Materials and methods analysis [55] increasing amounts 0.3–5.0 nM of
G . Guerriero et al. Brain Research 880 2000 92 –101
93
than in males [59]. Aromatase activity AA has been the anterior commissure. Hypothalamic regions were
detected in the preoptic area, hypothalamus and amygdala pooled into three groups of about 20 animals each and
of Rana catesbeiana [11], and aromatization seems to be stored in liquid nitrogen until use. Plasma was obtained
involved in the regulation of male sexual behavior in Rana after centrifugation of the blood at 8003g for 10 min, at
pipiens [7]. Autoradiography and immunocytochemical 48C and stored at 2208C until use. Ovaries were dissected
studies demonstrated, respectively, widespread distribution out and weighed.
of androgen and estrogen-concentrating nerve cells in hypothalamic and limbic structures of Xenopus laevis
2.2. Preparation of microsomal pellet, cytosol and [30,38] and in the urodele Taricha granulosa [14]. How-
nuclear extract ever, a biochemical approach to characterize and quantify
sex steroid receptors was not utilized in any of these All operations were carried out at 48C. Analytical grade
vertebrate studies. chemicals were used. Minced tissues were weighed and
Schlinger and Callard [56] demonstrated in quail that homogenized with 1:4 w v of TEMG 10 mM Tris–HCl,
hypothalamic aromatase regulates testosterone-induced 1 mM EDTA, 1 mM 2-mercaptoethanol and 10 glycerol,
aggressiveness by regulating the quantity of estradiol pH 7.8 containing 0.05 M NaCl homogenization buffer.
available for receptor binding. This was the first demon- The suspension was centrifuged at 8003g for 10 min. The
stration of a quantitative relationship between aromatiza- recovered supernatant was centrifuged at 105,0003g for
tion and estrogen receptor occupancy. It is not currently 60 min. The pellet of this centrifugation was employed for
known whether this relationship exists in the brain of AA detection and is indicated as microsomal pellet, the
amphibians. In Rana esculenta, a distinctive feature of supernatant constituted the cytosol. The 8003g pellet
female reproductive endocrinology is the relatively high crude nuclear pellet was suspended and washed three
level of circulating androgens during the reproductive times with homogenization buffer. Smears of the last pellet
cycle [42,44]. Although some hypotheses have been were inspected by a phase-contrast microscope to assess
advanced to explain the role of androgens in the female the presence and integrity of nuclei. Thereafter, the pellet
frog [16], the role of aromatization has not been addressed. was suspended in 1:4 w v TEMG containing 0.7 M KCl
Thus, the present study was undertaken in order to test the extraction buffer. The suspension was left 1 h in an ice
hypothesis that testosterone acts through conversion to bath, with continuous stirring. It was then centrifuged at
estradiol and subsequent binding to nuclear estrogen 105,0003g for 60 min. The supernatant constituted the
receptors. For this reason, we investigated both the pres- nuclear extract.
ence and amount of both AA and estradiol binding
3
molecules in the hypothalamus of the female frog, Rana 2.3.
H-estradiol binding assays esculenta, during the reproductive cycle.
3
[2,4,6,7] H-estradiol 90 110 Ci mmol was purchased from Amersham Buckinghamshire, UK. For Scatchard
3
2. Materials and methods analysis [55] increasing amounts 0.3–5.0 nM of
H- estradiol were incubated with aliquots 200 ml of cytosol
2.1. Animals and nuclear extract in the presence or absence of 100-fold
excess of unlabeled 17b-estradiol Sigma, St Louis, MO Adult female frogs of Rana esculenta were collected
to determine total and non-specific binding, respectively. from the surrounding of Naples during the following
Incubations were carried out for 16 h at 48C. Bound and periods of the reproductive cycle: prebreeding February to
free steroids were separated by adding a suspension of March; breeding from April to June; recovery, the
charcoal-dextran 0.5–0.05 w v Pharmacia, Piscata- period when vitellogenesis resumes in the ovaries from
way, NJ. The mixture was vortexed and kept in ice bath October to January. About 60 adult females were captured
for 5 min [41]. It was then centrifuged at 8003g for 10 for each of the above reported periods. Animal mainte-
min. The supernatant was transferred to vials containing nance and all surgical and experimental procedures were
4.5 ml Maxifluor scintillation fluid Packard, Milan, Italy. conducted in accordance with the European Communities
Radioactivity was measured in a Packard spectrometer Council Directive of 1986, 86 809 EEC. Soon after
Packard 1600 at 45 counting efficiency. Data of capture, the animals were anesthetized with MS 222
specific binding were plotted according to Scatchard Sigma, St Louis, MO. Animals were bled with a heparin-
graphical procedure. For steroid specificity experiments, coated glass capillary inserted into the heart. Successively
aliquots 200 ml of cytosol and nuclear extract were
3
frogs were sacrificed by decapitation, brains were immedi- incubated with 5 nM
