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Equipe d’Endocrinologie Moléculaire de la Reproduction, UPRES-A Centre National de la Recherche Scientifique 6026, Université de Rennes I, 35042 Rennes, France
Address all correspondence and requests for reprints to: Dr. Farzad Pakdel, Equipe d’Endocrinologie Moléculaire de la Reproduction, UPRES-A Centre National de la Recherche Scientifique 6026, Université de Rennes I, 35042 Rennes Cedex, France. E-mail: farzad.pakdel{at}univ-rennes1.fr
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(ER) and ERß,
which are members of the nuclear receptor family (2). Ligand-activated
ER regulates gene expression directly by binding to a specific
cis-element called the estrogen-responsive element (ERE) or
indirectly by interaction with other transcription factors implicated
in specific gene transcriptional pathways. On the basis of sequence
homologies, ER was divided into six distinct regions (A–F), which have
been shown to represent functionally independent domains. The ER
trans-activation functions, namely AF1 and AF2, were mapped
respectively at the N-terminal (A/B) and C-terminal (E/F) regions.
Interactions between these regions and specific coactivators,
possessing histone acetyltransferase activity may result in the
modification of target gene expression (3, 4, 5).
In mammals, in addition to ER and -ß forms generated from two
distinct genes, several isoforms of these two subtypes were recently
reported. These are transcribed either by alternative exon splicing or
usage of different promoters of a single gene (6, 7, 8, 9, 10). The existence of
multiple forms of the receptor may explain the pleiotropic actions of
estrogens in diverse tissues or species.
In fish, estrogens play a key role in gonadal sex differentiation, liver vitellogenesis, and fat metabolism. Although the liver and the ovary appear to be major targets of estradiol, the liver from previtellogenic female fish contains much higher ER levels than the ovary (11). For this reason, ER complementary DNA (cDNA) from several fish species were cloned from the liver and, in all of them, the A domain was lacking compared with that in mammals, birds, or amphibian ER (11, 12, 13, 14, 15). We report here the cloning and functional characterization of a new ER isoform possessing an extra 45 amino acid residues in-frame with the previously cloned rainbow trout ER.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Construction and screening of a trout ovary cDNA library
Two micrograms of polyadenylated RNA isolated from ovaries
containing vitellogenic follicles with a mean diameter of 1.2 mm were
used to prepare double stranded cDNA as described previously (16) with
Amersham Pharmacia Biotech’s cDNA synthesis system
(Arlington Heights, IL). The cDNA was ligated to the
EcoRI/NotI linker (Pharmacia) and then
phosphorylated by polynucleotide kinase for 45 min at 37 C.
Double-stranded cDNA was fractionated by gel filtration on a Sepharose
4B column (Pharmacia). The largest fractions were pooled and ligated to
gt10 vector, which had been dephosphorylated and digested with
EcoRI (Stratagene, La Jolla, CA) at a molar
ratio of 1:1 (cDNA:phage DNA). The ligation products were packaged
in vitro as recommended by the manufacturer (Promega Corp., Madison, WI; Biotec’s packaging system). Recombinants
(1.1 x 107) were obtained, and
approximately 5 x 105 recombinant phages
were screened. Duplicate filters (Hybond N, Amersham Pharmacia Biotech) were hybridized with either
32P-labeled rainbow trout ER (rtER) cDNA under
high stringency hybridization conditions (17) or with a 600-bp
SacI/SacI fragment of rat ERß hormone-binding
domain (18). With this latter probe, hybridization was performed with
lower stringency conditions using 25% formamide instead of 50% as
described by Thomas (17). Several positive clones were obtained, and
one of them, O2–512, which was positive with both probes and
contained a 3.5-kb cDNA fragment, was purified. The cDNA fragment was
subcloned into the EcoRI site of pBluescript II KS
(Stratagene) and sequenced on both strands. Nucleotide
sequences were analyzed by the Genejockey computer program. The
corresponding sequences have GenBank/EMBL Data Bank accession no.
AJ242740 and AJ242741.
S1 nuclease assay and primer extension
Modified S1 nuclease protection and primer extension procedures
were used as previously described (19, 20). These two methods involve
the use of biotinylated, single stranded DNA templates to prepare
highly labeled, single stranded DNA probes or long primers by extension
from specific primers with the T7 DNA polymerase in the presence of
[-32P]deoxy-CTP (3000 Ci/mmol). These probes
or long primers are then hybridized with the appropriate RNA sample and
subjected to either S1 nuclease digestion or reverse transcriptase
extension, respectively.
