General and Comparative Endocrinology 102, 342 – 350 (1996)
Article No. 0077
3b-Hydroxysteroid Dehydrogenase/Isomerase and
Aromatase Activity in Primary Cultures of
Developing Zebra Finch Telencephalon:
Dehydroepiandrosterone as Substrate
for Synthesis of Androstenedione
and Estrogens
Ana Vanson,* Arthur P. Arnold,*,† and Barney A. Schlinger†
Departments of *Psychology and †Physiological Science and the Laboratory of Neuroendocrinology of the Brain
Research Institute, University of California, Los Angeles, California 90024
Accepted January 25, 1995
3b-hydroxysteroid dehydrogenase/D50D4 isomerase
(3b-HSD) activity was measured in primary dissoci-
ated cell cultures prepared from telencephalons of de-
veloping zebra finches. 3b-HSD activity was confirmed
after cultures were incubated with [7-3H]pregneno-
lone (Preg) or (1,2,6,7-3H-) dehydroepiandrosterone
(DHEA) and 3H-progesterone (Prog) and 3H-andro-
stenedione (AE) were detected in the medium. Product
identity was confirmed by recrystallizations and by
HPLC analysis. When DHEA was used as substrate,
3
H-estradiol and 3H-estrone were also detected in the
culture medium, presumably derived from the aroma-
tization of 3H-AE or 3H-T produced from 3H-DHEA. To
test this idea, cultures were incubated with 3H-DHEA
together with radioinert AE or with fadrozole HCl, a
potent and specific aromatase inhibitor. In the pres-
ence of radioinert AE, 3H-AE increased but metabolites
of 3H-AE decreased in the media; in the presence of
fadrozole, 3H-estrogens decreased but 3H-AE and its
androgenic metabolite 3H-5b-androstanedione in-
creased. These data demonstrate 3b-HSD activity in
the songbird brain. The presence of Prog and estradiol
in these cultures suggest that Preg and DHEA can po-
342
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tentially serve as substrates for the ultimate formation
of active sex steroids in the songbird telencephalon.
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3b-hydroxysteroid dehydrogenase/isomerase (3b-
HSD) is a critical enzyme of steroidogenic tissues such
as the gonads, adrenals, and placenta where it catalyzes
the oxidation and isomerization of the D5-3b-hydroxy
steroids [pregnenolone (Preg) and dehydroepiandrost-
erone (DHEA)] into D4-keto steroids [progesterone
(Prog) and androstenedione (AE), respectively] (Sam-
uels et al., 1951; Fig. 1). 3b-HSD activity or its mRNA
have also been detected at lower levels in a variety of
other tissues including liver, kidney, skin, and brain
(Bain et al., 1991; Zhao et al., 1991; Dalla Valle et al.,
1992; Keeney et al., 1993). Although its role in these
tissues is not firmly established, it is likely to catalyze
reactions that locally activate or inactivate circulating
substrates (La Brie et al., 1988). The presence of 3b-HSD
activity in brain (Knapstein et al., 1968; Weidenfeld et
al., 1980; Jung-Testas et al., 1989; Bauer and Bauer, 1989;
Mensah-Nyagan et al., 1994)) is especially interesting.
In mammals, there is evidence that brain can accumu-
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3b-HSD in Avian Brain Cell Cultures
FIG. 1. Simplified diagram illustrating principle enzymes that catalyze the conversion of cholesterol to estradiol. Steroids are given in bold;
enzymes are underlined and placed in parentheses (P450scc Å cytochrome P450 side-chain cleavage; P45017a Å cytochrome P450 17a-hydroxy-
lase/C17-C20 lyase; HSD, hydroxysteroid dehydrogenase).
late Preg or DHEA or their conjugates, possibly through
local synthesis (Corpechot et al., 1981, 1983; Robel et al.,
1987). Consequently, substrate may be available for 3b-
HSD-catalyzed synthesis of Prog or AE, steroids that
are either active in brain or that can be metabolized by
brain into active compounds (McEwen et al., 1983; Paul
and Purdy, 1992). Recently, 3b-HSD activity and immu-
noreactivity were detected in the brain of an amphibian
(Mensah-Nyagan et al., 1994), but little is known of its
activity in other vertebrates.
