Cell type- and region-specific expression of aromatase mRNA in cultured brain cells

MOLECULAR BRAIN RESEARCH ELSEVIER

Molecular Brain Research 24 (1994) 153-158

Research Report

Cell type- and region-specific expression of aromatase m R N A in cultured brain cells Sumiko Abe-Dohmae a, Ryo Tanaka a, Nobuhiro Harada b,. a Department of Biochemistry, Nagoya City University Medical School, Mizuho-ku, Nagoya 467, Japan b Division of Molecular Genetics, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi 470-11, Japan

(Accepted 18 January 1994)

Abstract

The expression of aromatase mRNA in cultured mouse brain cells was measured by a quantitative reverse transcription-PCR method using an internal standard. Aromatase mRNA was expressed in the cultured neurons prepared from diencephalon at 0.037 + 0.005 attomol//xg total RNA. However, the mRNA was detected in neither the neurons from cerebral cortex nor astrocytes. These results demonstrate that expression of aromatase m R N A is regulated in cell type- and region-specific manners in cultured brain cells. The aromatase mRNA levels in neurons obtained from diencephalon were not affected by administration of testosterone, estradiol, dexamethasone, forskolin, or 12-O-tetradecanoyl 13-acetate. The results are in apparent disagreement with previous reports concerning regulation by androgens of brain aromatase activity in vivo and may suggest that aromatase expression in brain neurons is not directly induced by androgens. Androgen induction of brain aromatase may be mediated by several steps including cell-cell (neuron-neuron a n d / o r neuron-glia) interaction. Key words: Aromatase; Estrogen synthetase; Neuron; Diencephalon; Reverse transcriptase-polymerase chain reaction technique;

Culture

I. Introduction

Expression of sexual patterns of reproductive behavior and gonadotropin secretion in rodents is well known to be imprinted by neonatal exposure to sex steroids [1]. Many studies have revealed that androgens are involved in the sexual differentiation of the neuron number and size in Specific brain regions, and in the regulation of reproductive function and copulatory behavior [1]. Conversion of androgenic precursors to estrogens in the neurons of specific brain regions is believed to be crucial for many effects of androgen on these biological events [12,19]. Therefore, aromatase (estrogen synthetase; E C 1.14.14.1), which catalyzes aromatization of androgens to estrogens, is actually the decisive limiting factor for physiological actions of androgens in the brain. Aromatase has b e e n found not only in gonads but

* Corresponding author. Fax: (81) 562-93-8833. 0169-328X/94/$07,00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0169-328X(94)00014-6

also in extra-gonadal tissues, such as the liver [26], brain [20], skin [25], and adipose tissue [17]. A r o m a t a s e expression has been shown to be regulated in a tissuespecific m a n n e r by numerous factors including cAMP, dexamethasone, phorbol ester, insulin, and other growth factors [7,18,22]. A r o m a t a s e expression in the brain is unique. It is known that brain aromatase is not regulated by most of the regulatory factors of aromatase in other tissues. Androgens are the only factor known to control aromatase in the brain [23,24]. To elucidate the regulatory mechanism of aromatase expression in the brain, the expression levels of aromatase m R N A in cultured ceils p r e p a r e d from definite regions of mouse brain were investigated. Since the levels were too low to be determined by conventional Northern analysis, we employed a sensitive quantitation method that measured fluorometrically the P C R products which were obtained by reverse-transcription of aromatase m R N A and amplification of its c D N A with a fluorescent primer [9,10]. We show that aromatase expression in the brain cells was regulated in cell type- and region-specific manners.

