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, and Estrogen Receptor ß Show Distinct Patterns of Expression in Forebrain Song Control Nuclei of European Starlings1
Department of Neurobiology and Physiology (D.J.B., F.W.T.), Northwestern University, Evanston, Illinois 60208; Department of Psychology (G.E.B., G.F.B.), Johns Hopkins University, Baltimore, Maryland 21218; and Laboratory of Biochemistry (J.B.), University of Liège, B-4020, Liège, Belgium
Address all correspondence and requests for reprints to: Daniel J. Bernard, Ph.D., Department of Neurobiology and Physiology, Northwestern University, 2153 North Campus Drive, Evanston, Illinois 60208. E-mail: dbernard{at}nwu.edu
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Abstract |
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). The
recent cloning of a second form of the estrogen receptor in mammals,
ERß, raises the possibility that a second receptor subtype is present
in songbirds and that estrogenic effects in the song system may be
mediated via ERß. We therefore cloned the ERß complementary DNA
(cDNA) from a European starling preoptic area-hypothalamic cDNA library
and used in situ hybridization histochemistry to examine
its expression in forebrain song nuclei, relative to the expression of
AR and ER messenger RNA (mRNA), in the adjacent brain sections. The
starling ERß cDNA has an open reading frame of 1662-bp, predicted to
encode a protein of 554 amino acids. This protein shares greater than
70% sequence identity with ERß in other species. We report that
starling ERß is expressed in a variety of tissues, including brain,
pituitary, skeletal muscle, liver, adrenal, kidney, intestine, and
ovary. Similar to reports in other songbird species, we detected AR
mRNA-containing cells in several song control nuclei, including the
high vocal center (HVc), the medial and lateral portions of the
magnocellular nucleus of the anterior neostriatum, and the
robust nucleus of the archistriatum. We detected ER expression in
the medial portion of HVc (also called paraHVc) and along the medial
border of the caudal neostriatum. ERß was not expressed in HVc, in
the medial and lateral portions of the magnocellular nucleus of the
anterior neostriatum, in the robust nucleus of the archistriatum, or in
area X. In contrast, ERß mRNA-containing cells were detected in the
caudomedial neostriatum and medial preoptic area in a pattern
reminiscent of P450 aromatase expression in the same brain regions in
other songbirds. These data suggest that estrogenic effects on the song
system are not mediated via ERß-producing cells within song nuclei.
Nonetheless, the overlapping expression of ERß- and
aromatase-producing cells in the caudomedial neostriatum suggests that
locally synthesized estrogens may act via ERß, in addition to ER,
to mediate seasonal or developmental effects on nearby song nuclei
(e.g. HVc). |
Introduction |
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). Interestingly, sex differences in
singing behavior are paralleled by large sex differences in the
morphology of song control nuclei (e.g. Refs. 1, 2 ; cf.
Ref. 3). Although the mechanisms controlling sexual differentiation of
the song system are not clearly understood (e.g. Refs. 4, 5), treatment of genetic females with sex steroid hormones early in
development can masculinize song nuclei and singing behavior (6, 7). In
seasonally breeding species, both the incidence of singing behavior and
the volumes of song nuclei increase when birds are in breeding
conditions (8, 9, 10, 11, 12, 13). These changes in brain and behavior are regulated,
to a large extent (although not exclusively), by seasonal changes in
circulating testosterone (T) levels (14, 15, 16). In addition, in adults of
both sexes, in several species, exogenous T treatment increases the
incidence of singing behavior and alters the morphology of song nuclei
(e.g. Ref. 17).
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(ER)-expressing
cells (21, 27, 28, 31, 32, 33, 34, 35, 36, 37, 38). This pattern of steroid receptor
expression provides a substrate upon which steroids can act directly
within song nuclei to exert their effects. Many of T’s effects,
however, seem to be mediated via its estrogenic metabolites (39, 40).
Given the limited distribution of ER within song nuclei, most
estrogenic effects in the song system are thought to be
trans-synaptic. That is, estrogens bind to receptors within
paraHVc or other ER-expressing areas of the brain, to exert their
effects throughout the song system (41).
