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Biology of Reproduction 62, 8-15 (2000)
©Copyright 2000 Society for the Study of Reproduction, Inc.

Articles

Molecular Cloning and Functional Characterization of a Vasotocin Receptor Subtype That Is Expressed in the Shell Gland and Brain of the Domestic Chicken1

Fen-lai Tana, Stephen J. Lolaitd, Michael J. Brownsteine, Noboru Saitog, Veronica MacLeoda, Dennis A. Baeyensf, Philip R. Mayeuxc, Stacie M. Jonesb, and Lawrence E. Cornett2,a

a Departments of Physiology and Biophysics, b Pediatrics, c Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205 d Department of Medicine, Dorothy Crowfoot Hodgkin Laboratories, Bristol Royal Infirmary, Bristol, United Kingdom e Laboratory of Genetics, National Institute of Mental Health, Bethesda, Maryland 20892 f Department of Biology, University of Arkansas at Little Rock, Little Rock, Arkansas 72202 g Department of Animal Physiology, Nagoya University, Nagoya, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In chickens, oviposition is correlated with increased plasma levels of the neurohypophysial hormone vasotocin, and vasotocin stimulates contraction of uterine strips in vitro. A gene encoding a vasotocin receptor subtype that we have designated the VT1 receptor was cloned from the domestic chicken. The open reading frame encodes a 370-amino acid polypeptide that displays seven segments of hydrophobic amino acids, typical of guanine nucleotide-protein-coupled receptors. Other structural features of the VT1 receptor include two potential N-linked glycosylation sites in the extracellular N-terminal region, a conserved aspartic acid in transmembrane domain 2 that is found in nearly all guanine nucleotide-protein-coupled receptors, and two potential protein kinase C phosphorylation sites in the third intracellular loop and C-terminal tail. Expressed VT1 receptors in COS7 cells bind neurohypophysial hormones with the following rank order of potency: vasotocin {cong} vasopressin > oxytocin {cong} mesotocin > isotocin. In addition, the expressed VT1 receptor mediates vasotocin-induced phosphatidylinositol turnover and Ca2+ mobilization. In the chicken, expression of VT1 receptor gene transcripts is limited to the shell gland (uterus) and the brain. Thus, the VT1 receptor that we have cloned may mediate contractions of the shell gland during oviposition and activate reproductive behaviors known to be stimulated by vasotocin in lower vertebrates.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neurohypophysial hormones are nonapeptides that regulate various physiological processes related to water and electrolyte balance, blood pressure maintenance, and reproduction. In vertebrates, the neurohypophysial hormones can be divided into those that contain a basic amino acid residue in position 8 (vasopressin-like peptides) and those that do not (oxytocin-like peptides) [1]. The avian homologues of the mammalian hormones arginine vasopressin (AVP) and oxytocin (OT) are vasotocin (AVT) and mesotocin (MT), respectively [2].

Birds differ from mammals in that AVT, in addition to its antidiuretic and pressor activities [3–5], also has oxytocic activity. Several lines of evidence suggest that AVT released from the avian neurohypophysis plays a role in oviposition. Increased circulating levels of AVT during oviposition have been reported by several groups [4,6–10]. AVT has been shown to stimulate contraction of chicken uterine strips in vitro [10]. Finally, high-affinity, low-capacity binding sites for AVT have been identified in membrane preparations isolated from the shell gland (uterus) of chickens [10,11]. In the central nervous system of lower vertebrates, AVT can act as neurotransmitter or neuromodulator to affect reproductive and sexual behaviors (reviewed in [12]). For example, AVT enhances copulatory behavior in roosters [13]. The function of MT in birds, the oxytocic-like neurohypophysial hormone, remains unclear.

In mammals, the neurohypophysial hormone OT has important functions in both the periphery and the central nervous system. OT is released during parturition [14,15] and increases uterine contractility [16,17]. In the central nervous system, OT is involved in the initiation and maintenance of certain sexual behaviors [18–20]. In myometrium of the mammalian uterus, OT receptors couple to the phosphatidylinositol/Ca2+ signal transduction pathway [21]. Several mammalian OT receptor cDNAs and genes have been cloned [21–25]. In contrast to the situation with receptors for OT in mammals, which are relatively well characterized, little molecular information is available regarding AVT receptors in the chicken shell gland.

