enzymatic activity (hypothalamic homogenates), no significant differences exist between the formation of tritiated water and the actual production of estrogens. On the basis of these data, the tritiated water assay appears to be a sensitive and reliable method for the measurement of the aromatase activity. It must be pointed out that, in the present study, placental microsomes have been shown to form both estrone and estradiol, while the hypothalamus forms almost exclusively estrone. This last finding is in agreement with a previous report by Roselli and coworkers 31. It seems then that the tritiated water method provides a reliable evaluation of the aromatase activity, even when the ratio of the end products of such a process (estrone and estradiol) are different. Table II shows the aromatase activity present in cultured neurons, mixed gliai cells and type 1 astrocytes obtained from the fetal or neonatal brain as well as in oligodendrocytes isolated from the adult rat brain. It is apparent that only neurons are able to aromatize androgens with considerable yields; the capability of the glial cells and of type 1 astrocytes to form estrogens appears to be minimal and closely related to the sensiTABLE ! Formation of tritiated water or estrogens as products of the aromatase actit'ity in human term plao, nta microsomes and adult malt, rat hypothalamic homogenat,,s
Placental microsomes (0.017 mg of proteins) or hypothalamic homogenate (0.93 mg of proteins) were incubated for I h at 37°C with the same concentration (about 0.5 /.tM) of either [l/3-'~H]-andros tenedione (tritiated water production) or [I,2,6,7-N-3H]-andros tenedione (estrogen formation). Data are expressed as mean +S.D. In parentheses, the number of samples assayed. Products
Placental microsomes (nmol/mg protein) Hypothalamus (fmol/mg protein)
Tritiated water
Estrone + estradiol
3.71 + 0.32 (5)
2.96 + 0.53 (5)
27.14 + 4.53 (5)
19.41 + 2.95 (5)
330 40
TABLE !1 Aromatase actil'ity in cuhured neurons, mL~ed glial ('ells and type ! astrocytes and in isolated oligodendrocyws
in parentheses the number of determinations each consisting in a pool of two or more Petri dishes (about 0.1 mg of proteins/sample). The samples were incubated for 1 h at 37°C in the presence of [lfl--~H]-androstenedione (0.7/zM). Data are expressed as mean+ S.D.n.d. ffi not detectable.
I Km=52+20nM Vmax = 25.94 + 9.49 fmol3H20 1 mg protein I h
35 p.
.E 30
"~ 25 n
m
~' 2o ~ 0.10
_~ lO
Aromatase actii'ity fmol "~H,0 / mg protein
~e
0.05
5
Cultured Neurons
18.06 + 9.28 (19) !.01 + 1.75 (3) 1.01 + i.15 (7)
Mixed glial cells Type 1 astrocytes Isolated Oligodendrocytes
.;o -;o
0
0.0
o ,o
14S]
3'o " 4'o " s'o
i
i
l
l
0.2
0.4
0.6
0.8
,ml
.0
Androstenedione Odd)
Fig. 2. Michaelis-Menten constant (K m) and maximum velocity (Vm~x) of neuronal aromatase activity. Samples of pooled Petri dishes (about 0.1 mg of proteins) were incubated for 1 h at 37°C in the presence of [l/]-3H]-androstenedione (0.02-0.4 /~M). Inset: Lineweaver-Burke transformation of the data. V = fmol ~ H 2 0 / m g protein/h; [S] = [1 fl- 3H]-androstenedione concentrations (~t M). The data in the figure are derived from two independent experiments. K m and Vm,,x consequently represent the mean +S.D. of these two experiments. Moreover, curves perfectly superimposable to the one presented may be derived using the data of each experiment sepa rately.
n.d.
