Endocrinology Vol. 140, No. 1 112-117
Copyright © 1999 by The Endocrine Society
Androgenic Regulation of Hypothalamic Aromatase Activity in Prepubertal and Postpubertal Male Golden Hamsters1
Russell D. Romeo2,
Juli Wade,
Jane E. Venier and
Cheryl L. Sisk
Department of Psychology, Neuroscience Program, Michigan State
University, East Lansing, Michigan 48824
Address all correspondence and requests for reprints to: Cheryl L. Sisk, Department Psychology, Neuroscience Program, Michigan State University, East Lansing, Michigan 48824. E-mail:
sisk{at}pilot.msu.edu
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Abstract
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Doses of testosterone that fully activate male reproductive behavior in
castrated adult male hamsters fail to elicit mounting and intromissions
in prepubertal castrates, even when circulating levels of testosterone
are equivalent in the two age groups. We hypothesize that this
differential responsiveness to testosterone is mediated at least in
part by the efficacy with which testosterone in the hypothalamus is
aromatized to estradiol, an important hormone mediating male sexual
behavior. Therefore, hypothalamic aromatase activity, as measured by
the conversion of [3H]testosterone to
[3H]estradiol in tissue homogenates, was assessed in four
separate experiments: 1) intact prepubertal and adult male golden
hamsters, 2 and 3) castrated adult or prepubertal males that received
either a 0- or 2.5-mg dose of testosterone, and 4) castrated adult and
prepubertal males treated with the 2.5-mg dose of testosterone. These
studies demonstrate that hypothalamic aromatase activity is
significantly higher in adult males compared with prepubertal males,
and that hypothalamic aromatase activity is increased by testosterone
to the same extent in both the adult and prepubertal male hamster.
Therefore, the failure of testosterone-treated castrated prepubertal
male hamsters to engage in the full suite of male reproductive
behaviors is not due to the inability of testosterone to be converted
into estradiol in the hypothalamus. Differences in the ability of
testosterone to increase aromatase activity in other brain regions, or
differences in the action of testosterone and/or estradiol on other
cellular processes must account for the inability of testosterone to
facilitate male reproductive behavior in juvenile males.
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Introduction
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TESTOSTERONE (T) is converted
intracellularly to estradiol (E2) in peripheral and central
tissues by the aromatase enzyme. In the brain, estrogenic metabolites
of T play a major role in the expression of male sexual behavior (1, 2). For example, systemic injections of E2 benzoate to
castrated male hamsters induce mounting behavior (3), whereas aromatase
inhibitors decrease copulatory behaviors (4). The aromatase enzyme is
present in brain regions that mediate male sexual behavior, such as the
amygdala and hypothalamus (5, 6, 7), and appears to be regulated by
androgens in some regions of the hypothalamus (5, 7–10; but see Ref.
6).
The capacity to engage in steroid-dependent reproductive behavior
increases during pubertal maturation. Not only is there an increase in
circulating levels of T during this time, but responsiveness of neural
circuits to the behavioral actions of T increase as well. For example,
in male hamsters, doses of exogenous T that fully activate male
reproductive behavior in castrated adult male hamsters fail to elicit
mounting and intromissions in castrated juveniles, even when
circulating levels of T are equivalent in the two age groups (11). We
hypothesize that this increased behavioral responsiveness to T in
adults is mediated, at least in part, by the efficacy with which T is
aromatized to E2 in the hypothalamus. This hypothesis leads
to two related predictions. First, in intact males, aromatase activity
within the behavioral neural circuit should be greater in adults than
in juveniles. Second, T treatment of castrated males should increase
aromatase activity to a greater extent in adults than in juveniles,
which would result in higher local concentrations of E2 to
activate male reproductive behavior in adults. As a test of this
hypothesis, aromatase activity, as measured by the conversion of
[3H]T to [3H]E2, was assessed
in hypothalamic homogenates obtained from intact and from castrated and
T-treated adult and prepubertal male golden hamsters.
