| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Regular Article |
a EMBL, D-69117, Heidelberg, Germany
b Roslin Institute (Edinburgh), Roslin, Midlothian EH25 9PS, United Kingdom
c Endocrinologie Moléculaire de la Reproduction, UMR CNRS 6026, 35042 Rennes Cedex, France
d The University of Chicago, The Ben May Institute for Cancer Research, MC6027, AMB N660B, Chicago, Illinois 60637
ABSTRACT
Estrogen plays a key role in the control of reproductive behavior and in the regulation of the neuroendocrine system. To elucidate the mechanisms by which it controls these functions it is important to understand how estrogenic effects are mediated. We have investigated the distribution of the two isoforms of the chicken estrogen receptor alpha (cER-
) protein; the previously characterized cER- 66 and a new N-terminal truncated isoform, cER- 61. Immunolocalization demonstrated the presence of cER- 66 protein in hypothalamic areas, principally the nucleus septalis lateralis, bed nucleus striae terminalis medialis, nucleus preopticus medialis, and nucleus infundibuli hypothalami, and in the anterior pituitary gland. When the distribution of ER- immunoreactive cells was compared using the antibodies H 222 (directed against the hormone-binding domain) and ER 221 (directed against the 21-amino acid N-terminus), no apparent differences could be detected. Because this immunocytochemical approach was not able to distinguish whether full-length cER- 66 is the only isoform observed in the ER-positive regions or whether both cER- receptor isoforms are present, SI nuclease assays were performed to compare the relative abundance in these regions of the two distinct classes of cER- mRNA variants (A1-D and A2), which encode the cER- 66 and cER- 61 protein isoforms, respectively. In cockerels and hens, both variants of cER- mRNA are expressed in the anterior pituitary gland and basal hypothalamus with a dominance of the mRNA that encodes cER- 66, whereas the mRNA that encodes cER- 61 was not detectable in the anterior hypothalamus. Therefore, because both receptor isoforms differ in their ability to modulate estrogen target gene expression in a promoter and cell type-specific manner, these differences may mediate the pleiotropic actions of estrogen in reproductive behavior and neuroendocrine functions.
estradiol, estradiol receptor, steroid hormone receptors
INTRODUCTION
Estrogen plays a central role in the control of reproductive functions [1] including vitellogenesis in oviparous vertebrates [2]. In birds, in addition to the effects of estrogen at peripheral sites of action, estrogen also acts in the brain to regulate reproductive behavior [3–5] and the control of GnRH secretion [6].
One mechanism by which estrogen induces these physiological changes is via modifications in the expression patterns of specific genes, mediated through intracellular proteins, the and ß forms of estrogen receptor (ER- and ER-ß) [7–17]. It has been suggested that ER- and ER-ß perform different functions [14]. These receptors are ligand-inducible transcription factors that belong to the superfamily of nuclear hormone receptors [18–20]. Structure-function analysis of these receptors revealed that they contain a DNA binding domain (DBD), nuclear localization signals, a ligand binding domain (HBD), and several transcription activation domains (AFs) [21].
It is obvious that the elucidation of the molecular mechanisms controlling the tissue-specific expression of the ER- and ER-ß genes should provide information on how the pleiotropic effects of estradiol are integrated into a wide range of physiological processes. Previous studies on the structure and organization of the ER- gene in a variety of species showed that it is a complex genomic unit exhibiting alternative splicing and promoter usage in neuroendocrine [13, 16, 22–24] and peripheral tissues [8, 9, 25–33]. In the domestic chicken, two variants (classes I and II) of cER- mRNA have been demonstrated [30, 31]. The first class of transcripts, A1-D cER- mRNA, have different 5' untranslated regions, but all encode a common 66-kDa cER- protein (cER- 66) [30]. The second class (class II) of cER- mRNA variants (A2 cER- mRNAs) has been reported in the liver of chicken, rainbow trout, and Xenopus laevis species but has not been shown in mammals [31]. (It should be noted that in earlier publications cER- 66 has been called cER- form I [31]. In a similar manner, because of the increasing complexity of cER- isoforms, cER- form II has been renamed cER- 61.) This new class of cER- mRNAs is transcribed from positions downstream of the cER- 66 translation start site and encode a new ER- protein isoform (cER- 61), which lacks the N-terminal 41 amino acids present in cER- 66 and thus gives rise to a 61-kDa protein. Importantly, both receptor isoforms differ in their ability to modulate estrogen target gene expression in a promoter and cell type specific manner [31]. Whereas cER- 66 activates or represses in a strictly estrogen-dependent manner, the truncated cER- 61 is characterized by a partial transactivation or repression activity in the absence of ligand. Preliminary results have shown that the truncated cER- 61 is mainly expressed in chicken liver, whereas cER- 66 predominates in oviduct tissue [31].