A second form of the estrogen receptor, ERß, was cloned in
mammals and, more recently, in a nonsongbird, the Japanese quail
(Coturnix japonica) (42, 43, 44, 45). ERß shows a different
pattern of expression from ER in brains of rats and quail (42, 45, 46, 47). If songbirds also express this recently identified estrogen
receptor, then ERß may be expressed in song control nuclei, thereby
providing a direct substrate for estrogen action. To examine this
possibility, we: 1) cloned the ERß complementary DNA (cDNA) in a
temperate zone songbird, the European starling (Sturnus
vulgaris); and 2) examined AR, ER, and ERß expression in
forebrain song control nuclei using in situ hybridization
histochemistry. We selected starlings for these analyses because the
effects of sex steroids on the song system and singing behavior have
been examined extensively in this species (7, 8, 14, 48). The results
show that starlings express ERß and that AR, ER, and ERß show
different patterns of expression in song control nuclei and in other
parts of the brain.
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Materials and Methods |
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RNA extraction
Total RNA was extracted from starling telencephalon,
hypothalamus, cerebellum, pituitary, heart, skeletal muscle, liver,
adrenal, kidney, spleen, intestine, pancreas, and ovary using Trizol,
following the manufacturer’s instructions (Life Technologies, Inc., Gaithersburg, MD) and was resuspended in TE buffer (10
mM Tris, pH 8.0, 1 mM EDTA). RNA used in RT-PCR
analyses (see below) was treated with RQ1 ribonuclease-free
deoxyribonuclease (Promega Corp., Madison, WI), extracted
with phenol/chloroform, precipitated with ethanol and 0.3 M
sodium acetate (pH 5.5), and resuspended in TE. RNA concentration was
estimated at 260 nm. Samples with
A260/A280 < 1.6 were not used.
RT-PCR
Starling whole-brain total RNA was reverse transcribed into cDNA
using Maloney murine leukemia virus reverse transcriptase in the
presence of random hexamer oligonucleotides and deoxynucleotide
triphosphates (Promega Corp.). A fragment of starling
ERß was amplified by PCR using 2.5 U Amplitaq polymerase
(Perkin-Elmer Corp., Branchburg, NJ) and synthetic
oligonucleotide primers designed from conserved regions of the DNA and
ligand binding domains of the quail and rat ERß cDNAs (42, 43) (see
Table 1 and Fig. 2). PCR was performed on one fifth of the
RT reaction under the following conditions: 94 C for 3 min followed by
35 cycles of 94 C for 30 sec, 60 C for 30 sec, and 72 C for 1 min.
After a final extension step at 72 C for 7 min, the amplified product
was gel purified and then cloned into pCR-Script AMP SK+ using the
manufacturer’s instructions (Stratagene, La Jolla,
CA).
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were also amplified and
cloned as described for ERß. Primer sequences were derived from the
canary AR (GenBank Accession L25901; Ref. 49) and zebra finch
ER cDNAs (GenBank Accession L79911; Ref. 37). See Table 1
for primer
sequences. For ER, the annealing temperature was 55 C, and the
extension step was increased from 1 to 2 min. All other reaction
conditions were the same as for ERß.
To assess the tissue distribution of ERß expression, RT-PCR was
performed on the following starling tissues: telencephalon,
hypothalamus, cerebellum, pituitary, heart, skeletal muscle, liver,
adrenal, kidney, spleen, intestine, pancreas, and ovary. ERß and
ß-actin (loading control) were amplified in separate analyses, each
using one fifth of the RT reaction. The ß-actin primers used were
derived from chicken (Gallus gallus) ß-actin cDNA sequence
(GenBank Accession X00182; Ref. 50) and are indicated in Table 1.
Primers for ERß were designed from 3'-untranslated region (UTR)
sequence obtained in the course of library screening (see
below). The positions of the primers are indicated in Fig. 2.
In all RT-PCR analyses, specificity of amplified products was confirmed by including control reactions containing water alone or RNA from which the RT enzyme was omitted. All PCR primers were obtained commercially (IDT, Coralville, IA).
