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Program in Neuroscience (M.D.M.), Department of Cell Biology, Neurobiology, and Anatomy (L.L.D.C.), Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois 60153
Address all correspondence and requests for reprints to: Lydia L. DonCarlos, 2160 South First Avenue, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois 60153. E-mail: ldoncar{at}luc.edu
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Abstract |
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Introduction |
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Our laboratory has demonstrated a sex difference in AR messenger RNA (mRNA) content in the developing BSTpr and MPO of the rat, with males having more AR mRNA expression than females by postnatal day (PND) 10 (15). Further, exposure to the male gonadal hormone testosterone during the postnatal period is important for establishing this neonatal sex difference in AR mRNA content, because gonadectomy of males on the day of birth (PND-0) or PND-5 eliminates the sex difference in AR mRNA at PND-10 (16). In addition, exogenous treatment with testosterone maintains AR mRNA expression at male-like levels in animals gonadectomized on PND-0. Because testosterone may be metabolized to either androgenic or estrogenic metabolites, and AR expression is regulated by both androgens and estrogens in the adult rat (17, 18), regulation of AR mRNA gene expression during development may occur via an AR or an ER mediated mechanism. Thus, the purpose of these experiments was to determine whether initial sex differences in AR mRNA content are due to up-regulation of AR mRNA in males by androgenic or estrogenic metabolites of testosterone.
To determine the relative roles of androgens and estrogens in the production of masculine AR mRNA expression, we have used a 35S-labeled riboprobe and in situ hybridization to perform two experiments. In the first experiment, male rats were gonadectomized on PND-0 and treated from PND-0 through PND-9 with either diethylstilbestrol (DES), a synthetic estrogen, or dihydrotestosterone proprionate (DHTP), a nonaromatizable androgen, and AR mRNA hybridization density was examined on PND-10. In the second experiment, intact male rats were treated with flutamide, an androgen receptor antagonist, from PND-0 through PND-9, and AR mRNA content was examined on PND-10. In both experiments, analysis of AR mRNA focused on the BSTpr and MPO, which have been shown to exhibit a sex difference in AR mRNA expression by PND-10 (15). The ventromedial hypothalamus, an area that demonstrates no sex difference in AR mRNA expression during the perinatal period (15), was examined as a control in both experiments.
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Materials and Methods |
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Treatment groups
Exp 1 had five different treatment groups: intact males (males),
intact females (females), males gonadectomized on PND-0 (GDX-0), males
gonadectomized on PND-0 and given DHTP replacement daily from PND-0
through PND-9 (GDX + DHTP), and males gonadectomized on PND-0 and given
DES replacement daily from PND-0 through PND-9 (GDX + DES). A previous
study had demonstrated that, in males gonadectomized on PND-0 and
killed on PND-10, daily oil injections did not affect expression of AR
mRNA (16); therefore, a gonadectomized plus oil treatment was not
repeated.
Exp 2 also had five different treatment groups: males, females, males given sesame oil vehicle from PND-0 through PND-9 (males + oil), males given 40 µg flutamide daily from PND-0 through PND-9 (males + 40 µg Flut), and males given 300 µg flutamide daily from PND-0 through PND-9 (males + 300 µg Flut).
Surgeries
Bilateral gonadectomies were performed on male rats on PND-0 for
Exp 1. All animals were chilled on ice to induce anesthesia. Neonatal
orchidectomies were performed as previously described (16). After
gonadectomy, all pups were warmed under a heat lamp until a normal body
temperature and level of activity were regained, then they were
returned to their dam.
Hormone treatments
In Exp 1, dihydrotestosterone propionate (DHTP: 10 µg/0.1 cc
in sesame oil) or diethylstilbestrol (DES: 2 µg/0.1 cc in sesame oil)
was injected sc in the dorsum through a 20-gauge needle. Similar doses
of androgens and estrogen have been shown to masculinize various
parameters in the preoptic area (19, 20). DES was specifically selected
for use as an estrogen in these experiments because it has a relatively
long half life and is not bound by 
-fetoprotein (21). In addition,
although DES binds androgens and at low doses has positive effects on
characteristics generally thought to be androgen mediated,
e.g. prostatic growth (22), it apparently does not activate
androgen receptor target genes (23). A drop of collodion was placed
over the site of injection to prevent leakage. All treatments were made
daily beginning on the day of gonadectomy, PND-0, and continued through
PND-9, the day before rats were killed. The site of administration was
rotated each day to avoid damage to the skin as well as maximize uptake
of the hormone.
