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Laboratory of Neuroendocrinology, The Babraham Institute, Cambridge, United Kingdom CB2 4AT
Address all correspondence and requests for reprints to: Dr. Allan E. Herbison, Laboratory of Neuroendocrinology, The Babraham Institute, Cambridge, United Kingdom CB2 4AT. E-mail address: allan.herbison{at}bbsrc.ac.uk
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The medial preoptic area of the rodent represents one of the best
defined brain regions in terms of sexually differentiated neuronal
architecture. Sex differences in the morphology, size of distinct
nuclei, and numbers of specific, neurochemically defined neurons have
all been described in this brain region (1, 2, 8, 9, 10, 11, 12, 13, 14, 15). One
such sexually dimorphic neuronal population identified in the rat is
that synthesizing the neuropeptide 
-calcitonin gene-related peptide
(CGRP) (16, 17). These neurons are located almost
exclusively within the anteroventral periventricular nucleus (AVPV) and
medial preoptic nucleus (MPN) of the medial preoptic area and are more
numerous in the female than in the male (16).
Immunocytochemical and in situ hybridization investigations
suggest that testosterone exerts both organizational and activational
effects on these cells, which result in an almost complete suppression
of preoptic CGRP expression in the male (17, 18). Around
postnatal d 5, testosterone exerts an organizational effect to set the
maximum number of potential CGRP-expressing cells to approximately one
third of that observed in the female (17). Then, in the
adult male, testosterone exerts its activational influence to suppress
CGRP mRNA and protein expression in these neurons (18).
Although a subpopulation of preoptic CGRP neurons is known to be
activated after lordosis in the female rat (19), the
physiological role(s) of these neurons is not yet established.
Because of the robust sex difference that exists in CGRP cell number
and the clear dual organizational-activational effects of testosterone
on these neurons, we reasoned that these cells might represent a good
model to explore the precise impact of perinatal sexual differentiation
on activational responses in the adult. In particular, it is not always
clear whether activational sex differences are dependent upon sexually
dimorphic molecular imprints from the perinatal period or, more simply,
defined by the differing gonadal steroid levels of adult males and
females. Based upon our earlier findings (17, 18), we
hypothesized that activational sex differences in the regulation of
CGRP expression may depend solely upon male-female differences in
circulating testosterone concentrations. We have examined these
activational effects in the present study by administering male levels
of testosterone to gonadectomized male and female rats and by examining
the AR- and ER-dependent pathways involved in each sex. To establish
whether any effects of androgens might be direct, we have also
undertaken a series of dual labeling immunocytochemical studies
examining the relationship between ARs and preoptic area CGRP neurons.
A previous study had identified a sexually dimorphic pattern of ER
expression in preoptic CGRP neurons (20).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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90 d of age
for each sex) from the Babraham colony were maintained in a light- and
temperature-controlled environment (lights on at 0500 h, off at
1900 h; 22 C) with food and water freely available. All surgical
procedures were carried out on animals anesthetized with Avertin
(2% tribromoethanol; 1 ml/100 g BW, ip), and animals were treated in
accordance with United Kingdom Home Office regulations under Project
80/1005.
Exp 1: effect of gonadal steroid manipulation on CGRP mRNA
expression
Twenty-four male and 24 female adult rats were bilaterally
gonadectomized under Avertin anesthesia. Four weeks later, animals
of each sex were divided into 4 equal groups (n = 6) and implanted
sc with SILASTIC brand capsules (SILASTIC medical grade tubing; od,
0.062 in.; od, 0.125 in.; Dow Corning Corp., Midland, MI)
containing testosterone crystals (30-mm-long capsule; group 1), DHT
crystals (20 mm-long capsule; group 2), 17ß-estradiol (50 µg/ml
sesame oil in 14-mm-long capsule; group 3), or sesame oil alone
(14-mm-long capsule; group 4) for 1 wk. All steroids were obtained
from Sigma (Poole, UK). These gonadal steroid replacement
paradigms have been shown previously to achieve the plasma
concentrations of testosterone (3 ng/ml), DHT (1.0 ng/ml), and
estradiol (20 pg/ml) that are within the physiological range for
male rats (21, 22, 23, 24). In addition to these 48 gonadectomized
animals, 6 intact males and 6 female animals exhibiting at least 2
consecutive 4-d estrous cycles were killed on the next day of
diestrus.