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2. Materials and methods

2.3. Cell culture

2.1. Experimental animals

The neurons of diencephalon and cerebral cortex were prepared from El5 brains. The fetuses were aseptically removed from the uterus and transferred into chilled medium A. The brains were excised in the medium with the aid of a dissecting microscope as follows: At first, a coronal cut at the mesencephalic flexure was made. Then, the cerebral hemispheres were separated from the dorsal surface of diencephalon and cut out at the level of the interventricular foramina. The meningeal layer and surface blood vessels were removed, and the resulting tissue was used as diencephalon. Cerebral cortex was obtained from the cerebral hemispheres as described above by collecting the portions adjacent to the lateral ventricles after removal of meninges and blood vessels. Neural cells were dissociated by the method of Hatanaka and Tsukui [11] with minor modifications. Tissues were cut into about 1 mm 3 pieces with a pair of scalpels. After papain digestion (0.15 U/ml, Worthington) for 30 min at 37°C, 5 ml of medium B were added to the mixture, and the tissue fragments were sedimented by gravity. The pellet was resuspended in medium B and the cells were dissociated by pippetting repeatedly. The resulting cell suspension was filtered through lens paper (Fuji Photo Film) to remove undissociated cells. The cells were plated at 2× 106 cells per 35 mm dishes precoated with poly-i.-lysine (MS-CI006L, S.B. Medical Co.) in 2 ml of medium B. They were cultured for 24 h in humidified 5% CO2-95% air atmosphere at 37°C, and a half volume of the medium was replaced with medium C in order to suppress the growth of glial cells. After

Timed pregnant ICR mice (SPF) were purchased from Nippon SLC Co. (Hamamatsu, Japan). Embryonic day 0 (E0) was designated as the day when a vaginal plug was detected. 2.2. Reagents The culture media were prepared as follows. DF medium: 50% (v/v) Dulbecco's modified Eagle's medium and 50% (v/v) Ham's FI2 medium containing 15 mM HEPES buffer and 365 mg/ml L-glutamine (Sigma) and supplemented with 1.2 mg/ml sodium bicarbonate (Sigma), 100 U / m l penicillin G (Meiji Seika), and 100 p~g/ml streptomycin sulfate (Meiji Seika). Medium A: 50% (v/v) DF medium and 50% (v/v) Dulbecco's phosphate-buffered saline (Sigma). Medium B: DF medium with 10% (v/v) bovine calf serum (Cell Culture Laboratories). Medium C: DF medium containing with 5/xg/ml bovine insulin (Sigma), 5 p~g/ml human transferrin (Sigma), 5 ng/ml sodium selenite (Sigma), 20 nM progesterone (Sigma), and 10 /xM cytosine arabinoside (Ara-C, Sigma). Testosterone, estradiol, dexamethasone, fi)rskolin, and 12-O-tetradecanoyl 13-acetate (TPA) were purchased from Sigma. They were dissolved in dimethylsulfoxide (Dojindo) at the concentration of 20 mM, 10 mM, 1 mM, 10 mM, and 1 mM, respectively, and were kept as stock solutions at -20°C.

Fig. 1. Phase micrography of neurons. Diencephalon was dissected out from pooled El5 fetal mouse brains, and the cells dispersed by papain were cultured for 6 days with Ara-C as given in Materials and methods. Bar = 100/xm.

S. Abe-Dohmae et al./ Molecular Brain Research 24 (1994) 153-158 the ceils had been cultured for another 72 h, and a half volume of the medium had been exchanged again with 1 : 1 mixture of medium B and medium C containing the tested compounds, they were harvested on the designated days for total RNAs preparation. The cultured cells were shown to be neurofilament-positive ( > 95%) by immunocytochemistry with rabbit anti-bovine neurofilament antibody (Chemicon International Inc.). Astrocytes were prepared from El5 mouse brains as we reported previously [13] with slight modifications. Briefly, the tissue fragments of diencephalon and cerebral cortex prepared as described above were dissected out with a pair of scalpels and pooled, and the cells were dissociated with 0.05% trypsin-0.53 mM EDTA (Gibco) for 15 min. at 37°C. The cells were seeded into tissue culture flasks (Falcon, surface area 25 cm2) in DF medium containing 20% fetal calf serum (FCS, Whittaker Bioproducts, Inc.) (20% FCS/DF) under the same atmosphere as above at 37°C for 48 h without disturbance. Then the medium was changed with 20% FCS/DF and the cells were cultured for another 5 days. The cells were subcultured into 100 mm tissue culture dishes (Falcon) in DF medium supplemented with 10% FCS (10% FCS/DF). After 20 h, the medium was changed with 10% FCS/DF to eliminate the neuronal cells, which were still floating in the medium, leaving behind the glial cells. The cells were incubated for another 6 days with twice changes of medium and the resulting confluent monolayer astrocytes were harvested for total RNA preparation. The cultured cells were shown to be glial fibrillary acidic protein-positive (> 95%) by immunocytochemistry with rabbit antibovine glial fibrillary acidic protein antibody (Biomedical Technologies Inc.).