We have been interested in the possible presence of
steroidogenic enzymes, including 3b-HSD, in the song-
bird brain. In some species of songbird, aromatase (es-
trogen synthetase) is expressed at very high levels in the
telencephalon (Vockel et al., 1990; Schlinger and Arnold,
1991; Schlinger and Arnold, 1992a; Shen et al., 1994,
1995), but the endogenous substrate for brain aromati-
zation is unknown. Aromatase is expressed also at ex-
tremely high levels in primary cell cultures of devel-
oping zebra finch telencephalon (Schlinger et al., 1994,
1995; Wade et al., 1995). Since cell culture of mammalian
brain has proven to be an especially effective condition
for studying other enzymes of steroid synthesis or me-
tabolism (e.g., Jung-Testas et al., 1989), we are interested
in determining if 3b-HSD is present in similar cultures
from the songbird brain. Our present report of 3b-HSD
activity in zebra finch telencephalic cultures implies
that the conversion of Preg to Prog or DHEA to AE
can occur in the songbird brain. AE synthesized from
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343
DHEA is then available as substrate for aromatization
since its metabolites, estrone (E1) and estradiol (E2), ac-
cumulate in these cultures. These data suggest that the
zebra finch brain may utilize Preg or DHEA from blood
as a substrate for synthesis of active androgens or estro-
gens. Alternatively, de novo steroidogenesis may be a
property of the zebra finch telencephalon. Some of these
data were reported previously in Abstract form
(Schlinger et al., 1992).
MATERIALS AND METHODS
Preparation of Primary Cultures
Male and female zebra finch chicks, 1 – 5 days after
hatching, were decapitated, and the entire brain or tel-
encephalon was dissected from the skull. To dissect
the telencephalon, a pair of forceps was inserted under
aseptic conditions horizontally between the telence-
phalic lobes and the optic lobes at the caudal pole of
the brain and passed rostrally so that they were at the
base of the brain at levels rostral to the optic commis-
sure. In this manner the telencephalon was pinched off.
Primary cultures of zebra finch brains were prepared
essentially by the methods described earlier (Schlinger
et al., 1994). Briefly, cells from several male and female
brains were dissociated by gently teasing the tissue
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344
through a nylon mesh (210 mm) into a petri dish con-
taining medium. The cells were then filtered through
two stainless steel mesh filters (230 mm then 140 mm
mesh, Bellco), centrifuged for 10 min at 1100 rpm, resus-
pended in medium, and plated into plastic culture
dishes precoated with polylysine or fibronectin. Cul-
tures were incubated at 377, 5% CO2 in Dulbecco’s Mod-
ified Eagle’s Medium (DMEM)/Ham’s F12 (1:1, Irvine
Scientific, Santa Ana, CA), supplemented with fetal calf
serum (10%), Hepes Buffer (3.38 g/liter), NaHCO3 (2.16
g/liter), gentamicin (0.09 g/liter), and D-glucose (3.6 g/
liter). Cells were plated at one of three densities: 20, 10,
or 5 paired telencephalons per 100 ml. After plating,
cells were left undisturbed for 3 days, after which the
medium was replaced every 2 or 3 days. All experi-
ments were conducted on cells grown for 7 to 30 days
in vitro (d.i.v.).