In this study we attempted to determine the distribution of the two isoforms, cER- 66 and cER- 61, and their transcripts in another estrogen target site in birds—the neuroendocrine system.
MATERIALS AND METHODS
ER- Expression Vector Preparation
The expression vectors pSG cER- I and pSG cER- II were prepared by directionally cloning the cER- coding regions from +158 to +2038 and +308 to +2038, respectively, into vector pSG5 [34] as described previously [30, 31]. The complete human ER- cDNA (HEO) pSG5 expression vector [35] was a gift from P. Chambon.
In Vitro Transcription and Translation
In vitro transcription and translation was accomplished with the T7-TNT Coupled Reticulocyte Lysate System (Promega, Madison, WI). The recombinant expression vectors pSG cER- I, pSG cER- II, and HEO were used as templates to generate cER- 66 and 61, cER- 61, and human ER- proteins, respectively.
Western Blot Analysis
Western blot analyses were carried out as described previously [31]. In vitro transcription and translation mixes (5 µl) were resolved on 10% SDS-PAGE gels with Broad Range Protein Standards (Bio-Rad, Hercules, CA) and electrotransferred to nitrocellulose membrane. The membrane was blocked in TS (10 mM Tris pH 7.4, 0.5 M NaCl) containing 3% (wt/vol) nonfat dry milk powder. Membranes were incubated with primary antibody (1 µg/ml) in TS for 1 h at room temperature. Primary antibodies were rat monoclonal H 222 antibody directed against the hormone-binding domain of the human ER- [36], and rabbit polyclonal antibody, ER 21, directed against the 21-amino acid N-terminus of the rat ER- [37]. Incubation with secondary peroxidase-coupled goat anti-rat (H 222) or goat anti-rabbit (ER 21) was performed under the same conditions. ER- proteins were visualized by chemiluminescence using the enhanced chemiluminescence system (Amersham, Arlington Heights, IL).
RNA Isolation
Sixteen-month-old adult cockerels (n = 3) and laying hens (n = 3), maintained on a daily lighting schedule of 14L:10D with food and water freely available, were killed by cervical dislocation. All procedures involving animals were carried out in accordance with the provisions of the Animal (Scientific Procedures) Act 1987 of the United Kingdom. The neuroanatomical boundaries of tissue blocks dissected from the anterior and basal hypothalamus are shown diagrammatically in Figure 6. Hypothalami were dissected from the ventral surface of the brain after removal of the anterior pituitary gland and neural lobe. Bilateral cuts were made 2.5 mm on each side of the midline extending from the roots of the occulomotor nerves to the preoptic area. Subsequent transverse cuts were made caudal to the basal hypothalamus and rostral to the preoptic hypothalamus. The resulting tissue block was removed and separated into basal and anterior hypothalami by a transverse cut at the junction between the anterior median eminence and the optic chiasma. Tissues were frozen in liquid N2 and total RNA was extracted using TRIzol reagent (Gibco-BRL, Eggenstein, Germany).
|
Immunocytochemistry
Immunocytochemistry was performed using primary antibodies H 222 and ER 21 (three adult cockerels and three laying hens). Optimal immunolabeling for the monoclonal H 222 antibody was observed using perfused brain tissue, whereas the best results for polyclonal ER 21 antibody were achieved by fixing tissue sections after cutting. Brain fixed by perfusion was prepared from birds that were deeply anesthetized and perfused via the carotid arteries with heparin saline (10 U/ml 0.15 M NaCl), followed by 4% paraformaldehyde fixative in 0.1 M PBS (pH 7.4), and lastly, with a 20% sucrose -0.1 M PB solution. Perfusion pressure was 100 mm Hg, and flow rate was 25 ml/min for 20 min. Following dissection, brains were stored overnight in 20% sucrose -0.1 M PBS solution. Samples were frozen by immersion in isopentane chilled in liquid N2. Unfixed brains were dissected and frozen by immersion in isopentane chilled in liquid N2. Transverse brain sections were cut at 15-µm thickness on a cryostat in the plane of the basal hypothalamus (including anterior pituitary gland) and anterior hypothalamus. Sections were thaw-mounted onto microscope slides (Superfrost plus; Cellopath, Hemel Hempstead, UK). Unfixed brain sections were fixed in 4% paraformaldehyde for 5 min after mounting on glass slides.