Here, we report the cloning and sequencing of the gene encoding a chicken AVT receptor and the functional characterization of the cloned receptor expressed in COS7 cells. The AVT receptor that we have cloned and designated the VT1 receptor is expressed in the shell gland and brain. The molecular characterization of this avian AVT receptor constitutes an important step in understanding the role of AVT in oviposition in birds as well as establishing evolutionary relationships between AVT receptors in lower vertebrates with the AVP/OT receptor subtypes expressed in mammals.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation of AVT Receptor Genomic Clones

A partial genomic clone that coded for a segment spanning transmembrane domain 2 (TM2) through TM6 was obtained by homology-based polymerase chain reaction (PCR) from chicken genomic DNA (Clontech Laboratories, Palo Alto, CA) using sense and antisense degenerate primers corresponding to known AVP/OT receptor sequences (Table 1). Reactions included 100 pmol of each primer, 200 mM dNTPs, PCR buffer (Perkin-Elmer, Norwalk, CT) containing 1.5 mM MgCl2, 2.5 U Taq polymerase, 300 ng genomic DNA. Amplification occurred through 40 cycles of denaturation at 95°C for 45 sec, annealing at 54°C for 2 min, and extension at 72°C for 2 min, with a final extension at 72°C for 8 min. In order to obtain a complete clone, a gene walking strategy was employed (GenomeWalker Kit; Clontech Laboratories). Chicken genomic DNA digested with EcoRV, ScaI, DraI, PvuII, and SspI was subjected to PCR amplification in the 5' and 3' directions using adaptor primers and gene-specific primers (Table 1) according to the manufacturer's instructions. Amplified fragments were cloned into pGEM-T vector (Promega, Madison, WI). DNA sequencing was performed by the dideoxy chain termination method using Sequenase (U.S. Biochemical, Cleveland, OH).


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  		TABLE 1. Oligonucleotides used in PCR, gene walking, and 3'-RACE procedures

3'-Rapid Amplification of cDNA Ends (3'-RACE)

The genes encoding all known members of the AVP/OT receptor family include a single intron that interrupts the coding sequence downstream of TM6. Genomic clones that we obtained by gene walking in the 3' direction contained consensus sequences for a 5'-splice site. Therefore, 3'-RACE (Gibco-BRL, Gaithersburg, MD) using gene-specific primers (Table 1) was performed using total cellular RNA isolated from chicken shell gland according to the manufacturer's instructions. Amplified fragments were cloned into the pGEM-T vector (Promega) for DNA sequencing.

RNA Preparation and Ribonuclease Protection Assay

Total cellular RNA was prepared from freshly dissected shell gland and whole brain of a White Leghorn hen by using Tri Reagent (Molecular Research Center, Cincinnati, OH) according to manufacturer's instructions. After linearization with XbaI of pGEM4z containing a fragment of the VT1 receptor corresponding to nucleotides 1597 to 2195, T7 polymerase was used to generate a 648-nucleotide antisense RNA (Promega) in the presence of [{alpha}-32P]UTP. Total cellular RNA (40 µg) was hybridized to the antisense RNA (~105 cpm) overnight at 43.5°C followed by digestion with RNase A/T1. Protected fragments were analyzed on 5% polyacrylamide gels with 8 M urea. Polyacrylamide gels were dried and exposed to X-OMAT (Eastman Kodak, Rochester, NY) film with intensifying screens at -80°C for 48–72 h.