The origin of probe A template was a genomic PCR product obtained by
amplification of the regions from -424 in intron 1 to +508 in exon 2
(see Fig. 2A
). The DNA fragment was subcloned in the PCRTM2.1 vector
(Invitrogen, San Diego, CA) downstream of T7 and upstream
of M13 reverse primer. A PCR reaction was then performed using a
biotinylated T7 primer with M13 reverse primer. To generate the
template used to make probe B, a PCR reaction was performed using
pBluescript KS plasmid containing the entire rtER short form
(rtERS) cDNA, with a biotinylated T7 primer and
primer I (5'-CTATATAAGGGTGTGGACGAGG-3'). Finally, the template for the
long primer 
was obtained by PCR using the 5'-biotinylated primer
III (5'-ATGTACCCTGAGGAGACACGCGGA-3') and primer I.
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were obtained by
extending primer II (5'-TCTCCAGGTAGTATGACTGGCTGG-3') annealed to the
different biotinylated, single stranded templates. After elution of the
single stranded DNA probes by alkaline treatment and magnetic
separation, 105 cpm of the probe or primer were
coprecipitated with 30 µg total RNA and then dissolved in 20–30 µl
hybridization buffer [80% formamide, 40 mM PIPES (pH
6.4), 400 mM NaCl, and 1 mM EDTA (pH 8)],
denatured at 70 C for 10 min and hybridized overnight at 55 C. The S1
digestions and the reverse transcriptase extension were carried out,
and the samples were electrophoresed through denaturing
polyacrylamide-urea gels.
Transcription start sites were identified by the PEETA (primer
extension, electrophoresis, elution, tailing and amplification) method,
as recently described (21). Briefly, the extension product of interest,
obtained from primer (see Fig. 2A
) extension, was cut out and
eluted from the gel with 500 µl 0.5 M ammonium acetate,
10 mM magnesium acetate, 1 mM EDTA (pH 8), and
0.1% SDS followed by a Qiaex II gel extraction (QIAGEN,
Valencia, CA) as recommended by the manufacturer. Poly(C) tail was
added to the extension product at 37 C for 30 min using 50 U terminal
deoxynucleotidyl transferase, the corresponding buffer [100
mM cacodylate buffer (pH 6.8), 1 mM
CoCl2, and 0.1 mM dithiothreitol;
Promega Corp.] and 4 mM deoxy-CTP in a final
volume of 20 µl. The reaction was stopped by heating for 10 min at 65
C. One microliter of the reaction was subsequently submitted to 35
rounds of PCR amplification using an oligo(dG) adapter-primer
(5'-GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-3') and primer IV
(5'-GAGTTTTGGGGGGGAGATGACGTG-3'), which hybridizes between primer
and the exon 2 acceptor site. The PCR product was then separated by
electrophoresis on a 1.5% agarose gel, eluted using a Qiaex II gel
extraction kit (QIAGEN) and subcloned in the TA cloning
vector pCRTM2.1 (Invitrogen). The DNA product was
sequenced by the dideoxy chain termination method.
RT-PCR analysis
RT and PCR amplifications were performed using the Titan
one-tube RT-PCR kit (Roche Molecular Biochemicals).
Briefly, 5 µg total RNA from estradiol
(E2)-treated trout liver were reverse transcribed
(30 min at 50 C) using 25 pmol downstream oligonucleotide, P3
(5'-CGCTGTCCTGTGCTCCAGG-3'), located in exon 5 of rtER. Specific cDNAs
were amplified using the common primer P3 in combination with either
primer P1 (5'-ATTGTGATGCGAAGCCAGAT-3'), located in exon 1, or primer P2
(5'-TGTGAATGTGATGCTGGTCA-3'), located in exon 2a. PCR parameters were
an initial denaturation (94 C for 2 min), followed by 40 cycles of 94 C
for 30 sec (denaturation), 58 C for 30 sec (annealing), and 68 C for 45
sec (primer extension). Amplified products (1/10th of each reaction)
were separated on 1% agarose gel electrophoresis and stained with
ethidium bromide. The gel was also transferred onto a nylon membrane
(Hybond N, Amersham Pharmacia Biotech) and hybridized with
a labeled rtERS cDNA probe.