Library construction and screening
Hypothalamic areas of photosensitive starlings (eight males and
six females) were used for messenger RNA (mRNA) extraction and library
construction. Birds were housed under an 11L:13D photoperiod. Brains
were extracted after decapitation, and the preoptic area (POA) was
rapidly dissected out and frozen on dry ice. To collect the POA, the
brain was inverted upon extraction, and a block of tissue
(approximately 5 mm x 5 mm x 5 mm) was cut, with a scalpel,
from the area dorsal to the pituitary gland. A total of 1 g of
tissue was ground under liquid nitrogen and homogenized, and poly(A)+
RNA was extracted via gravity elution (Poly A+ RiboSep kit;
Becton Dickinson and Co. Labware, Bedford, MA). A cDNA
library was constructed commercially in Uni-ZAP XR
(Stratagene) and subsequently was amplified according to
the manufacturer’s instructions.
The library was screened using standard techniques. Briefly, 500,000 plaques were plated and transferred, in duplicate, to nitrocellulose filters (Schleicher & Schuell, Inc., Keene, NH). After a 4-h prehybridization, the filters were hybridized overnight at 42 C with a random prime-labeled (32P-deoxycytidine triphosphate; Amersham Pharmacia Biotech, Arlington Heights, IL) starling partial ERß cDNA (generated above) in 50% formamide, 5x SSPE, 10% dextran sulfate, 1x Denhardt’s, 0.1% SDS, and 100 µg/ml denatured salmon sperm DNA. Filters were washed consecutively in decreasing concentrations of SSC (2–0.2x) and 0.1% SDS for 1 h each at 65 C and then were exposed to x-ray film (XOMAT, Eastman Kodak Co., Rochester, NY) for 2 days, at -80 C, with an intensifying screen. Duplicating plaques were further purified through secondary and tertiary screens. Phagemid DNA was purified from isolated plaques after in vivo excision and transformation into SOLR cells. Clones were sequenced as described below.
5' Rapid amplification of cDNA ends (RACE)
Amplification of additional 5' cDNA sequence was performed with
the 5' RACE System (V. 2.0, Life Technologies, Inc.),
following the manufacturer’s instructions. Briefly, starling brain
total RNA was reverse transcribed into cDNA with a gene-specific primer
(GSP1): 5'-TTCGTAGCACTTCCTGAGCC-3'. Next, the cDNA was tailed with
deoxycytidine triphosphate and terminal transferase, and PCR was
performed with the 5' RACE Abridged Anchor Primer and a second
gene-specific primer designed 5' of GSP1 (GSP2/2):
5'-ACTGCAGACTGCACAGAAGTG-3'. The PCR reaction was then diluted and a
second round of PCR was performed with the Universal Amplification
Primer and a second nested gene-specific primer located 5' of GSP2/2
(GSP5): 5'-GGGCTTGTACAGTCATTGCCA-3'. Amplified products were purified
from a 1% low-melt agarose gel and sequenced directly using various
primers (see below).
DNA sequencing
DNA was sequenced using BigDye Terminator Cycle Sequencing (ABI
Prism, Foster City, CA) and T3 and T7 promoter primers (Promega Corp.) or gene-specific primers (IDT). Sequences were aligned
using Sequencher (v. 3.0, Gene Codes Corp., Ann Arbor, MI) on a
Macintosh 9500/150 computer (Apple, Cupertino, CA).
Genomic DNA extraction and Southern blot analysis
Starling liver was homogenized briefly in DNA digestion buffer
[100 mM NaCl, 10 mM Tris (pH 8.0), 25
mM EDTA (pH 8.0), 0.5% SDS, 100 µg/ml proteinase K] and
incubated overnight, at 50 C, with shaking (200 rpm). After two
extractions with phenol/chloroform and precipitation with ethanol, DNA
was resuspended at a concentration of 1 µg/µl in TE. Twelve
micrograms of DNA were digested overnight with BamHI,
EcoRI, HindIII, NcoI, or
PstI and were electrophoresed through a 0.8% agarose gel.