In Exp 2, flutamide injections were administered sc at doses of either 40 µg or 300 µg (in 0.1 cc of sesame oil). These doses have previously been shown to block organization of specific patterns of masculine behavior (6, 24). All injections were given sc in the dorsum through a 20-gauge needle. Intact males received injections daily beginning on the day of birth, PND-0, and continuing through PND-9. Control males were injected with sesame oil. All animals were killed on PND-10.
In situ hybridization
In situ hybridization was conducted as previously
described (15). Briefly, androgen receptor (AR) mRNA was detected using
a 35S-labeled complementary RNA probe transcribed from a
rat AR complementary DNA (cDNA) corresponding to nucleotides
3350–3840. The complementary RNA probe was diluted with hybridization
buffer to a final activity of 1.5 x 107 cpm/ml. The
tissue was prepared for hybridization by acetylation, delipidation, and
dehydration. Each slide was hybridized with 100 µl of hybridization
solution for 20 h at 60 C. Following hybridization, slides were
rinsed in sodium chloride-sodium citrate (SSC), treated with RNase, and
rinsed again to a final stringency of 0.1 x SSC at 60 C.
Following the rinses, slides were dehydrated, allowed to air dry, and
apposed to Hyperfilm Betamax (Amersham Pharmacia Biotech,
Arlington Heights, IL) to produce film autoradiograms for analysis.
Analysis
Film autoradiograms were examined using a Macintosh IIci
computer with a Scion videocard (Scion Corp., Walkersville, MD)
attached to a Sony video camera (Imaging Research, Inc.,
St. Catherines, Ontario, Canada) and NIH IMAGE analysis software
(developed at the U.S. National Institutes of Health available at
http://rsb.info.nih.gov/nih-image/). Three sections per animal were
analyzed bilaterally per region of interest. The analysis focused on
two regions that were previously shown to have a sex difference in AR
mRNA at PND-10 (15), the principal portion of the bed nucleus of the
stria terminalis and the medial preoptic area. The ventromedial
hypothalamus, a region with no sex difference in AR mRNA expression at
PND-10 (15), was also examined. Each cell group was analyzed based on
detectable signal as previously described (15). The entire area of
label was outlined regardless of signal intensity. The average pixel
value of the outlined region was measured and expressed as a mean gray
level. These mean gray levels represent a semiquantitative index of
steady state levels of AR mRNA.
To eliminate differences due to nonspecific hybridization, five
background measures were taken from the caudate-putamen of each animal.
The mean background measure for each animal was subtracted from each
individual mean gray level to obtain a corrected gray value. The effect
of hormone treatment on mean corrected gray values was analyzed by
one-way ANOVA for each area, with planned post hoc tests (Fisher’s
PLSD and Scheffé’s multiple comparison test). Differences were
considered significant at P 
0.05. Individual
t tests, with P 0.01, were performed to
assess specific differences due to treatment. Body weight, anogenital
distance and seminal vesicle weight were also analyzed by one-way ANOVA
with planned post hoc tests for each experiment.
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Results |
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. There was no effect of treatment on
body weight in either experiment. Ano-genital distances were
significantly lower in intact females when compared with all other
treatments (P 0.01). Gonadectomy plus hormone replacement with
DES significantly increased ano-genital distance (P 0.01).
Neither gonadectomy, gonadectomy plus DHTP replacement, nor flutamide
treatment had any effect on ano-genital distance because these were
equivalent when compared with the intact males.
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0.01). Treatment of gonadectomized
males with DES or DHT maintained seminal vesicle weights at levels not
significantly different from the intact males. Flutamide treatment
significantly lowered seminal vesicle weights in comparison to intact
males (P 0.01).
Exp 1: Does treatment with DHTP or DES maintain
male-like AR mRNA content in male rats gonadectomized on
PND-0?
In this experiment, male rat pups were gonadectomized on PND-0 and
given hormone replacement on PND-0 through PND-9 with either
dihydrotestosterone propionate (DHTP), a nonaromatizable androgen, or
diethylstilbestrol (DES), a synthetic estrogen. AR mRNA content was
then measured in PND-10 animals. If higher levels of AR mRNA expression
are dependent on androgen-mediated regulation, then replacement with
DHTP should maintain AR mRNA expression at masculine levels. However,
estrogenic regulation may also be sufficient to up-regulate AR mRNA
expression, and if so then replacement with DES should maintain
male-like AR mRNA content.
In the BSTpr and MPO, gonadectomy at PND-0 decreased AR mRNA expression
at PND-10 to female-typical levels (Figs. 1 and 2).
Treatment of gonadectomized males with DHTP did not maintain AR mRNA at
male-typical levels in the BSTpr or MPO (Figs. 1 and 2). Instead,
gonadectomized males treated with DHTP exhibited steady-state levels of
AR mRNA in the BSTpr and the MPO that were not significantly different
from the AR mRNA levels in the corresponding areas of intact females.