Male and female rats were killed 1 wk after capsule implantation
alongside intact rats (all animals were 90 d of age) by cervical
dislocation and decapitation between 1000–1200 h. The brains were
quickly removed, frozen on dry ice, and stored at -70 C until
processing for in situ hybridization experiments. Trunk
blood from each rat was collected into a heparinized beaker and
centrifuged, and the plasma supernatant was stored at -20 C until used
for analysis of testosterone levels by RIA.
Exp 2: effects of testosterone and combined estrogen and DHT
treatment on CGRP mRNA expression in female rats
Eighteen adult female rats were ovariectomized under Avertin
anesthesia and 4 wk later were divided into three equal groups of six
animals and implanted with SILASTIC capsules. Rats in the first group
received a 2-mm-long capsule filled with testosterone crystals; rats in
the second group received a 14-mm-long capsule filled with estradiol
(50 µg 17ß-estradiol crystals/ml sesame oil) as well as a
20-mm-long capsule filled with DHT crystals, and rats in the third
group received a 20-mm-long capsule filled with sesame oil. Female rats
were killed 1 wk after capsule implantation by cervical dislocation and
decapitation between 1000 and 1200 h. The brains were quickly
removed, frozen on dry ice, and stored at -70 C until processing for
in situ hybridization experiments. Trunk blood from each rat
was collected into a heparinized beaker and centrifuged, and the plasma
supernatant was stored at -20 C until used for analysis of
testosterone levels by RIA.
In situ hybridization analysis of CGRP mRNA
expression. Fresh-frozen sections (15 µm thick) were cut in the
coronal plane through the entire preoptic area on a cryostat and
thaw-mounted onto Vectabond (Vector Laboratories, Inc., Peterborough, UK) coated slides. Sections were kept at
-70 C until used. In situ hybridization for CGRP mRNA was
undertaken as described previously (18), In brief, three
antisense oligonucleotides (31–35 mer) complementary to bases
316–350, 510–541, and 643–676 of the rat CGRP cDNA were
synthesized and 3'-end labeled with
[35S]deoxy-ATP (1000–1500 Ci/mmol; NEN Life Science Products, Boston, MA) to a specific activity of
approximately 109 cpm/µg.
Frozen sections from all animal groups were processed simultaneously.
One complete set of sections in Exp 1 and 2 underwent hybridization
using an equimolar cocktail of the three labeled CGRP probes diluted in
hybridization buffer (final concentration, 6 x
103 cpm/ml). After an overnight hybridization at
37 C, sections were washed in 1 x SSC (standard saline citrate)
at room temperature, three times in 1 x SSC at 55 C (30 min
each), and again in 1 x SSC for 1 h at room temperature.
Slides were then dipped in Ilford K-5 nuclear track emulsion
(Ilford Imaging UK, Cheshire, UK) and exposed for 17 d in
light-tight boxes. At the appropriate exposure time, as
determined by test slides taken off at weekly intervals, all slides
were photodeveloped with Ilford phenisol and lightly counterstained
with methylene blue. Signal specificity was assessed by use of
competition experiments in which the three radiolabeled probes were
applied to sections in the presence of a 25-fold excess of each
unlabeled probe.
Analysis. The analysis of relative CGRP mRNA expression was undertaken by an investigator blind to the experimental groups of the rats. Expression was determined by computer-assisted analysis (Seescan, Cambridge, UK) of silver grain density overlying individual cells and also by cell counts of positively labeled cells within the AVPV and MPN, as reported previously (18). The boundaries of the AVPV and MPN were defined by reference to the characteristic landmarks of the preoptic area and cell densities as reflected in the methylene blue counterstaining, with the rostro-caudal extent of the AVPV and MPN corresponding to plates 17–19 and 20–23 of Swanson (25), respectively. The delineation of these nuclei was further assisted by the distribution of CGRP mRNA-expressing cells, which at these coronal levels are found almost exclusively in the AVPV and MPN (16).
For the silver grain analysis, the mean silver grain density overlying individual hybridized AVPV and MPN cells was determined in control excess unlabeled probe sections, and in experimental sections, only those cells expressing numbers of silver grains 5 times the control value were used for analysis (18). In each animal the silver grain density of at least 30 AVPV and at least 30 MPN cells counted from both sides of the brain and from 2–3 different sections were analyzed. For each rat, an average silver grain density per cell was determined, and these values combined to give experimental group means. The number of cells expressing CGRP mRNA in each animal was assessed by counting the total number of positively hybridized cells located within the AVPV and MPN in a minimum of 4 hemisections from each animal. Section counts were used to provide individual animal averages and then were combined to provide group means. In all cases statistical analysis was undertaken using nonparametric ANOVA with the Student-Newman-Keuls post-hoc significance test.