Immunocytochemistry was performed simultaneously on brain sections (four to eight per anatomical region) from pairs of cockerels and hens. Sections were sequentially incubated at room temperature in the following solutions: 0.1 M PBS containing 0.2% Triton-X 100 (PBS-T; three times for 20 min each), freshly prepared 1% hydrogen peroxide in PBS-T (30 min), PBS-T (10 min), and blocking solutions (20% normal goat serum in PBS-T, 60 min followed by 30 min in PBS-T containing 1% BSA). Sections were incubated for 40 h in one of the primary antibodies diluted in PBS containing 1% goat serum, 0.1% gelatin, and 0.5% Triton-X 100 at 4°C in a humidity chamber. Primary antibodies, H 222 and ER 21, were used at a 1:50 dilution of a 1 µg/ml immunoglobulin G (IgG) stock solution. Following incubation in primary antibody, sections were immunolabeled using the avidin-biotin complex method (Vectastain Elite kit, Vector Laboratories, Burlingame, CA). Final reaction product was developed by treatment for 1 min with the chromogen 3, 3'-diaminobenzidine (Vector Laboratories). Sections were washed in water for 5 min, dried overnight, mounted in nonaqueous DPX mounting media (Merck Ltd., Poole, England) and examined by light microscopy. Control sections were included omitting primary or secondary antibody from the immunocytochemical procedure. Antibody absorption studies were not carried out because it has been previously shown that virtually all immunostaining is eliminated when the H 222 antibody is preabsorbed with a cellular extract enriched in ER- [38] or when the ER 21 antiserum was preabsorbed with the peptide fragment (21 amino acid N-terminus) from which it was produced [39]. Moreover, no cross-reactivity with ER-ß was observed for H 222 or ER 21 antibodies (unpublished observations).
SI Nuclease Mapping Analysis
A modified SI nuclease mapping procedure was followed as described by Flouriot et al. [40]. The method involves the use of biotinylated single-stranded DNA templates in order to prepare highly labeled single-stranded DNA probes by extension from a specific primer by the T7 DNA polymerase in the presence of [-32P]deoxy-CTP (3000 Ci/mmol). Probes are then hybridized with the appropriate RNA sample and subjected to an SI nuclease digestion.
The cER- SI probe templates (from +158 to +892, in Fig. 5A; numbering is according to Nestor et al. [25]) and the chicken ß-actin probe were generated by reverse transcription-polymerase chain reaction (RT-PCR). The cER- 5' and 3' primers used for the amplification were S1 (5'-ACTGCCAGCTGCCGATCTTG-3') and S2 (5'-ATAGTACACTGGTTAGTGGCAG-3'), respectively. ß-Actin primers were A1 (5'-TGCCCCGAGGCCCTCTTCCAGCCATCTTTC-3') and A2 (5'-AATCCAGACAGAGTACCTGCGCTC-3') [41]. RT-PCR products were subcloned downstream of T7 and upstream of M 13 reverse primer in the TA cloning vector pCR 2.1 (Invitrogen, San Diego, CA). A PCR reaction was then performed using a biotinylated T7 primer with M13 reverse primer. Biotinylated PCR products were bound to streptavidin-coated magnetic beads (Dynal, Great Neck, NY) and the nonbiotinylated DNA strands were removed in 0.1 M NaOH. The cER- and ß-actin SI probes were generated by extending their respective primer (S2 and A2) annealed to the corresponding biotinylated single-stranded template. Following elution of single-stranded DNA probes by alkaline treatment and magnetic separation, 105 cpm probe was coprecipitated with 40 µg total RNA and dissolved in 20 µl of hybridization buffer (80% formamide, 40 mM PIPES pH 6.4, 400 mM NaCl, 1 mM EDTA, pH 8), denatured at 70°C for 10 min, and hybridized overnight at 50°C. SI nuclease digestions were performed in the presence of 300 units of SI nuclease for 1 h at 30°C, precipitated, and resolved on 4% polyacrylamide-urea gels and exposed to film.