Gonadectomized males treated with DES had masculine levels of AR mRNA
in the BSTpr and the MPO (Figs. 1 and 2). In the VMH, neither
gonadectomy nor gonadectomy followed by treatment with DHTP or DES had
an effect on AR mRNA expression (Fig. 3).
Thus, estrogen, but not a nonaromatizable androgen, is sufficient to
up-regulate AR mRNA content in the developing male rat forebrain.
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Flutamide treatment did not alter AR mRNA expression in either the
developing BSTpr or MPO (Figs. 4 and 5). Similarly, daily vehicle injections
to intact males had no effect on AR mRNA (data not shown). Both doses
of flutamide were biologically effective in that both doses
significantly decreased seminal vesicle weights (Table 1
). Males that
received 40 µg/day or 300 µg/day of flutamide had levels of AR mRNA
expression in BSTpr and MPO that were not significantly different from
levels in corresponding areas of the intact males (Figs. 4 and 5). In
addition, there was no effect of flutamide treatment on AR mRNA
expression in the VMH (Fig. 6), an area
that demonstrates no sex difference in AR mRNA content. Thus, AR mRNA
expression in the BSTpr, MPO, and VMH was unaffected by blocking
androgen receptor mediated action with the antagonist, flutamide.
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Discussion |
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Sex differences in ER mRNA expression arise as early as PND-0 (27), whereas sex differences in AR mRNA do not develop until sometime between PND-4 and PND-10 (15). Neonatally, ER mRNA content is down-regulated by estrogens (20), whereas AR mRNA is up-regulated by estrogen. Androgens do not appear to regulate ER (20) or AR mRNA during the perinatal period. Thus, one role of estrogen during development may be to shift the balance of the impact of testosterone toward its role as an androgen, supporting the hypothesis that sequential phases of estrogenic and androgenic stimulation demarcate specific phases of the sexual differentiation process (see Refs. 28, 29 for reviews). This hypothesis was initially formulated based on evidence that the developmental organization of masculine sexual behaviors in male ferrets is sensitive to estrogen prenatally and androgen postnatally, as well as evidence that the enzyme responsible for estrogen synthesis in the brain, aromatase, is regulated by androgens (30).
In adult rats, testosterone and DHT stimulate aromatase activity in the hypothalamus (31). It might be expected that androgen or androgen antagonists that affect estrogen synthesis would also affect AR mRNA expression in neonates because DES increased AR mRNA levels. However, DHT did not increase, nor did flutamide reduce, AR mRNA in the present experiments. It remains to be determined whether the DHT or flutamide treatments given here would actually affect estrogen synthesis to any great extent given that, in adults, DHT is less effective than testosterone or DHT plus estradiol in stimulating aromatase activity (31). Moreover, flutamide had no effect on aromatase mRNA levels in the perinatal rat hypothalamus (32), although the androgen receptor antagonist, cyproterone acetate, decreased aromatase mRNA expression in the perinatal mouse hypothalamus (33). Therefore, interpretation of the lack of effect of DHT or flutamide on neonatal AR mRNA expression will require more information on perinatal regulation of aromatase activity.
Responsiveness to androgens is greater in the adult male rat than in the adult female (11, 34, 35). Conclusions about whether more robust responses to androgens depend on a sexually differentiated ability to express androgen receptors are mixed. In support of a permanent sex difference in the capacity to produce AR are studies of gonadectomized male and female rats, in which androgen binding levels in some brain regions remain higher in males despite the lack of circulating hormone (36). However, female mice, when treated with testosterone, express AR at levels equivalent to males (37); it is possible that, as in mice, female rats retain the ability to produce male-typical levels of androgen receptor in response to testosterone, particularly if estrogen receptors participate in this regulation in adulthood.