Expression of short and long forms of rtER (rtERS and
rtERL) in rabbit reticulocyte lysate
The in vitro translation reaction was performed using
1 µg pBluescript II KS plasmids containing the entire coding region
of both ER cDNAs and T7 RNA polymerase in a rabbit reticulocyte lysate
(methionine depleted) with 20 µCi
[35S]methionine. Reactions were carried out at
30 C for 60–120 min, as recommended by the supplier (TNT Reticulocyte
Lysate System, Promega Corp.). Translation products of
synthetic mRNA were analyzed by SDS-PAGE and autoradiography.
Western blot analysis
Western blots were performed with slight modifications of a
previously described method (22). Routinely, 25–30 µg protein in
nuclear extracts from trout liver or whole cell extracts from yeast
cells were fractionated on 10% polyacrylamide-SDS gels and transferred
to Immobilon-P type polyvinylidene difluoride membranes
(Millipore Corp., Bedford, MA). Blots were incubated for
12 h with the purified anti-rtER antibody (raised against
C-terminal domains of rtER), diluted 1:100. Then the blots were
incubated for 1 h with the secondary antibody, which was either
goat antirabbit IgG-horseradish peroxidase phosphatase conjugated
(dilution of 1:1000 of commercial stock, TEBU, Le
Perray-en-yvelines, France) or goat antirabbit IgG-alkaline
phosphatase conjugated (dilution of 1:5000 of commercial stock, Tropix,
Bedford, MA). After several washes, the blots were revealed by either
4-chloro-1-naphthol (Sigma) or the CSPD chemiluminescent
detection system from a Tropix kit by autoradiography for 1–2 min. All
incubations and manipulations were performed at 22-25 C as previously
described (22).
Plasmid constructions, yeast expression, and
ß-galactosidase assays
The entire coding region of rtERL was
amplified by PCR and inserted into the unique BamHI site of
the yeast expression vector YEpucG, which was also used to express the
entire coding region of rtERS as described
previously (23). The reporter genes used in this study were
3EREc-Cyc-lacZ (23) and FP3-EREp-Cyc-lacZ (24). Briefly, 3EREc-Cyc-lacZ
reporter plasmid was constructed with three tandem copies of the
consensus ERE linked to the yeast cytochrome (Cyc) proximal promoter
located upstream the lacZ gene in the yeast vector PLGÆ178
(25). The reporter FP3-EREp-Cyc-lacZ was constructed with 100 bp of the
trout ER gene promoter encompassing the major cis-acting
element involved in the estrogen regulation of the gene and containing
an imperfect ERE (26). This 100-bp fragment (FP3-EREp) was subcloned
into the XhoI site of the Cyc promoter of PLGÆ178 yeast
vector (24). All of the constructions were verified by sequencing.
The yeast strain used in this study was BJ2168 (a leu2 trp1 ura3–52 prb1–1122 pep4–3 prc1–407 gal2; Yeast Genetic Stock Center, Berkeley, CA). As described previously (23), yeast cells were stably transformed with 2 µg of each plasmid using a standard lithium acetate method and were selected by growth on complete minimal medium [0.13% dropout powder lacking uracil and tryptophan, 0.67% yeast nitrogen base, 0.5% (NH4)2SO4, and 1% dextrose]. ß-Galactosidase assays were performed as previously reported (23) in the presence of 17ß-estradiol or different hormones (for 4 h at 30 C) for the trans-activation assays. ß-Galactosidase activity was measured at 420 nm using the o-nitrophenyl ß-D-galactopyranoside substrate. The formation of colored product was quantified with a spectrophotometer. ß-Galactosidase activity was expressed in Miller units.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), contains 171 nucleotides in the
5'-untranslated region and possesses one in-frame ATG codon, located at
nucleotide 172, which adds 45 amino acid residues at the N-terminal
region of the short isoform rtER (Fig. 1B). This cDNA open reading
frame encodes a protein of 622-amino acid residues with a calculated
molecular mass of 68.1 kDa. The complete cDNA sequence of
rtERL was deposited at the EMBL database
(accession no. AJ242740).