Radioenzymatic Assays for 3B-HSD Activity
3b-HSD activity was determined by measuring the
conversion of [7-3H]-Preg (NEN; sp act 25 Ci/mmol) to
3
H-Prog or the conversion of (1,2,6,7-3H-) DHEA (NEN;
sp act 86.6 Ci/mmol) to 3H-AE. Cultures of zebra finch
telencephalon were incubated with 3H-Preg (65, 130, or
430 nM) for 2.5, 5, 6, 24, or 48 hr or with 3H-DHEA
(62.5 or 130 nM) for 2.5 or 24 hr. Radiolabeled substrates
were purified by thin layer chromatography (TLC)
prior to use (see below). To ‘‘trap’’ formed products, in
some experiments radioinert product was included in
the reaction media. Control wells containing radiola-
beled substrate in media without cells were used to
calculate background values. Procedural losses were es-
timated by adding approximately 100,000 cpm of 3H-
Preg, (1,2,6,7-3H)AE (85 Ci/mmol), or (6,7-3H-)E1 (60
Ci/mmol) to parallel control wells.
To measure product formation, media were extracted
three times with ethyl ether (2 ml). In experiments using
3
H-Preg as substrate, extracts were chromatographed
twice on TLC plates in chloroform:ethyl acetate (3:1)
with radioinert Preg, Prog, 17a-Preg, 17a-Prog, AE, and
testosterone (T) as carriers. Primulin and long-wave ul-
traviolet light were used to visualize preg. Prog was
visualized under short-wave ultraviolet light. In experi-
ments using 3H-DHEA as substrate, ether extracts were
subjected to double phenolic partition with CCl4 and
0.1 N NaOH (1:1). CCl4 fractions were chromato-
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Vanson, Arnold, and Schlinger
graphed twice on TLC plates in chloroform:ethyl ace-
tate (4:1) to separate DHEA, AE, T, 5b-androstanedione,
and 5a-androstane-3b,17b-diol. AE and T were rechro-
matographed in ether:hexane (3:1) and visualized with
short-wave ultraviolet light. NaOH fractions were ex-
tracted three times with ethyl acetate (2 ml). Extracts
were then chromatographed with radioinert E1 and es-
tradiol (E2) twice on TLC plates in ether:hexane (3:1).
Estrogens were visualized by exposure to iodine va-
pors. Silica bands on TLC plates were scraped and
eluted with 15% methanol. The eluate was added to
scintillation vials containing Biofluor (New England
Nuclear) and radioactivity was counted using a Pack-
ard Tri-Carb Model 1900 CA Liquid Scintillation Ana-
lyzer. Cells were harvested to determine protein con-
tent using the Bradford protein assay (Bradford, 1976).
Data are reported as amount of steroid produced/mg
cell protein. We used 2X background as the criterion
for detectable product formation.
We used two methods to verify product identity.
First, radioactivity eluted from TLC plates was recrys-
tallized (three times in ethanol/dH2O) to constant spe-
cific activity. Second, radioactivity eluted from TLC
plates was shown to coelute with radioinert standards
using high pressure liquid chromatography (HPLC).
Separation of steroid hormones by HPLC was based on
procedures previously described (Edmond et al., 1991;
Erkoc et al., 1989). For HPLC, radioactivity and radioin-
ert steroid (10 – 100 mg) in ethanol were evaporated un-
der N2 and redissolved in aqueous acetonitrile (50% for
AE, E1 or E2; 10% for Prog). A total volume of 50 ml
was applied to the HPLC column after equilibration
with the appropriate solvent mixture. The column con-
taining AXXI-CHROM C-18 [ODS], 5-mm particle size,
and was 4.6 mm i.d. 1 250 mm long (Cole Scientific,
Calabasas, CA) and was fitted with a compatible RP-
18 Newguard precolumn, 3.2 1 15 mm (Brownlee Labs,
Santa Clara, CA). The column was operated at 347 in an
HPLC system composed of a model 110A Altex pump,
Rheodyne injection valve with 100 ml injection loop,
Hitachi model 100-30 spectrophotometer, Shimadzu
model C-RIA Chromatopak, and Eldex column temper-
ature control unit (Cole Scientific). Solvent was pumped
through the column at a flow rate of 0.6 ml/min. Ra-
dioinert steroids were detected at 220 nm. Fractions (1
ml/min) were collected in glass vials using an LKB
model 212 Redirac fraction collector. All HPLC proce-
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3b-HSD in Avian Brain Cell Cultures
FIG. 2. Percentage of original substrate remaining after incubation
of primary, dissociated cell cultures of developing zebra finch whole
brain with Preg or telencephalon with DHEA or Prog. Data for Preg
(closed circles); DHEA (open circles) and Prog (plus signs) were de-
rived from separate experiments using cells grown in 75-cm2 flasks
(Preg), or in 24-well plates (DHEA, Prog), at final concentrations of
62.5 nM (DHEA), 65 nM (Preg), or 230 nM (Prog).