DNA was then depurinated and denatured before being transferred to a
nylon filter (Nytran, Schleicher & Schuell, Inc.) by
capillary action. Filters were hybridized with a random prime-labeled
PCR-derived cDNA fragment corresponding to 636-bp of the starling ERß
3'-UTR (see Fig. 2). Blots were hybridized under both high [50%
formamide, 1 M NaCl, 1% SDS, 10% dextran sulfate, 100
µg/ml denatured salmon sperm DNA at 42 C] and low-stringency
conditions [25% formamide, 1 M NaCl, 1% SDS, 10%
dextran sulfate, 100 µg/ml denatured salmon sperm DNA at 37 C].
Filters were washed in 2x SSC/1% SDS at 55 C (low) or 65 C (high) for
1 h and exposed to Biomax film (Eastman Kodak Co.)
with two intensifying screens at -80 C.
Northern blot analysis
Total RNA was extracted using Trizol from pooled kidneys of
female starlings photostimulated with long days (16L:8D) for 3 weeks.
Poly(A)+ RNA was further purified using the PolyATtract System
(Promega Corp.). Approximately 6 µg poly(A)+ RNA were
electrophoresed through a 1.5% agarose, 3.3% formaldehyde gel. RNA
was then transferred overnight to a nylon membrane (Nytran) by
capillary action. The filter was hybridized overnight at 42 C with the
same probe used for Southern blotting (above) in 50% formamide, 5
x SSC, 1 x Denhardt’s, 20 mM NaPO4 (pH
6.8), 1% SDS, 5% dextran sulfate, and 100 µg/ml denatured salmon
sperm DNA. The filter was washed for 30 min each in 2x SSPE/0.5% SDS,
at room temperature and at 65 C, and then exposed to Biomax film at
-80 C with two intensify screens.
In situ hybridization histochemistry
Frozen 20-µm coronal sections from first-year, photosensitive
male starling brains were cut on a cryostat and mounted onto
gelatin-coated slides. Sections were fixed in 4% paraformaldehyde (pH
7.4), acetylated with 0.25% acetic anhydride in 1x
triethanolamine (pH 8.0), and dehydrated in a graded series of
alcohol. Adjacent sections were hybridized with antisense riboprobes
[complementary RNA (cRNA)] directed against starling ER, ERß, or
AR mRNA. cRNA probes were transcribed in vitro from
linearized plasmids containing partial cDNAs of the three receptors
(see above; note that the ERß probe corresponded to 636-bp of the
3'-UTR; see Fig. 2) with T7 or T3 RNA polymerase (MAXIscript,
Ambion, Inc. Austin, TX) in the presence of
33P-uridine 5'-triphosphate (2000 Ci/mmol, 10 mCi/ml;
NEN Life Science Products, Boston, MA) to a
specific activity of 3.2 x 108 cpm/µg. Probes were
applied to sections at a final concentration of 0.1 µg/ml in 50%
formamide, 300 mM NaCl, 10 mM Tris (pH 8.0), 1
mM EDTA (pH 8.0), 1x Denhardt’s, 10% dextran sulfate, 10
mM dithiothreitol, 500 µg/ml yeast transfer RNA, and 500
µg/ml poly(A)+ RNA. Coverslips were applied, and hybridization
proceeded overnight at 51 C.
Coverslips were removed in two changes of 4x SSC. Sections were then treated with 20 µg/ml ribonuclease A in 2x SSC at 37 C for 30 min, rinsed in 2x SSC for an additional 30 min at 37 C, and then incubated in 2x SSC for 2.5 h at room temperature. After a final stringent wash in 0.1x SSC at 65 C for 30 min, sections were dehydrated in a graded series of alcohol and then exposed to Biomax film at room temperature for 9 days. After developing the film, slides were dipped in NTB-2 emulsion (Eastman Kodak Co.), air dried, and stored with desiccant at 4 C for 5 weeks until developed using standard techniques. Control experiments using sense probes did not produce detectable signals in the tissues examined.
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Results |
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and 3).
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) based on
the presence of an ATG codon at nucleotide position 203 preceded
by an in-frame stop codon at nucleotide position 98 (51). The predicted
protein is 554 amino acids in length, with a calculated molecular mass
of 62.1 kDa, and it shares a high degree of sequence identity with
ERß in other species (see Fig. 3). Overall, starling ERß is greater
than 70% identical with rat, human, and quail ERß. Within the DNA
and ligand binding domains, the amino acid sequence is 98–100 and
89–98% identical, respectively. The full-length ER cDNA has been
cloned and sequenced in another songbird species, the zebra finch (37).