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). This process does
not modify the structure of the rtERS protein, as
illustrated in Fig. 2A. The first 227 nucleotides of the novel
rtERL cDNA, referred to as exon 2a, exhibited
100% sequence identity with the 3'-end of intron 1 of the trout ER
gene (data not shown). To confirm the transcriptional initiation of a
new class of rtER transcript within intron 1, primer extension and
S1 nuclease assays were performed with RNA from
liver, a tissue in which estrogens have been reported to have important
regulatory functions, such as the induction of vitellogenin (28). The
two single stranded DNA S1 probes, A and B, were designed to be
specific for rtERL and
rtERS mRNAs, respectively (see Materials
and Methods for the preparation of the probes). These two probes
shared a common region 3' to the acceptor splice site of exon 2 (Fig. 2A). After hybridization of probe A with liver RNA and S1 nuclease
digestion, several specific protected fragments were detected. No
protected fragments were seen with yeast RNA, which was used as a
negative control. The two smallest fragments (366 and 407 bp)
corresponded to a protection of rtERS mRNA 1 and
2 that remained homologous to probe A as far as the two acceptor splice
sites at the 3'-end of intron 1, and then diverged in their 5'-ends
from probe A complementary sequences. Protected fragments,
approximately 500–600 bases in size, indicated the existence of a
distinct class of rtER transcripts (rtERL mRNAs)
that are generated from intron 1 (Fig. 2B, probe A). It is worth noting
that several transcription start sites were detected for this new class
of transcripts. Likewise, the detection of a fragment corresponding in
size to a total protection of rtER-specific sequences of probe A
(without the 88 last nucleotides of the probe that correspond to the
vector sequence) indicated the existence of rtERL
transcripts that could start further in 5'. The results of the S1
nuclease mapping analysis with probe B showed, in addition to the
expected protected fragment detected at the acceptor splice site of
exon 2 (due to a partial protection of probe B by
rtERL mRNA and rtERS mRNA
2, 366 bases in size), two other protected fragments that corresponded
to the transcription start sites of rtERS mRNA 1
(Fig. 2B, probe B). The sizes of these fragments were 475 and 490
bases.
The existence of two classes of rtER transcripts was
strengthened by a primer extension experiment using primer , which
was designed to hybridize rtER mRNAs in a region downstream from the
splice site position in exon 2 and was thus able to be extended to all
of the 5'-extremities of rtER transcripts. The results showed several
extension products corroborating in size to the transcription start
sites of rtERL mRNA (fragment of 584 bases),
rtERS mRNA 2 (fragments from 470–490 bases), and
rtERS mRNA 1 (fragments from 510–530 bp)
determined above by S1 nuclease assay (Fig. 2B, primer ). The
identity of the longest extension product was further confirmed by the
PEETA (primer extension, electrophoresis, elution, tailing, and
amplification) method (21). Although 10 nucleotides shorter, the
sequence of this extension product was similar to the 5'-extremity of
the novel rtERL cDNA clone. The existence of two
classes of rtER transcripts was finally confirmed by RT-PCR analysis
(Fig. 3). Single stranded cDNAs were
synthesized from liver RNA using a rtER gene-specific primer (P3)
located in exon 5. The two rtER transcripts were amplified by PCR using
specific 5'-primers for exon 1 (P1) or exon 2a (P2) in combination with
the common downstream primer (P3) in exon 5 (Fig. 3). The size of the
amplified cDNAs was as expected (Fig. 3B, left side), and
Southern blot hybridization with labeled rtERS
cDNA confirmed the specificity of these PCR products (Fig. 3B, right side). Additionally, the cDNA fragment amplified from
P2/P3, which was subcloned and partly sequenced, showed a sequence
identical to that of the rtERL cDNA clone (data
not shown). Together, these results showed that trout liver expresses
two classes of rtER transcripts potentially encoding two different rtER
protein isoforms. Quantitative analysis of the S1 nuclease mapping and
primer extension results indicated that rtERS
mRNAs are the main rtER transcripts expressed in liver and that both
classes of rtER mRNAs are up-regulated by estradiol
(compare lanes Liver -E2 and Liver +E2 in Fig. 2B).
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, lane 2). The expression
levels of both receptors were virtually undetectable in the liver of
untreated male trout (Fig. 4A, lane 1), whereas they were strongly
induced by estradiol. Analysis of the proteins synthesized by in
vitro translation from cDNA rtERL and
rtERS in rabbit reticulocyte lysate also
evidenced two protein bands migrating at the same mol wt as those
detected by Western blot analysis in liver extract (Fig. 4B, lanes 2
and 3). Moreover, transformed yeast with an expression vector
containing rtERS cDNA exhibited only a 65-kDa
protein form (Fig. 4C, lane 2), whereas the rtERL
transformed yeast expressed the 71-kDa protein as the major ER form
(Fig. 4C, lane 3). These data indicate that both classes of rtER
transcripts (rtERS and
rtERL mRNAs) detected in the liver are indeed
translated in this tissue, giving rise to different ER isoforms (Fig. 4).