A) 40
tivity of the method. Moreover, oligodendrocytes are completely inactive in this respect. The kinetic parameters of the neuronal aromatase are shown in Figs. 1 and 2. The production of tritiated water is linear with increasing amounts of protein concentration up to 0.4 mg (R 2 - 0.99, Fig. IA) and is also directly correlated with the time of incubation up to 3 h (R 2 - 0.98, Fig. I B). In the experimental conditions adopted, the neuronal enzyme exhibits an apparent Michaelis-Menten constant (Kin) of 52 + 20 nM, with a maximum rate of aromatase activity (Vm,x) calculated to be 25.94 + 9.49 fmol/mg of proteins/h (Fig.
3O
I0
I
I
0,1
I
0,2 0,3 mg protein
I
I
0,4
05
B)
2). 40 35 30 25 20 15 10 5 ~" 0 0
DISCUSSION
RLo.976 i.
30
i
60
I
!
90 120 minutes
!
!
150
180
Fig. I. Linear regression analysis of the neuronal aromatase activity in function of protein content (A) and incubation time (B). Incubation conditions: A: samples from pooled Petri dishes (0.05-0.4 mg protein) were incubated for I h at 37°C in the presence of [I/3-3H]androstenedione ((I.5/zM). B: samples from pooled Petri dishes (0.18 mg protein) were incubated for different times at 37°C in presence of [ 1,8-3H]-androstenedione (0.6 p.M).
The results obtained in the experiments described here show that among the different cell populations tested only neurons seem to possess in a significant degree the enzyme involved in the aromatization of androgens. Moreover, the data also indicate that, in the present experimental conditions, the kinetics of the neuronal enzyme are linear with respect to the protein concentration and the incubation time; the formation of estrogens follows the Michaelis-Menten model. To the authors' knowledge, this paper provides the first direct evidence that, in the brain, the aromatase activity is practically localized only in neurons; in fact, the low levels of tritiated water recovered during the
331 incubation of androstenedione with mixed glial cells and with type 1 astrocytes are at the limit of the sensitivity of the method, and do not allow one to derive firm conclusions on the real presence of the enzyme in these cells. Moreover, oligodendrocytes seem to be completely devoid of aromatase activity. It must be pointed out that the total inactivity of oligodendrocytes might be due to the fact that these were obtained from adult rather than fetal-neonatal animals. Previous work had suggested that in the brain the aromatase activity decreases from embryonic life to adulthood ~3"2°. Moreover, oligodendrocytes were purified from the whole brain and not specifically from regions known to possess a rather elevated aromatase activity (e.g., amygdala, preoptic region, etc.). The present results agree with previous data by Canik and coworkers TM who used, however, a totally indirect approach. These authors described a 6-fold increase in the aromatase activity in primary cultures of fetal rat hypothalamic cells in which the neuronal component had been enriched by the addition to the medium of cytosine arabinoside, a factor which inhibits the proliferation of non-neuronal cells s. An analogous conclusion was reached by the same authors 7 on the basis of a reverse experiment; a dose-dependent decrease of the aromatase activity was found in hypothalamic cell cultures in which the neuronal component had been diminished by the addition to the cultures of kainic acid, a neurotoxic agent. As previously mentioned, prevalence of the aromatase within neurons has also been shown by immunocytochemical studies performed both in rats "~:~and in birds ~a. The aromatase present in neurons possesses a Michaelis-Menten constant (Km 52 4- 20 nM) which is not significantly different from that calculated for the human purified