Cell Culture and Transient Transfection

COS7 cells were grown in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum in a humidified 5% CO2 incubator. The VT1 receptor open reading frame (ORF), corresponding to nucleotides 1256 to 3588 with the intronic sequence excised, was subcloned into pcDNA3 (Invitrogen, Carlsbad, CA). This plasmid was designated pcDNA3/VT1. Two additional expression plasmids were prepared, pcDNA3/ß2AR and pcDNA3/{alpha}1BAR; pcDNA3/ß2AR and pcDNA3/{alpha}1BAR contain the rat ß2- and {alpha}1B-adrenergic receptor ORFs [26,27], respectively. For transfections, 0.8–1.2 x 106 cells were plated into 100-mm dishes. Twenty-four hours later, cells were transfected with 8 µg of plasmid DNA per dish using DEAE-dextran [28].

Radioligand Binding Assays

COS7 cells were transiently transfected with pcDNA3/VT1. Cells were harvested 48 h later by scraping in 50 mM Tris, 2 mM MgCl2, pH 7.4. Cells were disrupted using a Model F60 Dismembrator (Fisher Scientific, Pittsburgh, PA), and membranes were isolated as previously described [29]. Membranes were suspended in modified vasopressin receptor assay buffer composed of 100 mM KCl, 10 mM MgCl2, 2 mM EGTA, 20 mM Hepes, pH 7.4, 120 mM NaCl, and protein determined by the method of Bradford [30] using BSA as the standard. Binding assays were carried out with [3H]AVP (specific activity, 68.5 Ci/mmol; New England Nuclear, Boston, MA) as the radioligand as previously described [10]. Nonspecific binding was determined in the presence of 0.1 µM AVT. Data from saturation experiments were analyzed using LIGAND, a nonlinear, least-square curve-fitting program [31]. Inhibition constants (Ki) of unlabeled peptides were calculated as described by Cheng and Prusoff [32]. OT was purchased from Peninsula Laboratories (San Carlos, CA). All other peptides were purchased from Bachem Bioscience (King of Prussia, PA).

Determination of cAMP Production

COS7 cells were transiently transfected with either pcDNA3/VT1 or pcDNA3/ß2AR. Cells were treated with either 10-7 M AVT or 10 µM (-)-isoproterenol in the presence of 250 µM isobutylmethylxanthine (IBMX) for 10 min. Cellular cAMP levels were determined by RIA using the Biotrak CAMP Assay System (Amersham Life Science, Arlington Heights, IL; now Amersham Pharmacia Biotech, Piscataway, NJ).

Determination of Inositol Phosphate Production

COS7 cells were transiently transfected with either pcDNA3/VT1 or pcDNA3/{alpha}1BAR. Twenty-four hours later, the culture medium was replaced with medium that contained 3 µCi/ml myo-[3H]inositol (17 Ci/mmol; Amersham Pharmacia Biotech). After a 24-h labeling period, cells were incubated for 15 min at 37°C in 1 ml buffer A (150 mM NaCl, 5 mM KCl, 0.8 mM MgSO4, 1 mM CaCl2, 20 mM Hepes, pH 7.4, 5.5 mM glucose, 0.1% BSA) in the presence of 10 mM LiCl. After two washes with buffer A, cells were stimulated in the same buffer with either 10-7 M AVT or 10 µM (-)-epinephrine for 10 min. The reaction was stopped by the addition of ice-cold 20% perchloric acid. Cells were scraped, sonicated briefly, and centrifuged. Supernatants were neutralized to pH 7.5 with 10 M KOH. Inositol phosphates were isolated by anion-exchange chromatography [33], and radioactivity was determined using a Packard Tri-Carb 2100TR Scintillation Counter (Packard Instrument Company, Meriden, CT).

Measurement of Intracellular Free Calcium [Ca2+]i

COS7 cells were transiently transfected with pcDNA3/VT1. Twenty-four hours later, cells were harvested by trypsinization using 0.05% trypsin, 0.53 mM EDTA in Hanks' Balanced Salt Solution (Gibco-BRL) and prepared for determination of intracellular calcium levels as previously described [34]. Intracellular fura-2 fluorescence was monitored using a Hitachi F2000 spectrofluorometer (Hitachi Instruments, Danbury, CT). Measurements of the 340:380 nm emission ratio were made using an excitation wavelength of 510 nm. Maximum and minimum fluorescence were determined by the addition of Triton X-100 (0.2% final concentration) and EGTA (10 mM final concentration), respectively. [Ca2+]i was calculated by using the formula

where Kd = 224 nM [35].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Isolation of a Genomic Clone Encoding an AVT Receptor Subtype