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, the activation of reporter gene
by the rtERL was estrogen specific, and as
expected, no other hormones could stimulate transcriptional activity of
the receptor (Fig. 5A). Similar results were observed for
rtERS expressed in yeast Saccharomyces
cerevisiae (Fig. 5B). However, interestingly, in the absence of
exogenously added estrogens, rtERL did not
exhibit any transcriptional activity (Fig. 5A), whereas
rtERS consistently showed a significant
transcriptional activity representing 10–15% of the maximum
activity obtained with estradiol (Fig. 5B, see also Fig.
6).
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represents the dose-response curve
obtained in the ß-galactosidase assay from recombinant yeast cells
expressing either rtERS or
rtERL after exposure to various estradiol
concentrations. The lacZ reporter genes used for these
assays were 3ERE-Cyc, containing three ERE linked to the yeast
cytochrome c-proximal promoter (Fig. 6A), and FP3-EREp-Cyc,
which consisted of a 100-bp fragment of trout ER gene containing an
imperfect ERE linked to the yeast cytochrome c-proximal
promoter (Fig. 6B). Both reporter genes showed very low
ß-galactosidase activity in the absence of ER (<0.2 U; data not
shown). The level of ß-galactosidase expression was increased 50- to
100-fold (22 U, Fig. 6A; 13 U, Fig. 6B) by the presence of
rtERS in the absence of hormone (Fig. 6, A
and B). Upon addition of estradiol, the expression of both reporter
plasmids was further enhanced to reach almost 250 U (Fig. 6A) or 40 U
(Fig. 6B). In contrast, in the absence of E2, the
rtERL isoform did not exhibit any basal
transcriptional activity (Fig. 6), even with the reporter plasmid
FP3-EREp-Cyc, whereas rtERS showed a very high
E2-independent transcriptional activity (30% of
E2 induction; Fig. 6B). However, in the presence
of ligand, the stimulation of reporter gene expression by
rtERL was roughly half that achieved by
rtERS. These data indicate that although the
novel rtER isoform (rtERL) is a potent
trans-activator of estrogen target genes, this isoform
exhibited a clearly different hormone-independent
trans-activation function.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The sequence homologies and phylogenetic relationships of various ERs
suggest that rtER should be considered as an ER-like receptor, and
this is also true for most other teleost ERs described to date (14). It
is not clear why screening of the trout ovary cDNA library with the
hormone-binding domain (HBD) of rat ERß cDNA did not allow to clone a
trout ERß form. However, different hypothesis can be formulated. As
these experiments were performed under low stringent conditions, the
HBD of trout ERß-like might have lower sequence homology to rat ERß
HBD than that expected in comparison to this domain among ER cloned
from different species. In other words, the ligand-binding domain of
ERß may have been less conserved during evolution compared with the
-subtypes. In fact, phylogenetic analysis seems to indicate that
ER has been more conserved during evolution and that ERß would
represent a phylogenetically newer protein. However, the presence of
ERß subtype in fish cannot be excluded, because at least in
anguillomorph species, ß-like ERs are expressed (13, 14). Thus, in
future studies, the highly conserved DNA-binding domain of mammalian
ERß cDNA should be used to isolate this ER subtype in trout.
Analysis of the protein synthesized from both rtER isoform cDNAs revealed two protein bands at 65 and 71 kDa, which were also detected in liver nuclear extracts using a specific antibody raised against the C-terminal region of the receptor. Although the rtERS cDNA produces only a 65-kDa protein, expression of the novel rtERL cDNA in yeast S. cerevisiae demonstrated the production of a major 71-kDa protein that reacts strongly with the specific anti-rtER antibody. The hepatic endogenous 71-kDa protein form is probably encoded by the rtERL transcript, which is also present in this organ. However, we failed to detect ER proteins or transcripts in the ovary by classical assays such as Western or Northern blots (data not shown). Further experiments are needed to determine whether this is due to a lack of sensibility (probably much lower level of ER in this tissue) or if the receptor is only expressed during certain critical steps of ovarian maturation. On the other hand, unlike the liver, the ovary is not a homogenous tissue, and therefore, ER may be expressed only in some specific cells. To answer these questions and to investigate cell- and/or stage-specific expression of these two forms of rtER, in situ hybridization and/or immunocytochemistry with specific probes or antibodies for each ER form, S1 nuclease assays, and/or RT-PCR will be necessary.