enzyme 'v, and for the aromatase present in brain homogenates of different animal species 26"28'32'36. This indicates that the culture conditions adopted do not modify the kinetic characteristics of the aromatase. Moreover, preliminary results obtained in our laboratory (data not shown) indicate that a known aromatase inhibitor (4-hydroxy-androstenedione), when added to the neuronal cultures at the beginning of the aromatase assay, practically abolishes the formation of tritiated water. The amounts of tritiated water formed by neurons (as evaluated by the Vmax) appear to be lower than those described for the aromatase present in embryonic or adult male rat hypothalamic fragments ~3 or in particular nuclei of the adult male rat brain 2~. Therefore it might be hypothesized that the aromatase is not equally expressed in all neuronal population, but might be more concentrated in neurons localized in particu-
lar regions of the CNS. In this respect, Sanghera and coworkers aa have recently shown that a polyclonal antibody raised against a synthetic peptide corresponding to a 20 aminoacid segment common to the rat, human and chicken aromatase, is able to immunostain with decreasing intensities the amygdaloid structures, the supraoptic nucleus, the paraventricular and arcuate nuclei as well as the hippocampus. Neurons in the bed nucleus of the stria terminalis, the medial basal hypothalamus and the preoptic area show little aromatase immunoreactivity. On the contrary, high yields of immunoreactivity were detected in specific brain regions not previously recognized to contain the enzyme (i.e., the reticular thalamic nucleus, the olfactory tract and the piriform cortex) a3. Comparing the data of the present paper with those obtained in this laboratory on the 5a-reductase activity 23, it appears that the two main androgen metabolizing pathways coexist only in neurons, while the glial component of the brain apparently utilizes androgens only via the 5a-reductase pathway. It is interesting that androgen and estrogen receptors are present in neurons 3'22. The intracellularly formed estrogens and 5areduced androgens could either exert separate actions in the neurons, or synergize with each other in the regulation of various neuroendocrine functions. In this context, it may be underlined that, in the rat, estradiol administration to castrated animals is able to increase androgen receptor levels in specific brain areas, such as the medial preoptic nucleus and the medial amygdala ~5, in which the aromatase has been shown to be present 2~. On the other hand, the aromatase activity of the rat brain appears to be under androgenic control probably through a receptor-mediated mechanism 3°'3~. It is then possible to postulate that DHT formed via the 5a-reductase pathway might increase the intraneutonal formation of estrogens.
Acknowledgemous. Thanks are due to Dr. R. Maggi, Department of Endocrinology, University of Milan, for providing the Macintosh version of the 'ENZYME' program. The experimentsdescribed here were supported by grants of the Consiglio Nazionale delle Ricerche through the following target projects: 'Biotechnologyand Bioinstrumentation' N. 91.0192PF20, 'Factors of disease' N. 91.002t)7PF4, 'Aging' N. 91.00419PF40 and through the bilateral project ItalyYugoslavia N. 90.01445.04. Such support is gratefullyacknowledged. REFERENCES I Balthazart, J., Foidart, A. and Harada, N., immunocytochemical localization of aromatase in the brain, Brain Res., 514 (1990) 327-333. 2 Balthazart,J., Foidart, A., Surlemont, C., Vockel,A. and Harada, N., Distribution of aromatase in the brain of the Japanese quail, Ring dove, and Zebra finch: an immunocytochemical study, J. Comp. Neuroi., 301 (1990) 276-288.