Exp 3: immunocytochemical investigation of the presence of ARs in
preoptic CGRP neurons
Six male and six female adult rats were anesthetized with
Avertin, placed in a stereotaxic frame, and given an injection of 50
µg colchicine (2.5 µl of a 20 µg/µl solution in 0.9% saline)
into the third ventricle. Twenty-four hours later, the animals were
anesthetized with Avertin and perfused transcardially with heparinized
saline (25 IU/ml isotonic saline) followed by 4% paraformaldehyde in
0.1 M phosphate buffer (pH 7.6) for 15 min. The brains
were removed and postfixed for 1 h in 4% paraformaldehyde before
being transferred to a 30% sucrose Tris-buffered saline solution
overnight.
Immunocytochemistry. Brain sections (25 µm
thick) were cut in the coronal plane on a freezing microtome, and three
sets of sections encompassing the entire preoptic area collected. One
set of free-floating sections was then processed for AR
immunocytochemistry as described previously (26). Briefly,
sections were incubated in a polyclonal rabbit antisera directed
against the N-terminal region of the androgen receptor (PG-21, 1
µg/ml; gift of G. Prins, Chicago, IL) and then placed in biotinylated
goat antirabbit Igs (1:200; Vector Laboratories, Inc.,
followed by the Vector Elite Kit (1:100) for 90 min. AR
immunoreactivity was then visualized using a
glucose-oxidase-nickel-3,3-diaminobenzidine tetrahydrochloride
procedure. For the CGRP dual labeling, AR-stained sections were placed
in rabbit polyclonal antisera raised against CGRP (1:3000;
Amersham International, Little Chalfont, UK) for 48 h
at 4 C, followed by peroxidase-labeled goat antirabbit antibodies
(1:200; Vector Laboratories, Inc.) for 4 h at room
temperature. CGRP immunoreactivity was visualized using the same
glucose oxidase-3,3-diaminobenzidine tetrahydrochloride method as
above but without the nickel. The production and specificity of the AR
and CGRP antisera used in the present study have been reported
previously by others and ourselves (17, 26, 27, 28, 29). Control
experiments included the omission of either primary antiserum or the
use of AR antisera incubated overnight with a 20-fold molar excess of
the peptide AR-21 (0.8 g/ml) used to generate the PG-21 antibody. Both
procedures resulted in a complete absence of specific staining.
Analysis. All CGRP-immunoreactive (IR), AR-IR, and (CGRP+AR)-IR cell profiles were counted within the boundaries of the AVPV and MPN using a Orthoplan microscope (Leica Corp., Rockleigh, NJ) at x40–100 magnification. Cell profiles exhibiting black nuclear staining associated with brown cytoplasmic immunoreactivity were considered double labeled. Cell counting was carried out on a minimum of six hemisections per animal from both the AVPV and MPN, and counts were combined to give mean numbers of single and double labeled cells per hemisection for males and females in the AVPV and MPN. Statistical analysis was undertaken using the nonparametric Mann-Whitney U test.
RIA for testosterone. The testosterone concentrations of all plasma samples were assayed in a single RIA using a Coat-a-Count kit (Diagnostic Products, Los Angeles, CA). Samples of known concentration were used to produce a calibration curve from which sample testosterone concentrations were directly measured. The sensitivity of the assay was 0.01 ng/ml, and the intraassay coefficient of variation was 9.8%.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) and caudal and lateral aspects of the
MPN. No clusters of silver grains were detected in sections hybridized
with radiolabeled probes in the presence of excess unlabeled probes.
These findings are in excellent agreement with previous in
situ hybridization and immunocytochemical studies
(16, 17, 18) and indicate the specificity of the procedure for
detecting CGRP mRNA.
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and 3). The silver grain
density of cells was not different between intact males (0.90 ±
0.05 silver grains/µm2·cell) and intact
females (0.79 ± 0.06) in the MPN, but was higher in females
(0.76 ± 0.05) than in males (0.43 ± 0.04; P
< 0.01) in the AVPV (Figs. 2 and 3).