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Materials and Methods
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Animals and housing
Male golden hamsters (Mesocricetus auratus) were bred
at Michigan State University (E. Lansing, MI) from stock obtained from
Charles River Laboratories, Inc. (Kingston, NY). Animals
were weaned at 21 days of age and at that time housed singly in clear
polycarbonate cages (37.5 x 33 x 17 cm) with wood chips as
substrate (Aspen Chip Laboratory, Warrensburg, NY) and with
ad libitum access to both food (Teklad Rodent Diet No. 8640;
Harlan Bioproducts for Science, Inc., Madison, WI)
and water. The animal colony was maintained at 21 ± 2 C and the
light-dark schedule was 14 h light/10 h dark (lights on at
0600 h EST). All animals were treated in accordance with the NIH
Guide for the Care and Use of Laboratory Animals, and protocols were
approved by the Michigan State University All-University Committee for
Animal Use and Care.
Experimental design
Four experiments were conducted because the number of samples
that can be run in a single assay is limited. Exp 1 characterized the
amount of hypothalamic aromatase activity in untreated, intact
prepubertal and adult male hamsters. In this experiment, 63-day-old
(adult, n = 8) or 28-day-old (prepubertal, n = 8) male
hamsters were weighed and rapidly decapitated. Hypothalami, blood
samples, and testes were collected as described below. Exps 2–4
investigated the effects of T on hypothalamic aromatase activity before
and after puberty in male hamsters. Exp 2 assessed the effects of T on
aromatase activity in adult males. Adult males (60 days of age) were
castrated under methoxyflurane anesthesia and implanted with a 3-week
time-released pellet (Innovative Research of America,
Sarasota, FL) containing either 0 mg (n = 7) or 2.5 mg of T
(n = 7). One week after castration and implantation, hamsters were
weighed, rapidly decapitated, and hypothalami and blood samples were
collected as described below. Previous work has shown that aromatase
activity is maximally increased within a week of T treatment (12).
Furthermore, the difference in T-stimulated sexual behavior observed in
prepubertal and adult male hamsters is observed 1 week after T
treatment (11). Exp 3 assessed the effects of T on aromatase activity
in juvenile males. In Exp 3, prepubertal males (21 days of age) were
castrated and implanted with either a 0 mg (n = 7) or 2.5 mg
(n = 6) pellet of T. One week after treatment, tissues were
collected as in Exp 2. Exp 4 directly compared the effect of T on
aromatase activity in juvenile and adult males. Prepubertal (21 days of
age, n = 6) and adult (60 days of age, n = 8) males were
castrated and implanted with a 2.5-mg pellet of T. One week after
treatment, tissues were collected as in Exp 2.
Tissue collection
Animals were rapidly decapitated by a guillotine. Trunk blood
samples were collected and centrifuged. Plasma was removed and stored
at -20 C until RIAs were performed (see below). Brains were quickly
removed, and the hypothalamus was dissected on a stainless steel
surface on wet ice with a razor blade. Coronal cuts were made directly
anterior to the optic chiasm and at the posterior end of the
hypothalamus, just anterior to the mammillary bodies. Then a horizontal
cut was made just ventral to the anterior commissure as it crossed the
midline. Finally, the brain was placed on the dorsal surface and the
optic chiasm, and tissue lateral to the hypothalamus was removed. The
dissected hypothalamus was then snap frozen in dry ice and stored at
-70 C until the aromatase assays were performed (see below).
Assay for steroid metabolizing enzymes
Individual hypothalami were homogenized in 600 µl of 250
mM sucrose/50 mM potassium phosphate buffer.
Assays were conducted with minor modifications from those used in
lizard brain tissue (13). Initially, validation assays that varied the
incubation time and substrate concentration were run on adult male
hamster hypothalamic homogenates to determine the appropriate assay
conditions (details presented in Results). Once the assay
was validated, experiments were conducted using duplicate 200-µl
aliquots of hypothalamic homogenates incubated for 25 min with 250
nM substrate. The tissue homogenates were added to test
tubes in which [3H]T (New England Biolabs, Inc., Boston, MA) had been dried. In all cases, substrate was
repurified by TLC before use. Samples were incubated at 37 C with a
NADH/NADPH-generating system, and the reaction was terminated by
freezing the tubes in a methanol/dry ice bath.
Steroids were extracted from homogenates three times with diethyl
ether. Androgens were then separated from estrogens twice by phenolic
partition, and estrogens extracted three times with ethyl acetate.
Androgenic and estrogenic products were applied to TLC plates following
the addition of radioinert carrier steroids (Steraloids, Wilton, NH).