Overall, starling ERß and zebra finch ER share less than 45%
sequence identity (data not shown), but the DNA (97%) and ligand
binding domains (61%) are more highly conserved.
Southern blot analysis
To determine whether ERß is a single copy gene in starlings, we
digested genomic DNA with several restriction endonucleases and
performed Southern blot hybridization with a PCR-generated fragment
containing 636 bp of the 3' UTR of the starling ERß cDNA. As shown in
Fig. 4 (left panel),
high-stringency hybridization conditions yielded unique bands for all
of the enzymes used. An identical banding pattern was observed under
low-stringency conditions (see Fig. 4, right panel). The
blots were hybridized with additional probes corresponding to different
parts of the ERß cDNA, and similar results were obtained (data not
shown). These data are consistent with the proposition that ERß is a
single copy gene in starlings.
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). A low level of expression was
observed in cerebellum, heart, and spleen. We did not detect any
products in pancreas in ethidium bromide-stained agarose gels (see Fig. 5). We repeated the analysis with different primer sets and found
similar results (data not shown). RT-PCR, using primers directed
against ß-actin, produced products of the predicted size in all the
tissues examined. PCR with water alone or with RNA in the absence of RT
failed to produce detectable signals (see Fig. 5).
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). Four prominent transcripts were detected (2.1, 2.3, 4.4, and
7.5 kb), the most abundant at approximately 4.4 kb (see Fig. 6). A similar banding pattern,
although of lower intensity, was observed in ovary (data not shown).
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, and
ERß antisense riboprobes. As described in other species (20, 29), we
detected high levels of AR expression in several song control nuclei,
including lMAN, mMAN, HVc, and RA (see Figs. 7, 8, and 9). Contrary to previous reports, we also
detected AR mRNA containing cells within area X, although the signal
was not as prominent as in the other song nuclei listed above (data not
shown). ER mRNA was expressed in the caudomedial aspect of HVc (also
known as paraHVc; Ref. 30) but not in any of the other song nuclei
examined (see Figs. 7; 9; and 10, a,
b). We did not detect ERß mRNA in any forebrain song control nuclei.
The distinct patterns of AR, ER, and ERß expression are perhaps
most apparent in the case of HVc (see Figs. 7 and 9). Figure 9 provides
a schematic representation of the distribution of AR, ER, and ERß
expression at three different levels of the caudal neostriatum in the
vicinity of HVc. As can be seen, AR mRNA-containing cells occur
throughout the rostral-caudal extent of HVc. ER is selectively
expressed in the medial portion of HVc and along the medial border of
the caudal neostriatum. Although ERß does not seem to be expressed in
the song system, a low level of expression was observed in the
caudomedial neostriatum in a pattern distinct from ER in that part
of the brain (see Figs. 9 and 10). In addition, a relatively high level
of ERß mRNA was also detected in nucleus taeniae (see Figs. 9; and
10, c and d).
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(see Fig. 11). AR
mRNA was much less abundant, but still detectable, in adjacent
sections.
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Discussion |
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In addition to the cross-species sequence similarity, several pieces of
information converge to indicate that a second form of the estrogen
receptor exists in starlings, as it does in other vertebrates. First,
the reported sequence shares greater similarity to ERß in nonavian
species than it does to ER from another songbird species, the zebra
finch (37). Second, we cloned a partial cDNA for ER in starlings, in
the present study, that is more similar to zebra finch ER than it is
to starling ERß (data not shown). Third, in situ
hybridization analysis shows separate and distinct expression patterns
of ER and ERß in the starling forebrain.