2.4. Quantitative analysis of aromatase mRNA The total RNA fraction was isolated according to the method of Chirgwin et al. [6]. The aromatase mRNA levels were determined by reverse-transcription of total RNA and PCR amplification of the resulting cDNA with a fluorescent primer as previously described [9]. Usually, 10 p~g of total RNA and 0.5 attomol of internal standard RNA were used as the templates. PCR was repeated for 24 cycles. The detection threshold was 0.001 attomol//~g of total RNA.

3. Results 3.1. Regional and cellular distribution o f aromatase m R N A in the brain P ri o r to m e a s u r e t h e a m o u n t o f a r o m a t a s e m R N A , effect o f A r a - C on t h e p r o l i f e r a t i o n o f glial cells w e r e examined. Neurons from diencephalon and cerebral cortex w e r e p r e p a r e d a n d c u l t u r e d for 6 days as described in M a t e r i a l s a n d M e t h o d s w i t h or w i t h o u t Ara-C. Proliferation and formation of interconnecting n e t w o r k of n e u r a l p r o c e s s e s (Fig. 1) w e r e o b s e r v e d in all cases. T h e a b s e n c e o f A r a - C a ll o w e d glias to prolife r a t e b e n e a t h n e u r o n s ( d a t a n o t shown). O n t he o t h e r hand, few typical glial ceils or fibroblast-like cells app e a r e d in t h e m e d i u m c o n t a i n i n g A r a - C (Fig. 1). T o obtain t o t al R N A f r a c t i o n f r o m n e u r o n - e n r i c h e d cultures, accordingly, A r a - C was a d d e d to t h e m e d i u m for neurons thereafter. T o d e t e r m i n e w h e t h e r a r o m a t a s e m R N A is exp r e s s e d in cell type- a n d r e g i o n - s p e c i f i c m a n n e r in

155

Table 1 Cell type- and region-specific expression of aromatase mRNA Cell Neuron Astrocyte

Aromatase mRNA (attomol//.Lg of total RNA) Diencephalon

Cerebral Cortex

0.037 + 0.005 N.D,

N.D. N.D.

Diencephalon and cerebral cortex were dissected out from El5 mouse brains. Neurons and astrocytes from these tissue fragments were prepared as described in Materials and methods. Total RNA fractions of neurons and astrocytes were isolated after culture for 4 days and 2 weeks, respectively. The aromatase mRNA levels were determined by reverse transcriptase-PCR method. The data are shown as the mean +_S.E.M. of 4 experiments, each derived from the pools of 11 to 49 animals. N.D., not detected.

c u l t u r e d b r ai n cells, a r o m a t a s e m R N A levels w e r e m e a s u r e d in n e u r o n s a n d astrocytes p r e p a r e d f r o m d i e n c e p h a l o n an d c e r e b r a l cortex. A s sh o w n in T a b l e 1, a r o m a t a s e m R N A was e x p r e s s e d only in t h e n e u r o n s p r e p a r e d f r o m d i e n c e p h a l o n at 0.037 a t t o m o l / / x g total RNA, whereas those from cerebral cortex exhibited u n d e t e c t a b l e level o f a r o m a t a s e m R N A u n d e r t h e s a m e c u l t u r e co n d i t i o n s. T h e a r o m a t a s e m R N A level in astrocytes c u l t u r e d for 14 days was also l o w e r t h a n d e t e c t i o n t h r e s h o l d ( T a b l e 1).