332 3 Blaustein, J.D. and Turcotte, J.C., Estrogen receptor-immunostaining of neuronal cytoplasmic processes as well as nuclei in the guinea pig brain, Brab~ Res., 495 (19891 75-82. 4 Bradford, M.M., A rapid and sensitive method for the quantitalion of microgram quantities of protein utilizing the principle of protein-dye binding, Anal, Biochem., 72 (19761 248-254. 5 Callard, G.V., Petro. Z. and Ryan, K.J.. Phylogenetical distribution of aromatase and other androgen-converting enzymes in the central nervous system, Endocrinology, 103 (19781 2283-2290. 6 Callard, G.V., Petro, Z. and Ryan, K.J., Conversion of androgens to estrogens and other steroids in the vertebrate brain, Am. Zool., 18 (19781 511-523. 7 Canik, J.A., Vaccaro, D.E., Livingston, E.M., Leeman, S.E., Ryan, K.J. and Fox, T.O., Localization of aromatase and 5a-reductase to neuronal and non-neuronal cells in the fetal rat hypothalamus, Brain Res., 372 (1986) 277-282. 8 Canik, J.A., Vaccaro. D.E., Ryan, K.J. and Leeman, S.E., The aromatization of androgens in primary cultures of fetal rat hypothalamus, Endocrinology, 100 (19771 250-253. 9 Celotti, F., Melcangi, R.C. and Martini, L., The 5alpha-reductase in the brain: molecular aspects and relation to brain function, in L. Martini and W.F. Ganong (Eds.), Frontiers in Neuroendocrino/ogy. Vol. 13, Raven, New York, 1992, pp. 163-215. l0 Chen, S., Beaman, M.J., Sparkes, R.S., Zollman, S., Kiisak, I., Mohandaa, T., Hall. P.F. and Shively, J.E., Human aromatase: eDNA cloning, Southern blot analysis, and assignment of the gene to chromosome 15, DNA, 7 (19881 27-38. Ii Corbin, C.J., Graham-l.x~rence. S., McPhaul, M., Mason, J.I., Mendelson, C.R. and Simpson, E.R., Isolation of a full-length eDNA insert encoding human aromatase system cytochrome P450 and its expression in non steroidogenic cells, Proc. Nat/. Acad. Sci. USA, 85 (1988) 8948-8952. 12 Dunnett, C.W., A multiple comparison procedure for comparing several treatments with a control, J. Am. Stat. Assoc., 50 (19551 1096-1121. 13 George, F W. ,'rod Ojeda, S.R., Changes in aromatase activity in the rat brain during embryonic, neonatal, and infantile development, l':mh)crino/ox~.', I I I (1982) 522-529. 14 Goy, R.W. and McEwen, B.S.. Se~'ual Diffi,rentiation of the Brain, MIT, Cambridge, 1980. 15 tlanda. R.J., Rosdli, C.E., |torton, L. and Resko, J.A., The quantitative distribution of cytosolie androgen receptors in nil. crodisseeted areas of the male rat brain: effects of estrogen treatments, l"ndocrinologv, 121 (1987) 233-240. 16 llarada N., Cloning of a complete eDNA encoding human aromatase: immunocytoehemieal identification and sequence analysis, Biochem. Biophys. Res. Commun., 156 (1988)725-732. 17 Kellis, J.T and Vickery, L.E.. Purification and characterization of human placental aromatase cytochrome P-450, J. Biol. Chem,, 262 (1987) 4413-4420. 18 Larsson, K., Features of the neuroendocrine regulation of masculine sexual behavior. In C. Beyer (Ed.), Endocrine Control of Seruai Behavior, Raven, New York, 1979, pp. 77-163, 19 Lutz, R.A, Bull, C. and Rodbard, D., Computer analysis of enzyme-substrate-inhibitor kinetic data with automatic model selection using IBM-PC compatible microcomputers, Enzyme, 36 (1986) 197-206. 20 MacLusky, N.J., Philip, A., Hulburt, C. and Naflolin, F,, Estrogen formation in the developing rat brain: sex difference in aromatase activity during early post-natal life, Psyconeuroemh)crinolog~.,, 10 (1985) 355-361. 21 McCarthy, K.D. and DeVellis, J., Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue, J. Cell. Bit)L, 85 (1980) 890-902. 22 McEwen, B.S., Bigeon, A., Davis, P.G., Krey, L.C., Luine, V,N., McGinnis, M.Y., Paden, C.M., Parsons, B. and Rainbow, C.M., Steroid hormones: humoral signals which alter brain cell properties and function, Rec. Prot,,. thmn. Res., 38 (1982)41-92,