|
RESULTS
H 222 and ER 21 Antibodies Distinguish Between the Two cER- Protein Isoforms cER- 66 and cER- 61 Produced In Vitro
Antibodies available for the different regions of the human ER- protein were analyzed for their ability to cross-react with the cER- protein isoforms cER- 66 and cER- 61. In particular we searched for antibodies that distinguish between the full-length (cER- 66) and N-terminal truncated (cER- 61) chicken ER- protein isoforms. Expression constructs pSG cER- I, pSG cER- II and HEO (shown diagrammatically in Fig. 1A) encoding the cER- 66 and 61, cER- 61, and human ER- proteins, respectively, were in vitro translated by the rabbit reticulocyte lysate system. The expression vector pSG cER- II, containing sequences from +308 to +2038, had previously been shown to generate a 61-kDa cER- protein isoform, whereas vector pSG cER- I, composed of the original cER- cDNA (from +158 to +2038) produced cER- proteins of 66 and 61 kDa in size [31] due to leakiness in the initiation of translation. Protein extracts were subjected to SDS-PAGE, transferred onto a nitrocellulose membrane and immunoblotted with the rat monoclonal H 222 antibody [36] (see Fig. 1A for approximate antigenic site). Antibody H 222 recognized both 66- and 61-kDa isoforms of the cER- protein (pSG cER- I and pSG cER- II lanes in Fig. 1B, H 222 panel). A band of size 66 kDa corresponding to the human ER- was also visible (HEO lane in Fig. 1B, H 222 panel). The two cER- protein isoforms can be distinguished from each other only by the presence of the N-terminal 41 amino acids in cER- 66. We tested the ability of a polyclonal ER 21 antiserum raised in rabbits against the 21 amino acid N-terminus of the rat ER- [37], which is 87% conserved in the cER- protein, to cross-react with the cER- 66 (see Fig. 1A for epitope location). Figure 1B, ER 21 panel, shows an immunoreactive band 66 kDa in size corresponding to the full-length chicken and human ER- proteins (pSG cER- I and HEO lanes), confirming that this antibody cross-reacts with ER- proteins in chicken and human. Moreover, the absence of an immunoreactive band of 61 kDa in size (pSG cER- I and pSG cER- II lanes in Fig. 1B, ER 21 panel) demonstrates that the ER 21 antibody does not bind to the truncated cER- 61. As monoclonal H 222 antibody recognized cER- 66 and 61, and whereas polyclonal ER 21 recognized only the full-length receptor cER- 66, these antibodies can be used to immunochemically study the regional and cellular distribution of the two cER- proteins in adult chicken brain.
|
Immunocytochemical Studies Localize cER- 66 Protein Isoform in the Anterior and Basal Hypothalamus, and Anterior Pituitary Gland. None of These Areas Uniquely Express the Truncated cER- 61
In order to determine if there was a sex-related difference in the expression pattern of the cER- mRNA isoforms, ER- immunoreactive (ER-IR) cells were visualized in the anterior and basal hypothalamus and anterior pituitary gland in cockerels and hens with antibodies H 222 and ER 21 (Figs. 2–4). Reaction products were localized to cell nuclei. Omission of the primary antibodies resulted in the absence or marked reduction in immunolabeling (Figs. 2–4, [-] control). In anterior hypothalamus, cells immunolabeled with either ER 21 or H 222 were observed in the nucleus septalis (SL), bed nucleus striae terminalis medialis (BSTm), nucleus preopticus medialis and nucleus anterior medialis (AM) (Fig. 2). In the basal hypothalamus, ER-IR cells labeled with each antibody were detected in the nucleus infundibuli hypothalami (IH) and nucleus dorsalis medialis hypothalami (DVM) (Fig. 3). Cells labeled with either ER 21 or H 222 were distributed evenly throughout the anterior pituitary gland (Fig. 4). In cockerels and hens, a similar pattern of distribution of ER-IR was observed and there was agreement between three independent observations.
|
|
|
In summary, all of the regions in which ER-IR was evident using the ER 21 antibody also showed ER-IR using the H 222 antibody. It was therefore concluded that there are no areas in the regions studied that express only the truncated cER- 61. The use of immunocytochemical methods did not allow us to distinguish whether the full-length cER- 66 is the only isoform observed in these anatomical regions, or whether both cER- receptor isoforms are present. Moreover, the limitations of immunocytochemical procedure exclude quantitative comparisons of the data obtained using the different antibodies. Finally, although adequate for liver and oviduct nuclear extracts [31], Western blot analysis was not sensitive enough to detect cER- proteins in chicken hypothalamus and anterior pituitary nuclear extracts (data not shown). To overcome these difficulties, the level and pattern of distribution of cER- mRNA variants (A1-D and A2) that encode the cER- 66 and cER- 61 protein isoforms of the cER- gene were analyzed.