3.2. Effects o f various compounds on expression o f aromatase m R N A A s a r o m a t a s e in t h e b r ai n is k n o w n to be i n d u c e d in vivo by a d m i n i s t r a t i o n o f a n d r o g e n s [23,24], w e e x a m i n e d w h e t h e r this i n d u c t i v e e f f e c t o f a n d r o g e n s d e r i v e s f r o m t h e i r d i r e c t a c t i o n to n e u r o n s . T h e n e u r o n s f r o m d i e n c e p h a l o n w e r e c u l t u r e d with v a r i o u s c o m p o u n d s an d t h e a r o m a t a s e m R N A levels in t h e s e cells w e r e m e a s u r e d . A s sh o w n in T a b l e 2, n o t only e s t r a d i o l , d e x a m e t h a s o n e , forskolin, or T P A b u t also testost e r o n e as an a n d r o g e n gave no c h a n g e in t h e e x p r e s -

Table 2 Effects of various compounds on aromatase mRNA levels Additions (final concentration)

Aromatase mRNA (attomol//xg of total RNA) Protocol A

Protocol B

No addition Testosterone (20/aM) Estradiol (10/aM) Dexamethasone (1/xM) Forskolin (10/aM) TPA (1/aM)

0.031 0.031 0.031 0.031 0.030 0.029

0.029 0.029 0.029 0.028 0.028 0.027

Neurons from pooled 41 diencephala were divided to 16 groups. They were cultured for 4 days as described in Materials and methods and were exposed to various compounds indicated. Total RNA fractions were prepared after 24 h (protocol A) or 48 h (protocol B) stimulation, and processed for aromatase mRNA quantitation.

S. Abe-Dohmae et al. / Molecular Brain Research 24 (1994) 153-158

156 ~, z (3c

rv" ~

0.05 0.04

0.03 0.02

E -5

o.ol

< o

0.00

2E

2

4

6

Culture time

8

10

[day]

Fig. 2. Culture time-dependent decrease of aromatase m R N A in neurons from diencephalon. Diencephalon was obtained from pooled 49 E l 5 fetal mouse brains, and the cells dispersed by papain were cultured as given in Materials and mMethods. After 4-day culture, a half volume of the m e d i u m was exchanged with 1:1 mixture of medium B and medium C containing with (filled circle) or without (open circle) 2 0 / z M testosterone. The aromatase m R N A levels were determined at the days indicated.

sion levels of aromatase m R N A in cultures 24 h (protocol A) or 48 h (protocol B) after drug administration.

3.3. Changes of aromatase mRNA levels during culture As shown in Table 2, the aromatase mRNA levels in the neurons from diencephalon cultured for 5 days (protocol A) were almost same as those in the cells maintained for an additional 24 h (protocol B). Since the aromatase m R N A levels in the mouse brain are known to increase developmentally [9] during the perinatal period with a transient peak on neonatal days 3 to 4, the aromatase m R N A levels in the neurons from diencephalon of E l 5 mouse were measured at different times during cell culture. Unexpectedly, the aromatase m R N A levels decreased in a time-dependent manner (Fig. 2). The addition of 20 /zM testosterone, an in vivo inducer of brain aromatase, did not affect the aromatase m R N A levels in the cells.

4. Discussion

Aromatase m R N A was detected only in the neurons from diencephalon. As shown in Table 1, the aromatase m R N A level in diencephalic neurons cultured for 4 days was 0.037 + 0.005 attomol/tzg of total RNA, indicating that they have almost the same content of aromatase m R N A as the diencephalon of perinatal mouse [9]. By contrast, aromatase m R N A was not detectable in the neurons prepared from cerebral cortex and in astrocytes. The detection threshold of our quantitative analysis was 0.001 attomol//~g of total RNA. Inasmuch as the R N A content of typical mammalian ceils is thought to be about 1 /zg R N A / 1 0 5 cells, this assay can theoretically detect expression of