In order to clone chicken AVT receptors, we initially used a PCR strategy with genomic DNA and two degenerate primers corresponding to conserved regions within previously cloned mammalian AVP/OT receptors. Segments of an approximately 620-base pair (bp) product were found to share 57.4% and 61.8% identity with the corresponding nucleotide sequences in the human V1a- and V2-vasopressin receptor genes, respectively. This suggested that the 620-bp product represented a segment of a G protein-coupled receptor, possibly an AVT receptor. We were able to clone 3736 bp of the gene using a gene walking strategy that employed primers within the original 620-bp fragment. The cloned gene consisted of 1314 bp in the 5'-untranslated region (UTR), an ORF of 1110 bp, 220 bp in the 3'-UTR, and a single 1090-bp intron that interrupted the coding region (Fig. 1). Using the TMpred program [36], the 370 amino acid protein determined from the ORF featured seven hydrophobic segments composed of 20–25 amino acids that likely represent membrane-spanning domains (Fig. 2), a characteristic of all members of the G protein-coupled receptor superfamily. Also present were two potential N-glycosylation sites (Asn-Phe-Ser and Asn-Lys-Ser) in the N-terminal region, a conserved aspartic acid residue in TM2 that is found in all G protein-coupled receptors, an internalization sequence (Asn-Pro-Trp-Ile-Tyr), two cysteine residues through a disulfide linkage that may cross-link extracellular loops, and two potential protein kinase C sites (Thr-Val-Lys and Thr-Asn-Lys) in the third intracellular loop and C-terminal tail, respectively (Fig. 1). At the amino acid level, the ORF exhibited the following overall identities with other AVP/OT receptors: fish AVT receptor, 52.1%; rat V1a receptor, 51.9%; human V1a receptor, 51.6%; human V1b receptor, 49.5%; human OT receptor, 49.3%; rat V2 receptor, 46.6%; and human V2 receptor, 43.8%.



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  		FIG. 1. Nucleotide and deduced amino acid sequence of the chicken VT1 receptor gene (GenBank accession no. AF147743). In the receptor ORF, the predicted transmembrane domains are underlined. Open circles indicate potential glycosylation sites in the amino terminus; solid circles, potential protein kinase C phosphorylation sites; diamonds, conserved aspartic acid residue in TM2; shaded box, internalization sequence in the carboxy tail. Upstream of the ORF, a potential TATA box is indicated by shading. Lower panel) A summary of the cloning strategy and a schematic representation of VT1 receptor gene structure are shown. The boxes represent the two exons interrupted by a 1090-bp intron.



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  		FIG. 2. Hydropathicity profile of the VT1 receptor. Values were calculated using TMpred [36]. Hydrophobicity increases with increasing values. Putative transmembrane helices are numbered by the convention used with G protein-coupled receptor family members

Expression of the VT1 Receptor mRNA in Chicken Tissues

The distribution of VT1 receptor mRNA in chicken tissues was examined by ribonuclease protection assays. Using the 648-nucleotide antisense RNA, the expected 598-nucleotide protected fragment was observed in total cellular RNA prepared from brain and shell gland (Fig. 3). The density of the protected fragment in brain was approximately 10 times higher than that in shell gland. In contrast, a protected fragment was not observed in total cellular RNA prepared from liver, kidney, and heart (Fig. 3).