H-estradiol in the presence or ately dissected out and the hypothalamic region was
absence of 1,000-fold excess of the unlabeled steroids sectioned under a stereomicroscope using the following
diethylstilbestrol DES, testosterone T, progesterone P margins: anterior, septo-mesencephalic tract; posterior,
and corticosterone C Sigma, St Louis, MO. The where the third nerve enters the brain; dorsal, the level of
incubation and bound from free steroid separation pro-
94 G
cedure were carried out as reported before. Because of the acterized, was a polyclonal antibody raised in rabbit,
3
low levels of H-estradiol binding molecules in the brain, directed against human placenta aromatase, and purified by
Scatchard analyses were used for single binding assay [35]. ammonium sulfate fractionation and affinity chromatog-
Values were extrapolated from Scatchard analysis per- raphy [24]. About 63 mg of total protein was used for each
formed on three different pools of animals captured during lane. Samples were electrophoresed on a SDS–PAGE 8
the main periods of the reproductive cycle. Values of total acrylamide according to Laemmli [31], and electrotrans-
receptors were obtained from the intercept with the X-axis, ferred onto a 0.45 mm nitrocellulose membrane Schleicher
normalized for the total protein content and expressed as and Schuell, Keene, USA with a transblot cell apparatus
fmol mg protein. Bio-Rad. Membranes were blocked overnight by incuba-
tion in 10 non-fat milk powder suspended in 20 mM Tris 2.4. Measurement of AA
pH 7.6 containing 137 mM NaCl and 0.1 Tween-20 TBS. Membranes were then washed three times with
To measure AA, the microsomal pellet the pellet of the TBS and incubated for 60 min in TBS containing Harada’s
first ultracentrifugation at 105,0003g was sonicated in aromatase antibody 1 mg ml diluted at 1:5000. Parallel
1:30 w v phosphate buffers 10 mM KPO , 100 mM strips were incubated in normal rabbit IgG. After three
4
KCl and 1 mM EDTA, pH 7.4. The incubation mixture, washes in TBS, membranes were stained in horseradish
which contained NADPH-generating system and 0.3 mM peroxidase-labelled goat antirabbit IgG. Proteins were
3
[1b- H] androstenedione NEN Research Products, DuP- detected using enhanced chemiluminescence ECL Amer-
ont Co., Boston, MA; SA, 25.4 Ci mmol in 100 ml of sham, Life Science, Arlington Heights, IL. Human
phosphate buffer, was pre-incubated at 378C for 30 min to placenta was used as a positive control for the presence of
generate sufficient NADPH for the aromatase reaction. aromatase. The negative control included incubation with
Aliquots 100 ml of hypothalamic microsome samples and normal rabbit IgG. Quantitative analysis of the autoradiog-
human placental microsomes that were used as positive raphic bands of interest was performed using Multi-
controls, were then added and the incubation was carried Analyst software Bio-Rad.
out for 1 h at 258C. These incubation conditions were adapted from a previous study in frogs by Callard et al.
2.6. Androgens and 17b-estradiol measurement in the
3
[48], and were within the linear range for H O production plasma
2
data not shown. The reaction was stopped with 400 ml 10 trichloroacetic acid containing 20 mg charcoal ml.
The steroid content in plasma samples was assayed by
3
The H O generated was purified on Dowex minicolumns RIA methods, adapted to Rana esculenta plasma [18]. The
2
Bio-Rad Laboratories, Richmond, CA. Tubes containing intraassay and interassay coefficient of variation were 7
3
incubation buffer and a known amount of H O were
and 13, for androgens and 5 and 10 for 17b-es-
2
carried through the entire procedure to correct for re- tradiol, respectively. Testosterone antibodies reacted also
coveries mean6S.E.M., 95.863.6; n59. The tritium with dihydrotestosterone about 80, therefore the term
was counted at about 30 efficiency for sufficient time to androgens will be used throughout the paper. Dr Bolelli
3
give 5 counting error. Details of the H O assay used in CNR, Physiopathology of Reproduction Service, Uni-
2
our laboratory have been published previously [50]. To versity of Bologna, Italy provided androgen and 17b-
3
validate the H O assay for use in the frog brain, aliquots estradiol antibodies.
2
of hypothalamic microsomes were incubated with increas-
3
ing amounts of [1b- H] androstenedione e.g. 6.2–200 2.7. Measurement of proteins
3
nM and the amount of H O generated was measured and
2
corrected for counts in buffer blanks. The data obtained Proteins were determined by the method of Lowry et al.
were plotted as a saturation plot and the maximal velocity [33], using BSA as standards.
V and Michaelis constant Km were derived by
max
non-linear regression analysis. In addition, incubations 2.8. Statistical methods
containing serial dilutions of substrate from 12.5 to 200 nM were carried out in the presence of inhibitor 1,4,6-
Data were analyzed with one-way analysis of variance androstatriene-3,17 dione ATD at concentrations of 30
ANOVA. Duncan’s multiple range tests were applied to and 300 nM, with or without the substrate itself.
determine which means differed significantly. 2.5. Western blotting