Starling ERß shares additional features with ERß in other species. For example, RT-PCR analysis shows that starling ERß is expressed in a wide variety of tissues. A broad tissue distribution has also been observed in other species (42, 44, 53, 56, 57, 58, 59, 60). In addition, as described for humans, mice, and quail (44, 45, 53, 54), the starling ERß gene encodes multiple transcripts, as indicated by Northern blot analysis. In starling kidney, we detect at least four transcripts, the most abundant at 4.4 kb. The presence of multiple transcripts of ERß suggests that the receptor message may be alternatively spliced. Indeed, several isoforms of the receptor have been described for other species (e.g. Refs. 57, 58, 61, 62, 63, 64, 65). We have not systematically examined whether the starling ERß mRNA is alternatively spliced, but the presence of at least four transcripts in kidney is consistent with this possibility.
Our primary interest in cloning the ERß in a songbird was to examine
its expression in the brain, relative to AR and ER, particularly
within the song control system. Androgens and estrogens play important
roles in activating singing behavior in adult songbirds (39, 40). In
addition, though there is controversy regarding the role of estrogens
in sexual differentiation of the song system (4, 5), it is clear that
estrogen treatment early in life can masculinize the song system and
singing behavior in females (6, 7, 66). The effects of androgens may be
direct because AR are expressed widely throughout the song system
(e.g. Refs. 18, 20, 29). The mechanisms through which
estrogens regulate the song system and singing behavior are less
well-understood, however, because ER mRNA and estrogen binding have
only been observed in one forebrain song control nucleus, HVc
(e.g. Refs. 27, 28, 30, 35, 37, 38). As a result of this
pattern of receptor expression, many investigators have concluded that
many estrogenic effects in the song system are
trans-synaptic (e.g. 41). An alternative
possibility is that estrogens may act through a second receptor such as
ERß. By cloning this receptor, we were able to examine this
possibility directly.
The results of in situ hybridization histochemical analyses
indicate that ERß mRNA is not detectable in any of the forebrain song
control nuclei examined (i.e. mMAN, lMAN, area X, HVc, and
RA). Other parts of the song system in the midbrain and hindbrain also
concentrate steroids (e.g. Refs. 18, 20, 21, 26). We have
not yet examined these regions and, therefore, do not know whether
ERß is expressed in other parts of the song system. The lack of ERß
mRNA in HVc contrasts with ER expression in adjacent sections. Using
starling-specific cRNA probes, we detect ER mRNA- expressing
cells extending from the medial part of HVc ventrally along the medial
portion of the caudal neostriatum. A significantly lower level of ER
expression is visible in the more lateral subdivision of HVc. This
pattern of expression is similar to that described previously in other
species (e.g. Refs. 30, 31, 38). It should be noted that in
canaries (Serinus canaria), some analyses indicate that
estrogen receptors are located throughout HVc, including the lateral
portion (27, 28, 32, 34), whereas other studies in the same species
reveal a distribution consistent with our observations in starlings
(30).
Although ERß is not expressed in HVc, we detect ERß mRNA in the
caudomedial neostriatum in a pattern distinct from ER. Rather than
showing a high level of expression clustered medially along the
midline, ERß seems to be expressed at a uniformly low level extending
from midline, several millimeters laterally. The distribution of ERß
mRNA within this part of the neostriatum, and within nucleus taeniae
(the avian homolog of the amygdala), is similar to the distribution of
P450 aromatase mRNA and protein-containing cells within these brain
regions in zebra finches (Taeniopygia guttata) (67, 68, 69).
There are exceptions, however, to this overlapping expression profile.
Most notably, we do not detect ERß mRNA expression dorsal to the
lamina archistriatalis dorsalis, where there is a band of aromatase-
producing cells in zebra finches (67, 68, 69). Nonetheless, the
coexpression of aromatase and ERß in caudomedial neo-striatum
suggests that locally synthesized estrogens may exert some of their
effects through ERß expressed in the same or neighboring cells. In
addition, it is perhaps noteworthy that ERß expression in the
caudomedial neostriatum occurs in the same area in which the
immediate-early gene, ZENK, is expressed in response to song (70).