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  		FIG. 3. Detection of VT1 receptor mRNA by ribonuclease protection assay. Samples (20 µg) of total cellular RNA isolated from the brain, heart, uterus, kidney, and liver of a White Leghorn hen, as well as yeast RNA, were hybridized to a 648-nucleotide VT1 receptor cRNA (105 cpm) as described in Materials and Methods. The expected 598-bp protected fragment was observed in uterus and brain

Expression of the VT1 Receptor in COS7 Cells

To investigate the pharmacological characteristics of the cloned VT1 receptor, the ORF was subcloned into pcDNA3 for expression in COS7 cells. The radioligand [3H]AVP bound to membranes prepared from COS7 cells transiently transfected with pcDNA3/VT1 in a saturable manner with high affinity. Scatchard analysis of [3H]AVP-binding isotherms indicated the presence of a single class of high-affinity sites. In a representative saturation experiment, an apparent dissociation constant (Kd) of 0.32 nM and a binding site concentration of 2725 fmol/mg protein were obtained (Fig. 4). Competition experiments were performed in order to determine the binding specificity of a series of neurohypophysial hormones (Fig. 5). From the calculated inhibition constants, the rank order of potency was AVT {cong} AVP > OT {cong} MT >> isotocin (Table 2). The pharmacological profile of the expressed receptor was similar to that of the AVT receptor expressed in the chicken uterus [10,11].



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  		FIG. 4. Saturation binding of [3H]AVP to membranes prepared from COS7 cells that had been transiently transfected with pcDNA3/VT1. Saturation experiments were performed as described in Materials and Methods. A representative Scatchard plot from a single transfection is shown. Inset: Direct plot showing total binding (closed circles), nonspecific binding defined by 0.1 µM AVT (triangles), and specific binding (open circles)



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  		FIG. 5. Competition studies of [3H]AVP binding to membranes prepared from COS7 cells that had been transiently transfected with pdDNA3/VT1. Competition experiments were performed as described in Materials and Methods.


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  		TABLE 2. Pharmacological characterization of the chicken VT1 receptor

Binding of hormone to other members of the AVP/OT receptor family has been shown to result in either elevated cAMP or activation of phosphatidylinositol turnover [37,38]. Therefore, we considered the possibilities that the cloned AVT receptor was functionally coupled to either adenylyl cyclase and cAMP synthesis or phospholipase C and Ca2+ mobilization. In order to test these possibilities, COS7 cells were transiently transfected with pcDNA3/VT1; 2 days later, cells were harvested, and either intracellular cAMP levels, inositol phosphate levels, or Ca2+ levels were determined after treatment with AVT. With the addition of 10-7 M AVT, the cloned AVT receptor was not able to increase intracellular cAMP levels, even in the presence of the phosphodiesterase inhibitor IBMX (Fig. 6). As a positive control, cAMP levels were increased approximately 16-fold in COS7 cells transiently transfected with pcDNA3/ß2AR and treated with 10 µM (-)-isoproterenol and 100 µM IBMX as compared to cells treated either with IBMX alone or with vehicle (Fig. 6). In COS7 cells that had been transiently transfected with pcDNA/VT1 and then labeled with [3H]inositol, inositol phosphate accumulation significantly (P < 0.05) increased approximately 7-fold in the presence of 10-7 M AVT compared to that in cells treated with vehicle alone (Fig. 7). The half-maximal response (EC50) to AVT was approximately 3 nM (data not shown). Inositol phosphate levels in COS7 cells that were transiently transfected with pcDNA3/{alpha}1BAR were significantly (P < 0.05) increased approximately 8-fold in the presence of 10 µM (-)-epinephrine in comparison to levels in cells treated with vehicle alone (Fig. 7). Inositol production in response to AVT was undetectable in cells transfected with pGEM-7z (Fig. 7). Finally, basal (unstimulated) [Ca2+]i in fura-2-loaded COS7 cells transiently transfected with pcDNA3/VT1 was approximately 250 nM (Fig. 8). The addition of 0.1 µM AVT resulted in a rapid, transient rise in [Ca2+]i in pcDNA3/VT1-transfected cells (Fig. 8) but had no effect on [Ca2+]i in nontransfected, fura-2-loaded COS7 cells (data not shown). The kinetics of the rise in [Ca2+]i is consistent with a Gq-phospholipase C-mediated response in that the initial response was very rapid (within 10 sec) and returned to basal within 1 min. This type of response has been observed to occur with other Gq-coupled receptors [39,40].