23 Melcangi, R.C., Celotti, F., Ballabio, M., Castano, P., Massarelli, R., Poletti, A. and Martini, L., 5a-Reductase activity in isolated and cultured neuronal and glial cells of the rat, Brab~ Res., 516 (1990) 229-236. 24 Naftolin, F., MacLusky, N.J., Leranth, C.Z., Sakamoto, H.S. and Garcia-Segura, L.M., The cellular effects of estrogens on neuroendocrine tissues, J. StcroM Biochem., 30 (19881 195-207. 25 Negri Cesi, P., Celotti, F. and Martini, L., Androgen metabolism in the brain: role in sexual differentiation and in the control of gonado!ropin secretion, Monogr. Neural Sci., 12 (19861 7-16. 26 Negri Cesi, P., Celotti, F. and Martini. L., Androgen metabolism in the male hamster: 2. Aromatization of androstenedione in the hypothalamus and in the cerebral cortex; kinetic parameters and effect of exposure to different photoperiods, J. Steroid Biochem., 32 (19891 65-70. 27 Palkovitz, M., Isolated removal of hypothalamic or other brain nuclei of the rat, Braht Res., 59 (19731 449-450. 28 Roselli, C.E., Ellinwood, W.E. and Resko, J.A., Regulation of brain aromatase activity in rats, Endocrinology, 114 (19841 192200. 29 Roselli, C.E., Horton, L.E. and Resko, J.A., Distribution and regulation of aromatase activity in the rat hy0othalamus and limbic system, Endocrinology, ! 17 (1985) 2471-2477. 30 Roselli, C.E. and Resko, J.A., The distribution and regulation of aromatase activity in the central nervous system, Steroids, 50 (1987) 495-508. 31 Roselli, C.E., Salisbury, R.L. and Resko, J.A., Genetic evidence fur an androgen-dependent and independent control of aromatase activity in the rat brain, Endocrinology, 121 (1987) 2205221(}. 32 Roselli, C.E., Stadelman, H., Horton, L.E.' and Resko, J.A., Regulation of androgen metabolism and luteinizing hormone-releasing hormone content in discrete hypothalamic and limbic areas of male Rhesus macaques, Endocrinology, 120 (19871 97106. 33 Sanghera, M.K., Simpson, E.R., McPhaul, M.J., Kozlowski, G., Conley, A.J. and Lephart E.D., lmmunocytochemical distribution of ammatase cytochrome P-450 in the rat brain using peptidegenerated polyclonal antibodies, Endocrinology, 129 (1991)28342844. 34 Santncr, S.I., Rosen, H., Osawa, Y, and Santen, R,J., Additive effects of aminoglutethimide, testonolacton¢, and 4-hydroxyandrostenedione as inhibitors of aromatasc, J. Steroid Biochem., 2(1 (19841 1239-1242. 35 Schumaker, M. and Balthazart, J., Neuroanatomieal distribution of testosterone metabolizing enzymes in the Japanese quail, Braba Res., 422 (1987) 137-148. 36 Schumaker, M., Contenti, E. and Balthazart, J., Partial characterization of testosterone-metabolizing enzymes in the quail brain, Brain Res., 305 ( 19841 5 !-59. 37 Selmanoff, M.K., Bmdkin, L.D., Weiner, R.I. and Siiteri, P,K., Aromatization and 5a-reduction of androgens in discrete hypothalamic and limbic regions of the male and female rat, Endocrinok~gy, 101 (1977) 841-848. 38 Sholl, S.A. and Kim, K.L., Aromatase, 5a-reductase and androgen receptor level in the fetal monkey brain during early development, Neuroendocrinology, 52 (19901 94-98. 39 Simmons, D.L., Lalley, P.A. and Kasper, C.B., Chromosomal assignments of genes coding for components of the mixed-function oxidasc system in mice, J. Biol. Chem., 260 (19851 515-521. 40 Simpson, E.R., Merril, J.C., Hollub, A.J., Graham-Lorence, S. and Mendelson, C.R., Regulation of estrogen biosynthesis by human adipose cells, Endocr. Rec., 10 (19891 136-148. 41 Thompson Jr,, E.A. and Siiteri, P.K., Utilization of oxygen and reduced nicotinamide dinucleotide phosphate by human placental microsomes during aromatization of androstenedione, J. Biol. Chem., 249 (197415364-5372.