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). Each of the gonadal steroid treatments (testosterone, DHT, or
estrogen) returned the numbers of hybridized cells and their silver
grain densities to intact levels in both the MPN and AVPV
(P < 0.05 for all parameters; Fig. 2). Mean plasma testosterone concentrations were significantly reduced in gonadectomized, oil-treated males (<0.01 ng/ml) compared with those in intact animals (3.6 ± 0.6 ng/ml). Treatment with testosterone returned levels to normal (2.7 ± 0.2 mg/ml), while testosterone concentrations remained low (all <0.03 ng/ml) after the administration of either estrogen or DHT.
Regulation of CGRP mRNA expression in the female. Ovariectomy
had no effect on the numbers of hybridized cells detected in either the
MPN or AVPV (Fig. 3A) of the female rat.
However, testosterone administration to ovariectomized rats
significantly reduced cell numbers in the MPN (P <
0.05) while dramatically increasing cell numbers in the AVPV
(P < 0.01; Fig. 3A). The effects of testosterone were
not mirrored by DHT or estrogen treatment alone.
In terms of cellular silver grain density, ovariectomy increased
expression in the MPN (P < 0.01), and this was
returned to normal levels by testosterone, DHT, or estrogen
(P < 0.05 for each; Fig. 3B). In the AVPV, gonadectomy
was found to have no effect on silver grain density, but, like the cell
number data, testosterone alone increased cellular mRNA levels
(P < 0.01; Fig. 3B).
Mean plasma testosterone concentrations were 3.1 ± 0.2 ng/ml in ovariectomized, testosterone-treated females (not significantly different compared with intact males or gonadectomized, testosterone-treated males), 0.2 ± 0.2 ng/ml in gonadectomized DHT-treated animals, and less than 0.03 ng/ml in the vehicle- and estrogen-treated rats.
Exp 2: gonadal steroid receptor pathways mediating the effects of
testosterone on CGRP mRNA expression in female rats
As changes in CGRP mRNA expression were observed in the MPN of
females after testosterone treatment, but not DHT or estradiol alone,
we undertook a further study to examine whether DHT and estradiol may
be acting synergistically to replicate the effects of testosterone. In
this experiment we also used a lower dose of testosterone to
investigate whether a dose-response relationship existed for the
dramatic stimulatory effects of testosterone on CGRP mRNA expression in
the female AVPV. Testosterone concentrations in ovariectomized,
testosterone-treated female rats in this experiment were 0.9 ±
0.2 ng/ml, whereas levels in the oil-treated and combined estrogen- and
DHT-treated females were less than 0.03 ng/ml in all cases.
As found in Exp 1, testosterone caused a significant reduction in the
number of hybridized cells (P < 0.05) and their
cellular silver grain density (P < 0.001; Fig. 4) in female MPN. Combined DHT and
estrogen treatment was found to replicate the suppressive actions of
testosterone in the MPN (Fig. 4). In the AVPV, the lower dose of
testosterone still induced a marked increase in the numbers of CGRP
mRNA-expressing cells (P < 0.01; Fig. 4A), and this
effect was replicated by the administration of both DHT and estrogen
together. The lower levels of testosterone did not, however, alter
cellular silver grain density in the AVPV. Combined DHT and estrogen
was similarly without effect (Fig. 4B).
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). Although there was a
trend toward fewer AR-IR cells per section in the female in both
preoptic nuclei, this was not significantly different (Table 1).
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).
Dual labeled cells exhibited black nuclear-located AR staining combined
with light brown cytoplasmic CGRP immunoreactivity (Fig. 5) and were
identified in both the AVPV and MPN. Dual labeled cells were rare in
the male (9%) compared with the female; about 50% of CGRP neurons in
the female MPN expressed ARs (P < 0.01; Table 1).
Similarly, approximately 35% of CGRP neurons in the female AVPV
expressed AR immunoreactivity compared with only approximately 3% in
the male (P < 0.01; Table 1).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Regulation of preoptic CGRP mRNA expression in the male
Our earlier studies demonstrated that testosterone acts in the
early neonatal period as well as during adulthood to restrict and then
suppress CGRP biosynthesis, in line with the well accepted
organizational-activational model of sexual differentiation
(1, 2, 3, 4). In good agreement with our earlier observations in
the MPN (18), gonadectomy of adult male rats elevated CGRP
mRNA expression, so that the numbers of hybridized cells detected as
well as their CGRP mRNA content were significantly increased in the MPN
and AVPV. This clearly indicates that CGRP mRNA expression in both
preoptic nuclei is tonically inhibited by gonadal steroids in the male.