Evidence for Differential Distribution of mRNA Variants Encoding cER- 66 and cER- 61 in the Anterior and Basal Hypothalamus, and Anterior Pituitary Gland
Because an immunocytochemical strategy would not be able to distinguish regions expressing only the cER- 66 isoform from those expressing both cER- 66 and 61 isoforms, an SI nuclease mapping experiment was performed to study the corresponding mRNAs (Fig. 5). Sexually mature male and female chickens were used to dissect out tissue blocks from the anterior hypothalamus, basal hypothalamus, and anterior pituitary gland. The anatomical boundaries used during dissection are diagrammatically illustrated in Figure 6. RNA samples, prepared from these tissues and liver (a known positive tissue expressing both cER- 66 and 61 kDa isoforms), were analyzed using an SI probe, complementary to cER- mRNA A1 sequences from +158 to +892 as schematically depicted in Figure 5A. To distinguish between undigested probe and specific protected fragments, the SI probe contained an additional sequence in its 3' extremity, which originated from the vector used for the single-stranded probe preparation. RNA integrity was checked by an SI nuclease protection assay using ß actin mRNA. When total RNA prepared from adult male and female basal hypothalamus, anterior hypothalamus, and anterior pituitary gland tissues was hybridized to the chicken SI probe, a protected fragment of 734 nucleotides, corresponding in size to the fully protected cER- specific region of the probe (but not including vector sequence), was obtained. No signal was evident with yeast total RNA used as a negative control (Fig. 5B). The 734 nucleotide fragment results from a protection of class I cER- mRNA variants A1-D [30]. In addition, two other major specific products of 607 and 588 nucleotides in size were detected. Their 5' extremities are located in a region downstream from the initiator methionine position, at +285 and +304 in exon 1A. It is known from previous primer extension experiments that these two sites were the transcription initiation sites of A2 cER- mRNA (class II variants) [31].
Densitometry of these signals allowed a quantitative comparison of the expression levels of A2 cER- mRNA (class II) with those found for 
A1-D cER- mRNAs (class I). Summarized in a table below Figure 5B, these data are expressed as a percentage of the total cER- expression detected in each tissue. This comparative analysis revealed that class I and class II cER- mRNAs present a differential pattern of expression in the chicken brain tissues studied. The two classes were expressed in the anterior pituitary gland and the basal hypothalamus of both sexes, whereas only class I cER- mRNAs were detected in the anterior hypothalamus (Fig. 5B). However, it should be noted that this result does not exclude the possibility that cell types occur in the brain hypothalamus and anterior pituitary gland that contain only one class of cER- mRNA variants. Class I cER- mRNA was shown to be predominant in the basal hypothalamus of both sexes, accounting for approximately 85% of the total cER- mRNA expression. Finally, only the anterior pituitary gland presented a sex-related difference in the expression pattern of the cER- mRNA isoforms where class II cER- mRNA was fourfold to fivefold less expressed in males than in laying hens.
DISCUSSION
This study demonstrates that antibodies directed against different epitopes on the human ER- protein, H 222 (directed against the hormone-binding domain) and ER 21 (directed against the 21-amino acid N-terminus) are suitable for immunocytochemical localization of cells expressing the ER- gene in the chicken neuroendocrine system. The specificity of these antibodies, which was evaluated by Western blot analysis, confirmed that H 222 recognizes both 66 and 61 kDa isoforms encoded by the cER- gene, whereas ER 21 recognizes only the full-length cER- 66 receptor. Using each antibody, immunolabeled cells were visible in the same hypothalamic areas, principally the nucleus septalis lateralis, bed nucleus striae terminalis medialis, nucleus preopticus medialis, and nucleus infundibuli hypothalami, and in the anterior pituitary gland. The cellular localization of immunoreactive ER- cells in the hypothalamus described in this study is consistent with previous immunocytochemical studies using the H 222 antibody in a wide range of avian species [42–45]. The pattern also agrees with autoradiographic radiolabeled estradiol binding studies performed in chick embryos [45, 46]. When the distribution of ER-IR cells in the chicken anterior and basal hypothalami and anterior pituitary gland was compared using the H 222 and ER 21 antibodies, no apparent differences could be detected. There are therefore no obvious neuroanatomical areas that solely express truncated receptor, cER- 61. Unfortunately, it was not possible with this approach to distinguish between the possibility that there are cell types that coexpress cER- 66 and 61, and others that express only cER- 66.