TLC plates containing estrogens were run twice in ether/hexane (3:1),
and the products visualized by exposure to iodine vapors. Plates
containing androgens were run twice in chloroform/ethyl acetate (4:1),
and the products were visualized under UV irradiation following a
primulin spray. Regions containing the steroids of interest were
scraped from the plates, and after the addition of 400 µl
H2O, steroids were eluted from the silica-gel in 2 ml
methanol. A fraction of the eluate was mixed with Bio-safe cocktail II
(Research Products International, Mt. Prospect, IL)
and counted in a liquid scintillation counter (Beckman Instruments, Inc., LS6500, Fullerton, CA). Each sample was
corrected for counter efficiency, volume, and background counts in
tubes incubated with buffer and cofactors but no tissue. Results were
also corrected for recovery efficiency, which was determined by the
addition of a known quantity of [3H]E2 or
[3H]T (
150,000 dpm) to tubes processed in
parallel.
Protein content in each assay tube was determined with the method of
Bradford (14) (Bio-Rad Laboratories, Inc., Hercules, CA)
using BSA as the protein standard. To confirm their authenticity,
samples of all steroid products were recrystallized with radioinert
steroids (Steraloids) to constant specific activity using ethanol and
water (details presented in Results).
T RIA
Plasma concentrations of T were measured in two different assays
using the Coat-A-Count Total Testosterone Kit (Diagnostic Products, Los Angles, CA). This assay has been validated in our
laboratory for the measurement of plasma T concentration in the Syrian
hamster. The lower limit of detectability of both assays was 0.1 ng/ml.
The intraassay coefficients of variation were 5.8% and 4.1%, and the
interassay coefficient of variation was 10%.
Testis histology
Following storage in Bouin’s fixative, one testis from each
animal in Exp 1 was cut in half, dehydrated in increasing
concentrations of ethanol, cleared in xylene, and embedded in paraffin.
The tissue was sectioned at 10 µm, stained with hematoxylin and
eosin, and examined for the presence of mature sperm.
Data analysis
The data for each experiment were analyzed using two-tailed
t tests. Differences were considered significant when
P < 0.05. All data are reported as mean ±
SEM. Values for the Michaelis-Menten constant
(Km) and maximum velocity (Vmax) were
generated from a Lineweaver-Burk plot using a regression line in
Statview 4.1 (Abacus Concepts, Inc., Berkeley, CA).
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Results
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Validation of aromatase assay
Samples of E2 were twice verified by recrystallization
to constant specific activity in ethanol and water (93.6/85.8,
77.0/77.7; crystals(dpm/mg)/mother
liquor(dpm/mg)). In both cases, recovery of the specific
activity was 100%. This measure reflects the ratio of the specific
activity (disintegrations per minute per milligram) in the crystals
following the final recrystallization compared with that before the
first recrystallization (disintegrations per minute of assay product
used per milligram cold steroid used). Although androgens were
detectable, the incubation time and substrate concentration for these
assays were not optimized to measure the rate of androgen production.
Estrone was not detected in any sample. Therefore, quantification of
aromatase activity consisted solely of the rate of E2
production.
Time course and saturation curve
An initial time-course study was performed using a 222
nM substrate concentration (specific activity = 45.1
Ci/mmol). Pooled hypothalamic tissue was incubated for 10, 30, 60, or
180 min. E2 production increased linearly at incubation
times up to 60 min, at which point it slowed. Based on these results,
an assay used to generate a saturation curve was incubated for 35 min
using substrate concentrations ranging from 4–2225 nM
(specific activity = 45.1 Ci/mmol). The reaction rate increased to
the 224 nM concentration at which point E2
production began to level off. For this saturation curve, the
Km was 27.4 nM and Vmax was 7.58
fmol•mg-1•protein-1•min-1.
A second time course experiment was performed using a 250
nM (specific activity = 92.4 Ci/mmol) substrate
concentration at 15, 30, 60, and 120 min incubation times.
E2 production increased linearly through 30 min, then at a
slightly lower rate between the 30- and 120-min time points (Fig. 1
). Based on the results of these assays,
an incubation time of 25 min and a substrate concentration of 250
nM (specific activity = 92.4 Ci/mmol) were used for
the four experiments.
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Figure 1. [3H]E2 production after
incubation of hypothalamic homogenates with 250 nM
[3H]T for 15, 30, 60, or 120 min. E2
production increased at a lower rate between the 30 and 120 min time
points.