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  		FIG. 6. Agonist-stimulated cAMP production in COS7 cells transiently expressing either the VT1 receptor or the ß2-adrenergic receptor. COS7 cells transfected with either pGEM-7z, pcDNA3/VT1, or pcDNA3/ß2AR were incubated in the presence of 250 µM IBMX with either 10-7 M AVT or 10 µM (-)-isoproterenol for 10 min at 37°C, and cAMP was measured as described in Materials and Methods. Values are the means from duplicate determinations in a single experiment. n.d., Below the limit of detection in cAMP RIA. The experiment was repeated with separately transfected COS7 cells, and identical results were obtained



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  		FIG. 7. Agonist-stimulated phosphatidylinositol turnover in COS7 cells transiently expressing either the VT1 receptor or the {alpha}1B-adrenergic receptor. COS7 cells transfected with either pGEM-7z, pcDNA3/VT1, or pcDNA3/{alpha}1BAR were incubated in the presence of 10 mM LiCl with either 10-7 M AVT or 10 µM (-)-epinephrine for 10 min at 37°C, and accumulation of inositol phosphates was measured as described in Materials and Methods. Values are the means ± SE from four separate experiments



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  		FIG. 8. Representative tracing of the change in [Ca2+] in COS7 cells expressing the cloned VT1 receptor in response to AVT. COS7 cells were transiently transfected with pcDNA/VT1, and changes in fura-2 fluorescence were used to monitor [Ca2+]i in stirred suspensions as described in Materials and Methods. The addition of AVT (final concentration, 10 nM) was made at the arrow. Identical results were obtained with separately transfected COS7 cells in three additional experiments


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have cloned and expressed in COS7 cells a gene that encodes a chicken AVT receptor. Analysis for orientation and transmembrane domains showed that the amino terminus is extracellular and that the predicted amino acid sequence includes seven putative hydrophobic regions that are 20–25 amino acids in length. These features are typical of AVP/OT receptor family members [41,42]. For the VT1 receptor, the location of TM7 and consequently the length of the third extracellular loop are ambiguous. Using the TMpred program, TM6 and TM7 are predicted to be separated by a single amino acid, whereas by alignment with other members of AVP/OT receptor family, TM6 and TM7 are separated by 18 amino acids. The ambiguity arises because of the high percentage of hydrophobic amino acids in this region (Fig. 2) and can be resolved only by detailed epitope mapping or site-directed mutagenesis in conjunction with biochemical and biophysical analysis [43]. Transcripts encoding this receptor, which we have designated the VT1 receptor, are expressed in the shell gland or uterus as well as the brain. As for other members of the AVP/OT receptor gene family, the gene encoding the VT1 receptor consists of two exons and a single intron that interrupts the receptor ORF downstream of TM6 [41,42]. The deduced amino acid sequence of the chicken VT1 receptor we have cloned and that of the previously cloned fish AVT receptor [44] are 52.1% identical. The amino acid sequence identity with other members of the AVP/OT receptor family range from 51.9% with the rat V1a receptor to 43.8% with the human V2 receptor.

Although the sequence of the chicken VT1 receptor is similar to that of the fish AVT receptor, their pharmacological profiles are different. For the chicken VT1 receptor, the rank order of potency for agonists was AVT {cong} AVP > OT {cong} MT (see Fig. 4), whereas the rank order of agonist potency in activating a Cl- current in oocytes expressing the fish AVT receptor was AVT > OT > MT > AVP [44]. The pharmacological profile of the chicken VT1 receptor expressed in COS7 cells is consistent with the relative potencies of the same peptides in stimulating chicken uterine contractility and interacting with AVT-binding sites in uterine membrane preparations [10]. Thus, it appears that we have cloned the physiologically relevant AVT receptor that mediates the contractile response of the chicken shell gland to AVT during oviposition.