dures were first verified by analysis using radioinert
and radiolabeled tracers.
RESULTS
Conversion of Pregnenolone to Progesterone
3
H-Preg (65 nM in 2 ml) was metabolized extensively
by primary cultures prepared from whole zebra finch
brain after 7 d.i.v. (Fig. 2). Numerous reaction products
were formed during a 24-hr incubation, appearing as
numerous peaks of radioactivity on TLC plates after
chromatography using chloroform/ethyl acetate. Some
radioactivity was seen to comigrate with radioinert
Prog, but afterwards this radioactivity did not crystal-
lize together with radioinert Prog. When 3H-Prog (230
nM) was added to similar telencephalic cultures, we
found that it too underwent extensive metabolism (Fig.
2). Therefore, in subsequent experiments in 24-well
plates with 3H-Preg, radioinert Prog (1 to 3 mg) was
included in the incubation media (300 ml/well) to pro-
tect newly formed 3H-Prog from endogenous metabo-
lism. Under these conditions, radioactivity eluted from
regions of the chromatoplates corresponding with Prog
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345
was successfully recrystallized (Table 1). Using HPLC,
radioactivity presumed to be Prog (pooled from Expts.
1, 2, and 3) migrated as a single peak together with
radioinert Prog providing additional evidence that au-
thentic 3H-Prog was formed under these conditions (not
illustrated). This peak accounted for 87.3% of the radio-
activity injected into the HPLC column. Under these
cell culture conditions, maximum Prog accumulation
was seen in the presence of 3 mg cold Prog after incuba-
tion for 24 hr with 130 nM 3H-Preg or after 6 hr with
430 nM 3H-Preg (Table 1). No 3H-Prog was measured
after 2.5-h incubations with or without cold Prog.
Conversion of DHEA to AE and Metabolites of AE
Primary cell cultures of developing zebra finch telen-
cephalon (32 d.i.v.) also rapidly metabolized 3H-DHEA
(62.5 nM/300 ml medium in 24-well plate; Fig. 2) pro-
ducing numerous reaction products. Under these con-
ditions, 3H-AE accumulated in the presence or absence
of radioinert AE (Table 2). 3H-AE authenticity was con-
firmed by recrystallization to constant specific activity
(Table 2) and by HPLC analysis. Metabolites of 3H-AE
were also identified in some cultures incubated with
3
H-DHEA. These included 3H-E1 , 3H-E2 , and 3H-5b-
androstanedione (Table 3). The identity of these prod-
ucts was also confirmed by recrystallization to constant
specific activity (Table 3), and, in the case of the estro-
gens, by HPLC analysis. Radioactivity comigrating with
androstenediol was also present on TLC plates under
all conditions, suggesting that 17b-HSD was also ex-
pressed in these cell cultures. 3b-HSD can catalyze the
conversion of 3H-androstenediol to 3H-T which could
then serve as substrate for the formation of 3H-E2 . How-
ever, only small amounts of radioactivity ever comi-
grated with T, and the quantities were insufficient to
verify their identity.