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Tissue specificity
To confirm tissue specificity, whole cerebellar homogenates from
intact prepubertal (25 days of age) and intact adult (65 days of age)
males were run in parallel with hypothalamic tissue in Exp 3. Assay
conditions for the cerebellar homogenates were identical to those used
for the hypothalamic homogenates. No aromatase activity was detected in
either the prepubertal or adult cerebellar homogenates, consistent with
results in other mammalian species (15, 16, 17).
Exp 1: Hypothalamic aromatase activity in prepubertal and adult
males
Paired testis weight and plasma T were significantly greater
in adults compared with prepubertal males (t = 15.86
and t = 4.68, respectively, both P <
0.05, Table 1). Mature sperm were
observed in the testes of adult but not prepubertal males (Fig. 2, A and B). Furthermore, adults had
significantly higher hypothalamic aromatase activity compared with that
of the prepubertal animals (t = 4.04, P
< 0.05, Fig. 3). Specifically, adults
had a 2-fold increase in aromatase activity compared with that of their
prepubertal counterparts.
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Figure 2. Testicular histology from an adult (A) and
prepubertal (B) hamster in Exp 1. Note the presence of mature sperm in
the adult testis only (A). Bar, 100 µm.
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Figure 3. [3H]E2 production
(femtomoles per milligrams protein per minute) in hypothalamic
homogenates from intact prepubertal and adult male hamsters.
Asterisk indicates significant difference. Values are
means ± SEM.
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Exp 2: Androgenic regulation of hypothalamic aromatase activity in
adult males
No significant difference was found in paired testis weight on the
day of castration between the adults that were treated with either the
blank or 2.5-mg pellet of T (Table 1
). Plasma T concentrations were
significantly higher in castrated adults treated with the 2.5-mg pellet
of T compared with castrated adults treated with the blank pellet
(t = 6.50, P < 0.05, Table 1). Furthermore,
T-treated adults had significantly higher levels of hypothalamic
aromatase activity compared with castrated controls (t
= 2.98, P < 0.05, Fig. 4A). Specifically, adults receiving the
2.5-mg dose of T had greater than a 2-fold increase in hypothalamic
aromatase activity compared with castrated adults treated with a blank
pellet.
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Figure 4. [3H]E2 production
(femtomoles per milligrams protein per minute) in hypothalamic
homogenates from castrated adult male hamster treated with either 0 or
2.5 mg of T (A), castrated prepubertal male hamsters treated with
either 0 or 2.5 mg of T (B), and castrated adult and prepubertal male
hamsters treated with 2.5 mg of T (C). Asterisks
indicate significant differences. All values are means ±
SEM.
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Exp 3: Androgen regulation of hypothalamic aromatase activity in
prepubertal males
There was no significant difference in paired testis weight on the
day of castration between the juveniles that were treated with either
the blank or 2.5-mg pellet of T (Table 1). Plasma T concentrations were
significantly greater in prepubertal animals implanted with the 2.5-mg
pellet of T compared with those prepubertal animals treated with a
blank pellet (t = 8.17, P < 0.05,
Table 1). Moreover, the prepubertal males treated with the 2.5-mg dose
of T had significantly higher hypothalamic aromatase activity compared
with the prepubertal males treated with the blank pellet
(t = 3.54, P < 0.05, Fig. 4B). Similar
to the increase in hypothalamic aromatase activity observed in adults,
T-treated prepubertal males had over a 2-fold increase in hypothalamic
aromatase activity compared with their prepubertal placebo-treated
counterparts.
Exp 4: Comparison of androgenic regulation of hypothalamic
aromatase activity in prepubertal and adult males
Adults had significantly heavier paired testis weight on the day
of castration compared with the castrated prepubertal animals
(t = 29.62, Table 1). Plasma T levels and hypothalamic
aromatase activity were equivalent between the castrated adults and the
castrated prepubertal animals treated with the 2.5-mg dose of T
(t = -1.03 and t = -1.27,
respectively, Table 1 and Fig. 4C, respectively).
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Discussion
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This series of studies demonstrates that hypothalamic aromatase
activity is significantly higher in adult male hamsters compared with
prepubertal males, and that hypothalamic aromatase activity is
positively regulated by T in both the adult and prepubertal male
hamster. Furthermore, the ability of T to increase hypothalamic
aromatase activity appears to be equivalent before and after puberty.
The androgenic regulation of hypothalamic aromatase activity most
likely accounts for the developmental difference in enzyme activity
observed in intact males in Exp 1, because the prepubertal males in
this experiment had significantly lower levels of circulating T
compared with the adults.