even one molecule of aromatase m R N A in more than 100 cells. Thus, aromatase m R N A is expressed, if at all, at an extremely low level in the astrocytes and neurons obtained from cerebral cortex. We reported in a previous paper that aromatase m R N A was detected not only in the diencephalon but also in the cerebral cortex [9]. The absence of aromatase m R N A in the neurons prepared from cerebral cortex in this study (Table 1) might result from the preparation procedure of the cerebral cortex without amygdala (see Materials and Methods), in which the presence of aromatase activity [28] and immunoreactivity [2] were reported. The results obtained in this study provide the first evidence that aromatase m R N A is expressed in cell type- and region-specific manners. Although a trace of aromatase activity has been reported in cultured glial cells of rat brain [21], the present results are consistent with previous immunocytochemical studies that showed the presence of brain aromatase of mouse [2] and quail [3] only in the neurons of restricted regions. The aromatase m R N A level in the neurons from diencephalon did not respond to testosterone (Table 2). Testosterone has been considered to be responsible for the in vivo induction of brain aromatase [23,24]. Studies using catalytic assay in microdissection [24], immunocytochemistry, and quantitative analysis of aromatase m R N A [8,10] have revealed that aromatase expression in the quail brain is pretranslationally regulated by testosterone. We also found that the aromatase m R N A level in the diencephalon but not in other parts of forebrain was increased by intraperitoneal injection of testosterone to adult mice [29]. Therefore, the data obtained in this study suggest that testosterone may not directly regulate expression of aromatase m R N A during the neonatal period but may regulate it rather in an undirect way with step(s) including 1) cell to cell or cell to extra cellular matrix interaction, 2) humoral factors produced by neighboring cells, a n d / o r 3) stimulus from upper neurons via synaptic transmission. Alternatively, the expression of aromatase m R N A in the fetal brain may not respond to sex steroids as was observed in the brain of adult mouse. Indeed, the effects of gonadal sex steroids on prenatal developmental expression of brain aromatase are still largely unknown. Transplacental administration of the antiandrogen flutamide failed to reduce aromatase activity in ferret fetal brain except in the bed nucleus of the stria terminalis of males [27]. All other compounds examined including estradiol, dexamethasone, forskolin, and T P A did not affect the aromatase m R N A level in diencephalic neurons, either (Table 2). No other substance than androgens has been shown to increase brain aromatase. Although the compounds which increase intracellular cAMP concentration have been reported to induce [4] or suppress [5,14] aromatase activity in cultured brain cells, the effect on

S. Abe-Dohmae et al. /Molecular Brain Research 24 (1994) 153-158

brain aromatase is still controversial. In the present study, the treatment with forskolin for either 24 h or 48 h did not affect the aromatase m R N A levels in cultured diencephalic neurons (Table 2). The differences in species, developmental stages at which cells were prepared, and culture conditions may contribute to these discrepancies. We have previously demonstrated that the aromatase m R N A level in mouse brain is regulated developmentally with a peak at 3 - 4 days after birth [9], which was supposed to be the marginal period of neonatal imprinting of sexual differentiation [16]. Similar results were also obtained in rat brain [15]. However, the aromatase m R N A level in the neurons from E l 5 diencephalon was decreased in a time-dependent manner in vitro (Fig. 2). In this study, Ara-C was added to the neuronal cultures to suppress the glial proliferation. Ara-C also might have some cytotoxic effects on the neurons. However, the decrease in cell number and in the recovery of total R N A during the culture was not so significant as compared with that in aromatase m R N A level (data not shown). From these facts, it may be deduced that the observed decrease in the aromatase m R N A was caused rather from the lack of physiological cell-cell interactions in the culture than from the cytotoxic effects of Ara-C. Further study is now in progress to determine the inductive mechanisms of the aromatase m R N A expression in the brain.

[6]

[7]

[8l

[9]

[10]

[11]

[12] [13]

[14]

[15]

Acknowledgments We wish to thank Drs. Yasuyuki Takagi and Kazuyo Yamada for helpful discussions and encouragement during this study and Mrs. Noriko Yoshimura for technical assistance. This work was supported in part by a Grant-in-Aid for Research from Fujita Health University, and a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan.

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