Experiments assessing the impact of neonatal hormone manipulations on AR mRNA in neonatal and adult females remain to be performed, but, recently, Bakker et al. reported no difference in the total number of AR immunoreactive (AR-ir) nuclei between intact males and males treated neonatally with an aromatization inhibitor (38). Because blockade of the conversion of testosterone to estrogen had no apparent effect on the total amount of AR-ir, these authors rejected a role for estrogen in the development of putative sex differences in AR content. In contrast, our results suggest that estrogen is responsible for the sex differences in AR expression observed on PND-10. Several possibilities exist that could account for this apparent incongruity. First, intrinsic differences in the hormone responsiveness between rat strains (Wistar in their study and Sprague Dawley in ours), could account for the different findings, because gonadectomized Wistar male rats are more behaviorally responsive to androgen replacement than gonadectomized Sprague Dawley males (39). Second, in the study by Bakker et al., no female control animals were used; therefore, it is difficult to determine whether sex differences in AR-ir could be detected under their assay conditions. Finally, in the Bakker study, males were estrogen deprived neonatally and then allowed to grow to adulthood under a normal hormonal milieu. This would suggest that hormone exposure after the period addressed in our study could be sufficient to reestablish male-typical AR expression. Thus, the sex differences in AR mRNA observed in neonates may or may not represent the onset of sexual differentiation of AR expression and sensitivity per se; that is, permanent differences in the capacity to produce androgen receptors and respond to androgens. Regardless, developmental events that require androgen may depend in part on sex differences in AR availability at a specific time and in a specific place. The present studies provide a temporal framework upon which to design physiological and behavioral experiments to determine whether estrogenic up-regulation of AR mRNA during the neonatal period does indeed enhance sensitivity to androgens during that specific period and allow androgens to have a greater impact on brain development than generally recognized.
Interestingly, AR mRNA in the adult rat is regulated by both androgens and estrogens. DHTP down-regulates the expression of AR mRNA in the adult BST and MPO and reverses the effects of gonadectomy on AR mRNA expression in these areas (18). Further, although estrogen replacement was capable of restoring AR mRNA expression in long-term castrated males, DHTP was clearly more effective in regulating AR mRNA following gonadectomy (18). In contrast with the adult, regulation of AR mRNA content in the developing rat forebrain appears completely dependent on estrogenic metabolites of testosterone. This indicates that a shift in the ability of androgens to regulate AR expression potentially occurs sometime between development and adulthood.
Although androgens may not directly impinge on the development of sex
differences in AR mRNA content in the BSTpr and the MPO, there remains
strong evidence for a role of androgen receptors in the masculinization
process. For example, androgen action during the perinatal period is
integral to the development of open field (8) and play behavior (6, 7)
as well as the complete expression of male sexual behavior (11, 13, 40). Interestingly, ER- knockout mice have both markedly reduced
expression of male-typical sexual behavior (41, 42) and lower
expression of AR immunoreactivity in the bed nucleus of the stria
terminalis (43). In addition, in vitro studies have
identified specific roles for androgens vs. estrogens in the
development of neuronal connections; androgens enhance neurite
arborization and the expansion of neuronal receptive fields (44),
whereas estrogen increases neurite length and number of neuritic spines
and gap junctions (44, 45).
Further evidence for a role for androgens during sexual differentiation comes from numerous studies demonstrating that synergistic actions of androgens with estrogens may be crucial to the organization of neural circuitry and neurochemistry necessary for the expression of sexually differentiated functions. For example, when male rats are gonadectomized on the day of birth, injections of either testosterone or dihydrotestosterone plus estrogen, during the first five postnatal days, were sufficient to maintain the capacity to express ejaculatory behavior during adulthood (9). In contrast, males gonadectomized on the day of birth that received injections of estrogen or dihydrotestosterone alone did not demonstrate ejaculatory behavior in adulthood (9). Estrogen and androgens also synergize to activate the expression of sexual behavior and aggressive behavior in rodents. Specifically, male sexual behavior is reinstated more rapidly and to a further extent in gonadectomized males treated with dihydrotestosterone and estrogen than with either hormone alone (46). Estrogen and dihydrotestosterone work synergistically in the brain to differentiate the expression of mounting behavior and lordotic responses in ovariectomized females (35). Aggression is also affected synergistically by estrogen and dihydrotestosterone in mice. Gonadectomized male mice treated with estrogen and dihydrotestosterone show increases in number of attacks and cumulative fighting time (47), whereas gonadectomized male mice treated with dihydrotestosterone alone show female-like patterns of aggression (48), and gonadectomized males treated with estrogen alone show incomplete restoration of aggression (49).
Our results suggest that androgens and estrogens do not synergize in the production of higher, male-typical levels of AR mRNA during development because treatment with either testosterone or DES resulted in equivalent levels of AR mRNA expression. Further, flutamide had no effect on the of sex differences observed in AR mRNA expression during development. However, the studies above reinforce the potential importance of androgen action in the development of other aspects of male-typical neural circuitry and emphasize the need for further study into the putative role of androgens during the critical period for sexual differentiation.
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Acknowledgments |
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Footnotes |
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2 Present address: Department of Biology, Morrill Science Center,
University of Massachusetts-Amherst, Amherst, Massachusetts
01003.
Received October 5, 1998.
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References |
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-dihydrotestosterone.
Neuroendocrinology 53:79–84[Medline]
-reductase type I mRNA
in the male and female mouse hypothalamus. J Neuroendocrinol 10:267–274[CrossRef][Medline]
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