The present data do not support the hypothesis that increased
behavioral responsiveness to T in adults is mediated by the efficacy
with which T is aromatized to E2 in the hypothalamus.
However, we cannot rule out the possibility that aromatase activity is
differentially regulated in the hypothalamus of prepubertal and adult
males in more discrete hypothalamic nuclei by adult-physiological
levels of androgens. Indeed, Hutchison et al. (7) reported
that regulation of hypothalamic aromatase activity by T in the adult
male hamster is brain-region specific. The measurement of aromatase
activity in distinct hypothalamic nuclei of prepubertal and adult male
hamsters will help resolve this issue.
The present series of experiments has unequivocally demonstrated that
aromatase activity in whole hypothalamic homogenates is positively
regulated by T in both adult and prepubertal male hamsters, and this
regulation is equivalent at these two developmental stages. Thus, the
question remains as to why the same dose of T is unable to activate
reproductive behavior in juvenile males. Our experiments suggest that
the insensitivity of the juvenile male nervous system to steroid
hormone lies downstream of T’s action on androgen receptors (11) and
on aromatase activity (present data). One possibility is that the
aromatized E2 may have differential effects on the
hypothalami of prepubertal vs. adult male hamsters. For
instance, there is precedence in the literature suggesting that
estrogen receptor expression can be differentially regulated by
estrogens depending on the current physiological condition of the
animal (18, 19, 20). Therefore, increased local concentration of estrogen
in the hypothalamus may lead to an increase in steroid receptors in
adults but not in prepubertal males, leading to behavioral activation
in the adult males only.
Another possibility is that the increased estrogen levels resulting
from T-induced increases in aromatase activity could alter the
connectivity and/or morphology of the hypothalamic neurons differently
in the prepubertal and adult males. Estrogenic metabolites of T are
important in the prenatal and early postnatal differentiation and
development of the mammalian central nervous system (21, 22, 23, 24, 25).
Furthermore, the hormonal milieu has been implicated in the
morphological plasticity exhibited by both neuronal and nonneuronal
(e.g. glial cells) elements of the central nervous system in
adult animals (26, 27, 28, 29, 30, 31). However, the nature of the plasticity of the
juvenile brain in response to the hormonal milieu has not been
established. Thus, age-related changes in E2-induced
alterations in the hypothalamic cytoarchitecture may underlie the
pubertal increase in responsiveness to T and the maturation of male
sexual behavior.
In rats, Lephart and Ojeda (32) observed a pubertal decrease in
aromatase activity and hypothesized that the presumptive decrease in
E2 availability was responsible for the pubertal decline in
steroid negative feedback regulation of gonadotropin secretion (32).
However, Roselli and Klosterman (33) found a pubertal increase in
aromatase activity in male and female rats. Variations in experimental
methodologies and animal species used could account for the different
results in the two experiments mentioned above and the present data.
For example, tissue dissection was different in all of these
experiments. In the present experiments, a hypothalamic tissue fragment
containing both the preoptic area and the hypothalamus was used,
whereas Lephart and Ojeda (32) and Roselli and Klosterman (33) assayed
more discrete areas of the hypothalamus. It is also possible that the
pubertal decrease in responsiveness to steroid negative feedback does
involve a change in aromatase activity, whereas the pubertal increase
in responsiveness to behavioral activation by steroids does not.
In conclusion, the present series of studies has established that adult
male hamsters have approximately a 2-fold increase in hypothalamic
aromatase activity compared with prepubertal males, and that, in both
prepubertal and adult male hamsters, hypothalamic aromatase activity is
under androgen regulation. Therefore, the failure of T-treated
castrated prepubertal male hamsters to engage in the full suite of male
reproductive behaviors is not due to the inability of T to be converted
into E2 in the hypothalamus. Differences in the ability of
T to increase aromatase activity in other brain regions, or differences
in the action of T and/or E2 on other cellular processes
must account for the inability of T to facilitate male reproductive
behavior in juvenile males.
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Acknowledgments
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We thank Heather Richardson, Chung Lee, and Stefani Diedrich for
their technical assistance.
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Footnotes
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1 This work was supported by National Science Foundation (NSF)
Grant IBN-9602169 (to C.L.S.) and NSF Grant IBN-9733074 (to
J.W.). 
2 Supported by NIH Training Grant NS-07279.
Received June 5, 1998.
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Abstract
Introduction