In mammals, the neurohypophysial hormones AVP and OT have discrete physiological roles in the periphery (reviewed in [38]). AVP principally regulates water balance and serum osmolality, but it also plays a role in maintaining systemic blood pressure particularly in response to hemorrhage. OT stimulates contraction of uterine smooth muscle during parturition and the myoepithelial cells of the milk ducts during suckling. In the chicken, AVT regulates water balance and plasma osmolality, is released during hemorrhage-induced hypotension, and stimulates uterine contractility during oviposition. Because multiple receptor subtypes have been demonstrated for AVP and OT in mammals, it seems reasonable to hypothesize that multiple AVT receptor subtypes might exist in lower vertebrates as well. Besides different pharmacological profiles for the chicken VT1 receptor and the previously cloned fish AVT receptor, additional evidence exists to support the existence of multiple AVT receptor subtypes. In the frog, glomerular AVT receptors can be pharmacologically distinguished from AVT receptors expressed in either the skin or bladder [45]. Thus, we would predict that at least two additional AVT receptor subtypes exist in the chicken. One putative AVT receptor subtype, orthologous to the mammalian V2 receptor, may be present in the kidney and mediate the antidiuretic action of AVT. The second putative AVT receptor subtype, orthologous to the mammalian V1a receptor, may be expressed in vascular smooth muscle and mediate the compensatory hypertensive effect of AVT during hemorrhagic hypotension.

Besides expression in the shell gland, our data demonstrate that VT1 receptor transcripts are expressed in the brain. In addition to its effects on uterine contractility, AVT has been shown to influence sexual behaviors in a wide range of lower vertebrate species (reviewed in [12]). In roosters, i.p. injection of AVT enhances copulatory behavior [13]; in lizards, AVT administration enhances egg-laying [46]; and in the bullfrog, AVT alters the display of several sexually dimorphic behaviors [47]. Additional studies are necessary in order to establish the identity of the AVT receptor in the central nervous system that mediates the behavioral effects of AVT. However, it is interesting to note that in mammals, OT has effects on reproductive and sexual behaviors [18–20].

An aspect of comparative neurohypophysial endocrinology that has received considerable attention is the molecular evolution of the hormones. Based on phylogenetic distributions and similarities in amino acid and nucleotide sequences, mammalian OT and AVP have evolved from MT (expressed in amphibians, reptiles, and birds) and AVT (expressed in fish, amphibians, reptiles, and birds), respectively. It has been proposed that isotocin, expressed in fish, gave rise to MT [2]. The isotocin and AVT genes presumably arose from an ancestral peptide through gene duplication. On the basis of this evolutionary scheme, one would predict that MT should serve as the oxytocic hormone in birds. However, this does not appear to be the case. MT is not released during oviposition in the chicken [4,10]. Moreover, MT is significantly less potent than AVT in causing contraction of hen uterine strips in vitro [10]. By way of contrast, relatively little is known concerning the molecular evolution of the receptors for the neurohypophysial hormones principally because relatively few neurohypophysial hormone receptors other than mammalian AVP and OT receptors have been cloned. With the availability of the gene structure of the avian VT1 receptor, it should become possible to rapidly clone homologues in representative species of other vertebrate classes and eventually elucidate how the neurohypophysial hormones and their receptors co-evolved.

In summary, we have cloned the gene encoding an AVT receptor subtype that appears to be expressed in the shell gland and brain of the domestic chicken. This receptor may mediate contractions of the shell gland during oviposition and modulate reproductive behaviors known to be stimulated by AVT in lower vertebrates. It is likely that additional AVT receptor subtypes are present in the domestic chicken that mediate the other physiological actions of AVT. Additional analysis of these subtypes may reveal important information about the relationships between members of the AVP/OT receptor family.


   ACKNOWLEDGMENTS
 
Thanks to Susan Foreman for technical assistance and to Drs. Patricia Wight and Richard Kurten for comments on the manuscript.


   FOOTNOTES
 
First decision: 20 July 1999.

1 This work was supported by NSF IBN9727915 (L.E.C.) and the Wellcome Trust, U.K. (S.J.L.). Back

2 Correspondence: Lawrence E. Cornett, Department of Physiology and Biophysics, Slot #750, University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, AR 72205. FAX: 501 296 1469; cornettlawrencee{at}exchange.uams.edu Back

Accepted: August 18, 1999.

Received: May 24, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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