Presumably, the 3H-estrogens present in these cul-
tures were derived from 3H-AE (or 3H-T) synthesized
from 3H-DHEA. However, to assess this directly we
examined the ratio of 3H-AE to 3H-AE metabolites (3H-
E1 , 3H-E2 , and 3H-5b-A) produced from 3H-DHEA in
the presence or absence of cold AE (Fig. 3). In the ab-
sence of cold trap, metabolites of 3H-AE always pre-
dominated over 3H-AE in the incubation media. Cold
AE always reversed this ratio, accumulation of 3H-AE
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346 Vanson, Arnold, and Schlinger

TABLE 1
3b-HSD Activity in Telencephalic Cultures of Developing Zebra Finches: 3H-Pregnenolone Conversion to 3H-Progesterone
3
H-
3
Experiment Pregnenolone Incubation H-Progesterone Recrystallizations
No. concentration time (hr) Cold ‘‘trap’’ (fmol/mg protein) (dpmcrystals/dpmmother liquor)

1 65 nM 24 1 mg Prog 734.4 373.1/352.6

(86.0% recovery)
2 130 nM 24 1 mg Prog 1200.0
3 mg Prog 4600.0 180.9/177.9
(62.8% recovery)
3 430 nM 6 3 mg Prog 2300.0 378.2/393.1
(77.9% recovery)
24 3 mg Prog ND
48 3 mg Prog 1050.0

increased and metabolites of 3H-AE were either absent
or present at very low levels (Fig. 3).
As a further test of the androgenic substrate of aroma-
tization in these cultures, we incubated duplicate cul-
tures with the aromatase inhibitor fadrozole hydrochlo-
ride (Ciba-Geigy). We have previously found that this
compound is a potent and highly specific inhibitor of
aromatase in these cell cultures (29). In the presence of
fadrozole and 3H-DHEA, estrogen synthesis was con-
siderably reduced after 2.5- or 24-hr incubations (Fig.
4). Because 3H-AE was present in greater abundance in
the presence of fadrozole, it is likely that 3H-AE, pro-
duced by the action of 3b-HSD on 3H-DHEA, was the
ultimate substrate for aromatization.
DISCUSSION
These data demonstrate the presence of 3b-HSD ac-
tivity in cell cultures derived from the telencephalon of
developing zebra finches. They suggest that this en-
zyme is expressed naturally in the brain of these birds
during development and perhaps also in adulthood. To
the best of our knowledge, these are the first observa-
tions of this enzyme in the brain of any bird species.
Since activity, mRNA or immunoreactivity have been
detected previously in the mammalian and amphibian
brain (Jung-Testas et al., 1989; Bauer and Bauer, 1989;
Mensah-Nyagan et al., 1994; Dupont et al., 1994), these
observations suggest that the expression of 3b-HSD is
a conserved property of the vertebrate brain.
The coexpression of aromatase and 3b-HSD together
in these cell culture preparations provides a mechanism
for the use of DHEA as substrate for the formation of
active estrogens. This observation is especially interest-
ing to us. Estrogens have significant organizational and
activational effects on the songbird brain (Arnold,
1992). Aromatase is expressed at very high levels in the

TABLE 2
3b-HSD Activity in Telencephalic Cultures of Developing Zebra Finches: 3H-Dehydroepiandrosterone conversion to 3H-Androstenedione
3
H- Recrystallizations
3
Experiment H-DHEA Incubation Androstenedione (dpmcrystals/
No. concentration time (hr) Cold ‘‘trap’’ (fmol/mg protein) dpmmother liquor)

1 62.5 nM 2.5 5 mg AE 56.2 420.3/427.5

(87.2% recovery)
2 130.0 nM 24 0 mg AE 420.8 450.1/467.4
(95.1% recovery)
30 mg AE 332.4 407.2/416.7
(97.6% recovery)
3 130.0 nM 24 30 mg AE 408.2 487.7/489.6
(100.0% recovery)
4 130.0 nM / dbcAMP 24 30 mg AE 2932.0 620.0/631.7
(82.6% recovery)

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3b-HSD in Avian Brain Cell Cultures
TABLE 3
3
H-Estrogens Crystallized from Cultures Incubated with 3H-Dehydroepiandrosterone
3 3
Experiment H-DHEA Incubation
No. concentration time Cold ‘‘trap’’
1 130 nM 24 hr
telencephalon of adult and in developing birds (Vockel
et al., 1990; Schlinger and Arnold, 1991, 1992a), but it
is expressed poorly in nonneural tissues of males
(Schlinger and Arnold, 1991, 1992a). Since the estrogen
content of blood is enriched by its passage through the
brain in males, we assume that the brain is a major
source of circulating estrogens in these birds (Schlinger
and Arnold, 1992b, 1993). However, since castration re-
duces circulating testosterone (Vockel et al., 1990; Ad-
kins-Regan et al., 1990), but not circulating estrogens
(Schlinger and Arnold, 1991; Adkins-Regan et al., 1990),
apparently the brain can receive aromatizable androgen
from sources other than the gonads.