As replacements with testosterone, DHT, and estrogen to physiological
concentrations were all equally effective in returning preoptic CGRP
mRNA levels to intact levels, it would appear that the suppressive
effect of testosterone on CGRP can occur through the AR or the ER.
The observation that either the AR or ER can mediate the effects of testosterone on CGRP mRNA expression in the male is unusual. Relatively little work has been undertaken to define the steroid receptors underlying the activational effects of gonadal steroids on sexually dimorphic neuronal populations. However, published work examining both ER and AR pathways often indicates the involvement of a just a single receptor type. For example, we have previously demonstrated that testosterone acts through the ER, and not the AR, to regulate GnRH mRNA expression in the male rat using exactly the same steroid treatment paradigms employed in this study (30). In contrast, the expression of somatostatin mRNA in the sexually dimorphic somatostatin periventricular neurons is clearly stimulated exclusively by AR-dependent mechanisms in the adult rat (31, 32). The only other example where testosterone has been shown to act through either the ER or AR in brain sexual differentiation involves the perinatal influence of testosterone on growth-associated protein-43 mRNA (34). In contrast, dual ER/AR activation is required to correctly regulate vasopressin expression in the bed nucleus of the stria terminalis (33).
The involvement of either ER or ARs in the regulation of CGRP
mRNA expression in the adult male raises interesting questions
regarding the molecular nature of steroid action on these cells. On the
one hand, it is possible that the CGRP gene expression is suppressed by
either ER or AR directly. The observation of a near-perfect
estrogen response element (GGTCCcttTGACC) within the CGRP gene
(20) provides at least theoretical support for this
possibility. However, the finding here that relatively few CGRP neurons
in the male express ARs (<10%) or ER (33%; Table 1) suggests
that a direct genomic mechanism of gene regulation may not exist for
these cells. Thus, these dual suppressive effects through the ER and AR
may be indirect or occurring through nonclassical pathways. In this
light, it is interesting to note that estrogen regulates neurotensin
gene transcription in sexually dimorphic preoptic neurons through cAMP-
and PKA-mediated mechanisms (15, 35). Future studies will
be required to establish precisely how testosterone down-regulates CGRP
expression in the male rat. As we do not yet know whether these cells
express aromatase, it is also unclear which of these receptor pathways
might be most important in vivo under normal circumstances.
The marked uniformity in testosterone’s suppressive influence on
CGRP immunoreactivity and mRNA expression within the preoptic area,
spinal motor neurons (36), and pituitary gland
(27) makes this issue even more intriguing.
CGRP mRNA expression and regulation in the MPN of the
female
Our findings suggest that the mechanisms underlying the regulation
of CGRP mRNA expression in the MPN of the adult rat are sex specific.
The gonadectomy of female rats was not found to have any impact on the
numbers of hybridized cells observed in the MPN, but testosterone
clearly suppressed CGRP cell number. Thus, even though ovarian steroids
do not normally exert any tonic influence, the exposure of
ovariectomized females to male levels of testosterone was capable of
down-regulating CGRP cell number in a manner similar to that found in
the male. Although at first sight this suggested that male and female
CGRP neurons in the MPN used common mechanisms of gonadal steroid
regulation, results with DHT and estradiol clearly demonstrated that
this was not the case. We found that ER/AR coactivation was required to
suppress CGRP cell numbers in the female, while either ER or AR
activation alone was sufficient in the male.
Unexpectedly, we found that gonadectomy and steroid administration in the female rat resulted is divergent changes in the two measured parameters: cellular mRNA levels and the numbers of hybridized cells. A perfect correlation between changes in grain density and cell number always existed in the male. In the female, gonadectomy increased cellular silver grain density, but had no effect on CGRP cell number, whereas, to the contrary, testosterone treatment reduced cell number, but had no effect on cellular mRNA levels. Furthermore, it was apparent that either ER or AR activation was able to suppress cellular mRNA levels, whereas ER/AR coactivation was required to reduce the numbers of CGRP mRNA-expressing cells in the MPN of the female. These observations suggest that independent steroid-regulated cellular mechanisms may underlie the regulation of the numbers of potential CGRP-expressing cells and their cellular levels of CGRP mRNA in the female. Thus, the mechanisms for regulating cellular levels of CGRP mRNA in the MPN appear similar between males and females, whereas those determining the numbers of CGRP cells are sexually dimorphic.