The absence of ERß expression in forebrain song nuclei is, in many ways, not surprising. Previous studies, using in vivo autoradiography, failed to detect estrogen binding sites within forebrain song nuclei, with the exception of the medial part of HVc (30, 38). If ERß were expressed in song nuclei, then binding may have been observed in these regions. Yet, a failure to observe binding by in vivo autoradiography does not necessarily indicate a lack of binding sites. If receptor levels are very low, then detection may be below the sensitivity of the assay. In addition, the failure of previous studies, and our own, to localize estrogen binding sites or ERß within forebrain song control nuclei may be a product of the sex, age, season, and hormonal condition of the animals under investigation. For example, here we examined expression only in short-day photosensitive male starlings. Perhaps if we had examined expression in different photoperiodic conditions or throughout development, we may have observed a different pattern of expression.
Despite the lack of ERß mRNA in the song system, we detect a high
level of expression within the medial POA. In this region of the brain,
both ERß and ER are highly expressed. Thus, as has been described
in rats (46, 47, 71), the two forms of the estrogen receptor show both
overlapping and discrete patterns of expression in the starling brain.
As is the case in the caudomedial neostriatum, the medial POA also
contains a dense population of aromatase-expressing cells (67, 68, 69),
again providing a coincidence of the sites of estrogen synthesis and
binding. Of course, the present analysis only examines mRNA expression.
Further analyses are necessary to confirm the presence of ERß protein
in this and other brain regions.
Finally, we examined the distribution of the AR mRNA in the song system of starlings. In general, we observe the same pattern of expression that has been described in a variety of species (e.g. Refs. 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29). High levels of AR mRNA are observed in mMAN, lMAN, HVc, and RA. In addition, we detected a low level of AR expression in area X. Prior studies, using in vivo autoradiography, immunocytochemistry, and in situ hybridization in several species, have not reported AR within this nucleus (19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29). We are currently repeating our analysis to determine whether this discrepancy reflects interspecific variation or whether AR expression within area X is regulated by hormonal and/or photoperiodic condition.
In summary, we have cloned the ERß cDNA in European starlings and
have shown that it is expressed in a variety of tissues. Within the
brain, ERß mRNA is not detectable within forebrain song control
nuclei but is expressed in other parts of the brain previously shown to
express aromatase. The distribution of AR and ER mRNA-containing
cells in the song system is similar to that described in other species.
Further studies are needed to determine whether the pattern of ERß
expression is regulated developmentally or seasonally; both are
life-history stages when dramatic changes in song system morphology and
circulating sex steroid hormones are known to occur.
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Acknowledgments |
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Footnotes |
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Received December 21, 1998.
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K. K. Soma, N. A. Alday, M. Hau, and B. A. Schlinger Dehydroepiandrosterone Metabolism by 3{beta}-Hydroxysteroid Dehydrogenase/{Delta}5-{Delta}4 Isomerase in Adult Zebra Finch Brain: Sex Difference and Rapid Effect of Stress Endocrinology, April 1, 2004; 145(4): 1668 - 1677. [Abstract] [Full Text] [PDF]
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 C. Griffin, G. Flouriot, P. Sharp, G. Greene, and F. Gannon Distribution Analysis of the Two Chicken Estrogen Receptor-Alpha Isoforms and Their Transcripts in the Hypothalamus and Anterior Pituitary Gland Biol Reprod, October 1, 2001; 65(4): 1156 - 1163. [Abstract] [Full Text] [PDF]
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 A. Dawson, V. M. King, G. E. Bentley, and G. F. Ball Photoperiodic Control of Seasonality in Birds J Biol Rhythms, August 1, 2001; 16(4): 365 - 380. [Abstract] [PDF]
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 J. A. McLachlan Environmental Signaling: What Embryos and Evolution Teach Us About Endocrine Disrupting Chemicals Endocr. Rev., June 1, 2001; 22(3): 319 - 341. [Abstract] [Full Text] [PDF]
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 D. J. Bernard and T. K. Woodruff Inhibin Binding Protein in Rats: Alternative Transcripts and Regulation in the Pituitary across the Estrous Cycle Mol. Endocrinol., April 1, 2001; 15(4): 654 - 667. [Abstract] [Full Text]
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E. A. Brenowitz and K. Lent Afferent Input Is Necessary for Seasonal Growth and Maintenance of Adult Avian Song Control Circuits J. Neurosci., April 1, 2001; 21(7): 2320 - 2329. [Abstract] [Full Text] [PDF]
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