FIG. 3. 3H-AE and its metabolites (3H-E1 , 3H-E2 , and 3H-5b-A) pro-
duced after 24 hr from 3H-DHEA (130 nM ) in the presence or absence
of radioinert AE (30 mg/flask; 3 mg/well). Cultures (14 to 22 d.i.v.)
were prepared in flasks (75 cm2/ Exp. 1; 25 cm2/Exp. 2) or in 24-well
plates (Exp. 3; 3 wells pooled for analysis). In the absence of cold
trap, metabolites of 3H-AE always predominated over 3H-AE in the
incubation media. In the presence of radioinert AE accumulation of
3
H-AE increased and metabolites of 3H-AE were either absent or
present at very low levels.
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347
Recrystallizations
H-Estrogen (dpmcrystals/dpmmother
(fmol/mg protein) liquor)
None E1-1370.8 698.2/690.3
(100.0% recovery)
None E2-389.4 435.9/467.8
(100.0% recovery)
The data presented here provide evidence for two
possible sources of aromatizable substrate. One possi-
bility is that DHEA is secreted by the avian adrenals
and is used as substrate for the formation of active
androgens and estrogens by the brain. Alternatively,
aromatizable substrate might be synthesized directly
by the brain itself. Cytochrome P450scc has been de-
tected in the mammalian brain (Jung-Testas et al., 1989;
Le Goascogne et al., 1987; Mellon and Deschepper, 1993)
implying that pregnenolone can be made available to
brain from de novo synthesis. However, there is limited
evidence for expression of cytochrome P450 17a-hy-
droxylase/c17-20 lyase (P45017a) in brain (Compagnone
et al., 1995; but see Mellon and Descepper, 1993; Le
Goascogne et al., 1991) implying that in general, andro-
gen reaches the brain only after peripheral synthesis.
FIG. 4. Androgens, A (3H-AE, 3H-5b-A), and estrogens, E (3H-E1 ,3H-
E2), formed in telencephlic cell cultures (30 d.i.v. in 24-well plates)
incubated in duplicate with 3H-DHEA (130 nM ) in the presence or
absence of the aromatase inhibitor fadrozole HCl (1.3 mM ). Exposure
to fadrozole reduced estrogen and increased androgen levels after
2.5- and 24-hr incubations.
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348
We do not yet know if the songbird brain expresses
any steroid synthetic enzymes other than 3b-HSD and
aromatase. However, since steroids are metabolized at
high rates by the songbird telencephalon (Schlinger and
Arnold, 1991; Schlinger et al., 1994), we are eager to
search for the additional steroidogenic enzymes
P450scc and P45017a .
It is also possible that 3b-HSD might be expressed
in neural tissue to reduce the concentration of active
androgens. In the presence of a hydrogen donor, 3b-
HSD can catalyze the conversion of the potent andro-
gen, 5a-DHT, into the inactive androgen androstene-
diol (Bain et al., 1991). Elimination of active androgens
might protect androgen-sensitive brain regions from in-
appropriate exposure to androgen. Using similar telen-
cephalic cultures, we have found unusually high activ-
ity of both aromatase and 5b-reductase (Schlinger et al.,
1995; Wade et al., 1995). Because of their high affinity for
androgenic substrate (aromatase) and high abundance,
these enzymes effectively reduce 5a-reduction at physi-
ological concentrations of androgen. Since the metabo-
lites of these reactions are nonandrogenic, these reac-
tions also may be expressed in zebra finch brain to
interfere with androgen action. 3b-HSD might act fur-
ther to inactivate 5a-DHT that is formed.