Yeast expression systems were used by several groups to demonstrate
that steroid receptors can function as a ligand-dependent
transcriptional activator as in mammalian cells (31, 32, 33, 34, 35). These data
also indicated that the basic transcription apparatus has been
conserved among eukaryotes ranging from yeast to mammals. A previous
study from our laboratory showed that the short isoform of rtER
expressed in yeast exhibited low (10–20% of the total activity), but
consistent, hormone-independent transcriptional activity compared with
human ER (23). As the N-terminal region of these two receptors
exhibits very low homology, and the AF1 activity of ER seems to be
predominant in yeast (36, 37), we have speculated that this could be a
consequence of the differences in the structure/activity of AF1
functions within each receptor. Interestingly, expression of the novel
rtERL in yeast showed very low, if any,
hormone-independent activity compared with that of
rtERS. In a recent work, the region responsible
for rtER AF1 activity was mapped in the B domain, and in addition, we
showed that further deletion of the A domain from human ER induces a
ligand-independent activity, as found for rtERS.
Therefore, these data suggest that the presence of the A domain at the
N-terminal region of ER represses this hormone-independent activity of
the receptor (Métivier, R., F. Petit, Y. Valotaire, and F.
Pakdel, in preparation).
Wahli and collaborators (38) showed that in the liver of
Xenopus two forms of ER, a full-length and an N-terminal
truncated version, were produced. These two isoforms are generated from
a single mRNA species by use of two ATG initiator codons within the
same reading frame. Interestingly, the N-terminal truncated ER form
lacks the first 41 amino acids corresponding to the A domain. It is,
however, unknown whether specific transcripts encoding this N-terminal
truncated ER isoform also exist in Xenopus, as at least four
ER mRNA species were detected in the liver (39). Recently, a study in
chickens also showed the presence of an A domain-truncated ER generated
from a distinct hepatic mRNA species (Gannon, F., personal
communication). These data together with the results reported here
demonstrate that in addition to a full-length ER, the existence in all
oviparous species of an N-terminal-truncated ER isoform lacking the A
domain. This truncated ER exhibits significant hormone-independent
transcriptional activity compared with that of the full-length ER
variant or with human ER activity (23) (this study). Further
experiments should be performed using naturally occurring
estrogen-responsive reporter genes to elucidate whether these isoforms,
which differ only in their N-terminal region, may activate target genes
in a different manner. In parallel, it would be interesting to examine
possible differential properties of these ER isoforms for their
interaction with recently identified coactivators for steroid receptors
(40–42; for review, see Ref. 3). Although the physiological
significance of this truncated ER form is unclear, the fact that this
isoform was only found in the liver of different oviparous species may
implicate a role in vitellogenesis. The process of vitellogenesis,
common to all oviparous species (birds, reptiles, amphibians, and
fishes), is characterized by hepatic production of yolk lipoproteins
and their massive deposition within the oocytes, thus providing the
main nutritional reserves necessary for embryo development (28).
Vitellogenin, a complex glycophospholipoprotein synthesized by the
liver under estrogenic induction, is transported via the bloodstream to
the ovary, where it is sequestered by growing oocytes in a
cell-specific fashion. Therefore, in addition to its detoxification and
biotransformation activity, the liver of oviparous vertebrates also
represents a reproductive organ in which the ER gene is highly
expressed. One can speculate that unlike mammals, in all oviparous
animals the presence of a constitutively active ER form
(rtERS) may direct the hepatic tissue toward its
reproductive function during organogenesis. This hypothesis may be
reinforced by the fact that the constitutive activity of
rtERS can be stimulated by the orphan receptor
COUP-TF1 (24), which plays fundamental roles in embryogenesis and the
control of cell differentiation (43, 44). In vertebrates, COUP-TF
activity has also been associated with the transcriptional regulation
of several liver-expressed genes (45, 46, 47), including the trout ER gene
(24, 48).
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Acknowledgments |
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Footnotes |
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Received July 1, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3) in breast cancer and the consequences
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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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J. Bouma and J. J. Nagler Estrogen Receptor-{{alpha}} Protein Localization in the Testis of the Rainbow Trout (Oncorhynchus mykiss) During Different Stages of the Reproductive Cycle Biol Reprod, July 1, 2001; 65(1): 60 - 65. [Abstract] [Full Text] [PDF]
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