Together, our findings suggest that the gonadal steroid regulation of CGRP mRNA expression in the female MPN probably results from multiple different mechanisms, some of which appear to be in common with the male and others that are sex specific. The requirement for ER/AR coactivation to reduce CGRP cell number in the female may provide a mechanism that protects CGRP neuronal number from suppression in the adult. For example, during proestrus, the elevated circulating estrogen concentrations will not alter CGRP cell number, as androgen levels will not be sufficiently elevated. Thus, in terms of maintaining the marked numerical sex difference in CGRP neurons, the absence of male-like levels of testosterone as well as the sex differences in ER/AR coactivation requirements are both critical.
CGRP mRNA expression and regulation in the AVPV of the
female
The major unexpected finding of this study was the marked region-
and sex-specific effect of testosterone to increase CGRP mRNA
expression within the AVPV. To date, this is the only known situation
whereby testosterone stimulates CGRP expression in the rat brain
(17, 36) and is very clearly different from the situation
encountered in the male AVPV and female MPN. The female-specific
dependence on ER/AR coactivation for the increase in CGRP
mRNA-expressing cell numbers is, nevertheless, retained in the AVPV.
Although comparing across two separate CGRP in situ
hybridization experiments, the 2-fold induction in CGRP cell numbers
with about 1 ng/ml testosterone and the 3-fold induction with
approximately 3 ng/ml testosterone suggest a dose dependency in the
response. As gonadectomy has no effect on CGRP mRNA expression in the
AVPV of the female, and levels of testosterone are maximal at about
0.07 ng/ml in our intact female rats (Spratt, D. P., and A.
E. Herbison, unpublished data), it is unlikely that this increase is of
physiological importance in the normal individual.
The very marked sex- and region-specific differences in the gonadal steroid regulation of CGRP expression revealed here in the AVPV serve to underlie the remarkable sexual dimorphism of this structure. As one of the few examples of a sexually dimorphic brain nucleus that is larger in females than in males (11, 37, 38), the AVPV expresses abundant gonadal steroid receptors and is known to contain several sexually dimorphic neuronal phenotypes that are themselves more numerous in the female (12, 14, 15, 16). Although the AVPV is believed to have a critical role in the sexually differentiated transmission of gonadal steroid information to the GnRH neurons (39, 40), a full understanding of the ontogeny and functional significance of the sexually dimorphic nature of the AVPV is lacking. In terms of differences between the AVPV and the MPN, subtle differences in the effects of gonadal steroids have sometimes been reported (41, 42), but the complete reversal encountered here appears unique. The sexually dimorphic nature of the AVPV is thought to result from testosterone’s ability to enhance cell death in the AVPV, resulting in a smaller nucleus with fewer cells in the postpubertal male (37). Thus, it is intriguing to speculate that the cells lost in this androgen-dependent perinatal organizational process are those capable of synthesizing CGRP in response to male-like levels of testosterone in the adult female.
Conclusions
It is important that the mechanisms underlying the
generation and maintenance of defined sexually dimorphic neuronal
populations are elucidated to aid our understanding of the functional
significance of sexually differentiated brain function
(43). The CGRP neurons of the preoptic area represent one
such sexually dimorphic neuronal population in which perinatal
testosterone exposure initially defines the female-dominant sex
differences in neuronal number. We now report here that novel sexually
differentiated region- and steroid receptor-specific activational
mechanisms function in association with the sex differences in
circulating testosterone levels to maintain the sexually dimorphic
nature of the CGRP neuronal population in the adult rat. Unlike other
characterized sexually differentiated neuronal populations, it appears
that either ER or AR activation is sufficient to suppress CGRP mRNA
expression in the preoptic area of the male, whereas ER/AR coactivation
is required for regulation in the female. The molecular mechanisms
underlying these novel events are not known. We show that ER and AR
expression by CGRP neurons is also highly sexually differentiated, but
this does not easily explain the sex differences observed in steroid
receptor activation patterns. A further apparently unique
characteristic is the ability of adult levels of testosterone to
markedly increase the numbers of CGRP mRNA-expressing cells in the AVPV
of the female. Although this response may be of dubious physiological
relevance, the phenomenon highlights further the marked sexually
differentiated, gonadal steroid-dependent plasticity of this unique
nucleus.
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Acknowledgments |
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Footnotes |
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Abbreviations: AVPV, Anteroventral periventricular nucleus; CGRP, calcitonin gene-related peptide; DHT, dihydrotestosterone; IR, immunoreactive; MPN, medial preoptic nucleus.
Received January 5, 2001.
Accepted for publication April 10, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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