It is also possible that the expression of these enzymes
in brain may have little to do with steroid action via
classic intracellular mechanisms, but may instead syn-
thesize steroids that act directly on membranes. For
example, two metabolites of progesterone, 5a- and 5b-
pregnan-3a-ol-20-one, bind directly to GABAA recep-
tors causing increased currents though the associated
Cl-channels (Lambert et al., 1990). Although we do not
know if the avian brain expresses 3a-hydroxysteroid
dehydrogenase, both 5a- and 5b-reductase are ex-
pressed widely in the songbird brain, including many
regions that lack sex-steroid receptors (Vockel et al.,
1990; Wade et al., 1995). These enzymes might partici-
pate in converting progesterone into neuroactive me-
tabolites. Moreover, at least one of these compounds,
5a-pregnan-3a-ol-20-one, strongly potentiates GABA
actions in neurons cultured from the developing zebra
finch telencephalon (Hales and Schlinger, unpub-
lished). Thus, it remains possible that progesterone,
synthesized in brain from Preg, serves as substrate for
the formation of neuroactive steroids. Since the zebra
finch telencephalic cultures show extensive metabolism
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Vanson, Arnold, and Schlinger
of Prog, Preg, and DHEA, it is possible that some of
these metabolites might be neuroactive steroids.
In these experiments, we focused on two principle
enzymatic reactions, 3b-HSD and aromatase. How-
ever, it is clear that the substrates for 3 b-HSD (Preg
and DHEA) as well as their products (Prog and AE)
underwent substantial metabolism. We have charac-
terized only a few of these products, concentrating on
those that might participate in classical sex-steroid-
dependent functions: Prog, T, and E2 . Because several
enzymes presumably act on Preg and DHEA, and
because we focused only on identifying active sex-
steroid metabolites, we do not know the true velocity
of 3b-HSD in these cultured cells. Because of these
multiple conversions, 3b-HSD activity is likely to be
greater than what we detected. Nevertheless, under
these conditions, 3b-HSD is a relatively minor reac-
tion. Only a fraction of the added radioactive sub-
strate appeared as products of 3b-HSD and many
other products were also formed. Thus, 3b-HSD may
be expressed by only a few cells or at low levels by
many cells. To understand the role of 3b-HSD in the
songbird telencephalon, we need to identify the cells
that express 3b-HSD immunoreactivity or mRNA,
and whether these cells also express aromatase im-
munoreactivity or mRNA.
In conclusion, these results contribute to the body of
evidence demonstrating the presence of 3b-HSD in the
vertebrate brain. The data collected in these studies
were all derived from cultures of developing zebra
finch telencephalon. We do not know if this enzyme is
expressed in the songbird brain in vivo or if the activity
we see in culture is the result of abnormal gene expres-
sion in these cells under these conditions. Also, we do
not know if 3b-HSD is expressed outside of the telen-
cephalon. However, in as much as the song control
circuitry, located predominantly in the telencephalon
of songbirds, is a rich target of steroid hormones
(Schlinger, 1994), these results invite further investiga-
tion of 3b-HSD and other steroid synthetic enzymes in
this brain region in vitro and in vivo.
ACKNOWLEDGMENTS
We thank Dr. John Edmond for use of his HPLC and for his assistance
with this procedure, Ciba-Geigy for donating fadrozole HCl, and Dr.
endoas AP: ENDO
3b-HSD in Avian Brain Cell Cultures
Jane Lubischer for comments on the manuscript. Supported by NSF
Grant IBN 9120776 (to B.A.S) and NIH Grant DC00217 (to A.P.A).
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