Analysis of the expression pattern of the cER- mRNA variants which encode cER- 66 and cER- 61 by SI nuclease mapping analysis revealed that class I cER- mRNAs (A1-D) is the only variant detectable in the anterior hypothalamus of both sexes, whereas in the basal hypothalamus and anterior pituitary gland, class I and II cER- mRNAs are expressed. Although this method is sensitive, it does not give any idea of the concentrations of mRNAs encoding cER- 66 and 61 in the different individual cell types within this complex tissue. The consequence at the protein level of this differential distribution pattern of the two classes of cER- mRNA should be that tissues expressing only the first class of cER- mRNAs will contain both cER- protein forms as a result of the leaky scanning mechanism [47], whereas tissues that coexpress A2 cER- mRNA transcripts will produce the truncated receptor as the main cER- protein. Gene expression of cER- in liver and oviduct was in complete agreement with this hypothesis [31]. Therefore, the presence of the A2 cER- mRNA class in the anterior pituitary gland and the basal hypothalamus tissues should allow the ratio between the two cER- proteins to be changed in favor of cER- 61. Unfortunately, attempts to confirm this prediction using Western blot analysis with H 222 and ER 21 antibodies were inconclusive due to the limiting amount of starting tissue.
The presence of cER- isoforms in the anterior and basal hypothalamus and anterior pituitary gland is consistent with the hypothesis that estrogen exerts a direct action on these neuroendocrine tissues and sexual behavioral control centers [5]. These results also corroborate the finding that estrogen acts directly on cultured pituitary cells to reduce their responsiveness to GnRH [48]. In the avian pituitary gland, estrogen was also shown to potentiate the action of the prolactin releasing hormone, vasoactive intestinal polypeptide [49, 50]. In the anterior hypothalamus, male sexual behavior, including appetitive and consummatory responses, is controlled by estrogen, acting via its receptors, produced within the preoptic medial nucleus (POM) by local aromatization of testosterone [5, 16, 51, 52]. Recent studies detected both ER- and ER-ß in the hypothalamus of the Japanese quail, including the POM [15, 17, 53]. The avian basal hypothalamus has been found to mediate the inhibitory steroid feedback control of gonadotropin release [54] and synthesis [55]. Observations in ER- knockout mice suggest that this gene plays an essential role in steroid feedback control of gonadotropin secretion, expression of female sexual receptivity, and male reproductive behaviors [13, 56, 57]. Further studies are needed to clarify the functional significance of ER- 66 and 61 in the chicken hypothalamus and anterior pituitary gland.
In fetal and newborn rat pituitaries, responsiveness to estrogen has been reported to be developmentally regulated by ER- mRNA splice variants lacking exon 3, exon 4, or both exons 3 and 4 [24]. It has been suggested that these short ER- isoforms are more abundantly detectable in the intermediate lobe. The truncated estrogen receptor product-1 (TERP-1) is also a naturally occurring rat ER- variant that is detected only in pituitary [58]. This isoform was shown to be dramatically and specifically modulated during the reproductive cycle in response to steroid hormones [59]. It remains to be determined whether expression of the truncated cER- 61 may similarly vary during chicken pituitary development and throughout estrous.
In conclusion, data from these experiments suggest that truncated cER- 61 is produced in the basal hypothalamus and anterior pituitary glands, whereas full-length cER- 66 is the predominant form expressed in anterior hypothalamus. A significant characteristic of cER- 61 is that it partially transactivates or represses target genes in the absence of hormone [31]. This functional property of the N-terminal truncated ER-, a form that is also expressed in other oviparous species [31, 60, 61], was shown to be the result of the absence of the A domain [62]. Altogether, these data may suggest therefore that this truncated receptor could mediate some neuroendocrine functions independently of estrogen. Further investigations are obviously necessary to determine the exact roles played by the two protein forms in mediating behavioral and endocrine functions in these discrete regions of the chicken neuroendocrine system.
ACKNOWLEDGMENTS
We thank Tim Boswell, Quishi Li, Peter Wilson, and Richard Talbot for technical assistance.
FOOTNOTES
First decision: 11 April 2001.
1 This study was funded in part by an EMBO fellowship. 
2 Correspondence: Caroline Griffin, MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK. FAX: 44 131 343 2620; cgriffin{at}hgu.mrc.ac.uk
Accepted: May 21, 2001.
Received: March 14, 2001.
REFERENCES
, and estrogen receptor ß show distinct patterns of expression in forebrain song control nuclei of European starlings. Endocrinology 1999; 140:4633-4643 isoforms during ontogeny of the pituitary gland. Endocrinology 1999; 140:2781-2789
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
