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
3. Results
All immunoblotting procedures were carried out at room
3
temperature. Dr
Harada Fujita
Health University,
3.1. H-estradiol binding activity assay
Toyoake, Aichi, Japan kindly furnished anti-aromatase
3
antibody. The anti-aromatase antibody, previously char- H-estradiol binding activity was detected in the cytosol
G . Guerriero et al. Brain Research 880 2000 92 –101
95
3
Fig. 1. Saturation A and Scatchard analysis B of H-estradiol binding in the cytosol of the female frog Rana esculenta hypothalamus. Only the specific binding is shown in the Scatchard plot. These results are representative of three different experiments carried out during each period of the
reproductive cycle pre-breeding, breeding and recovery. Kd values did not show any significant variation. j, specific binding; d, total binding; s, non-specific binding.
and nuclear extract of the female frog hypothalamus. for 17b-estradiol. In the nuclear extract 17b-estradiol was
Saturation was reached around a value of 2.5 nM of the only competitor. In the cytosol, 17b-estradiol was the
3
H-estradiol when the total protein content was kept at 2 best competitor, followed by DES, which competed,
210
mg ml. The average Kd value was 7.561.1310 M and
although at a lesser extent. T, P and C did not compete
210
6.160.9310 M in the cytosol and nuclear extract,
effectively in either the cytosol or in the nuclear extract
3
respectively Figs. 1 and 2. Kd values did not show any Fig. 3. H-estradiol binding specificity experiments were
significant variation throughout the reproductive cycle performed once for each period of the year prebreeding,
3
data not shown. H-estradiol binding moiety was specific breeding, and recovery and gave similar results.
3
Fig. 2. Saturation A and Scatchard analysis B of H-estradiol binding in the nuclear extract of the female frog Rana esculenta hypothalamus. Only the specific binding is shown in the Scatchard plot. These results are representative of three different experiments carried out during each period of the
reproductive cycle pre-breeding, breeding and recovery. Kd values did not show any significant variation. j, specific binding; d, total binding; s, non-specific binding.
96 G
3
Fig. 3. Specificity of H-estradiol binding in the cytosol open bar and nuclear extract black bar of the female frog Rana esculenta hypo-
thalamus. The percent binding is calculated as the specific binding in the presence of a 1,000-fold excess of competitor B divided by the specific
binding in the absence of competitor B 3100. The data represent the
3
means of duplicate determinations. H-estradiol binding specificity experi- ments were performed once for each period of the reproductive cycle and
Fig. 5. Competitive inhibition of AA in the microsomes of the female gave similar results.
frog Rana esculenta hypothalmus by 1,4,6-androstatriene-3,17-dione ATD at 30 and 300 nM. These results are representative of three
different experiments carried out during each period of the reproductive cycle prebreeding, breeding, recovery. Substrate concentration 1b-
3.2. Aromatase activity assay
3
Handrostenedione5300 nM.
3
The validity of the H O assay for use with the female
2
frog hypothalamus was tested by saturation analysis and competitive inhibition with ATD. Fig. 4 shows that
3.3. Western blotting aromatase activity in female frog hypothalamic micro-
somes exhibited typical Michaelis–Menten kinetics. The In Fig. 6 the immunoblotting of aromatase in the female
apparent Km was 4.2 nM and the V was 471 fmol
frog hypothalamus of Rana esculenta is reported. The
max 3
H O h mg protein. When microsomes were incubated in antibody cross-reacted with a single band with an apparent
2
two different concentrations of ATD, activity V was
mol.wt of 56 kDa both in the hypothalamus of Rana
max
decreased but the apparent Km was unaffected as would be esculenta in the three periods of the reproductive cycle
expected by competitive inhibition with 30 and 300 nM of lanes 1, 2, 3 and in the human placenta lane 4, used as
ATD Fig. 5. a positive control. No immunoreactive bands were detected
Fig. 4. Effect of substrate concentration on AA in the female frog Rana esculenta hypothalamus. Duplicate aliquots of microsomes were incubated for 1
3
h at 258C in 10 mM potassium phosphate buffer containing [1b- H] androstenedione. The reaction was terminated by the addition of 10 cold
3
trichloroacetic acid containing charcoal ml. Insert: Lineweaver–Burke plot of initial velocity of H O formed vs. concentration of androstenedione.
2
G . Guerriero et al. Brain Research 880 2000 92 –101
97
Fig. 6. Western blot analysis of Rana esculenta hypothalamus and human placenta as positive control. Female frogs captured in prebreeding lines
1; 5, breeding lines 2; 6, recovery lines 3; 7 periods; human placenta 4; 8. Samples were transferred onto nitrocellulose and incubated with
either the polyclonal antibody against aromatase lines 1, 2, 3, 4 or rabbit IgG as a control lines 5; 6; 7; 8. The position of marker proteins is
indicated on the left. Western blots were performed on four different pools of animals with consistent results. Insert, quantitative analysis of band
optical densities 1, prebreeding; 2, breeding; 3, recovery periods.
in samples when normal rabbit IgG was used in place of the specific antibody lanes 5, 6, 7, 8. To determine if the
level of aromatase protein expression changed over the reproductive cycle, quantitative analysis of sample relative
optical density was performed Fig. 6, insert. The results indicated an increase in expression between prebreeding
period band 1 and the breeding and recovery periods bands 2 and 3, ranging from 1.2 to 1.28, respectively. No
significant differences were measured between aromatase expression in the breeding band 2 and recovery band 3
periods.
3
3.4. Aromatase and H-estradiol binding activity and fluctuations throughout the reproductive cycle
3
Both aromatase and H-estradiol binding activity in the nuclear extract of the hypothalamus of the female of Rana
esculenta were low during the prebreeding period and increased during the breeding and recovery periods P,
3
0.05. H-estradiol binding activity in the cytosol was very
3
Fig. 7. Aromatase activity A and H-estradiol binding levels in the
low at any time of the reproductive cycle and did not show
cytosol B and nuclear C extracts of the female frog Rana esculenta
any significant variation Fig. 7.
hypothalamus during each period of the reproductive cycle prebreeding, breeding, recovery. Three groups of pooled hypothalamic regions were
analyzed for each period of the reproductive cycle. Asterisks indicate
3.5. Plasma steroid levels and ovarian weight
significant difference from the prebreeding period P,0.05. Values are
fluctuations
means6S.D.
The annual pattern of plasma steroids and ovarian weight variations throughout the reproductive cycle of
Rana esculenta are shown in Fig. 8. Plasma androgen showed the highest concentration during the recovery
levels attained the highest values during the prebreeding period. The ovarian weight was high during the prebreed-
and breeding periods. 17b-estradiol level was lower than ing period and decreased during the breeding period
androgens at any time of the reproductive cycle, and because of egg deposition. A progressive increase occurred
98 G
during the recovery period, due to vitellogenesis resump- tion.
4. Discussion
