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The avian neurohypophyseal hormone AVT is an important regulatory hormone involved in many physiological processes including oviposition; an age-related phenomenon, through its action on the shell gland. In this study, immunohistochemistry and in situ hybridization was performed to study the expression of immunoreactive (ir) AVT and its oxytocic-like receptor VT3 transcript in the ovary/shell gland of Japanese quail representing sexually immature, mature and old condition. Our results indicate that ir-AVT is present in the ovary of sexually active adult only, but in the shell gland it is observed in both sexually active adult and sexually quiescent old quail. Further, VT3 gene transcript although not detected in the shell gland of sexually immature birds, has been found abundantly in the myometrium of shell gland of sexually active adult quail with a slight decrease in old birds. It is concluded that in addition to the ovarian function and shell gland activity, the expression of AVT and VT3 receptor in the shell gland also varies with the age dependent reproductive/egg laying performance of the Japanese quail. Our findings also suggest that (i) local synthesis of AVT and the expression of its oxytocic-like VT3 receptors is estrogen dependent and (ii) shell gland AVT upregulates its VT3 receptor (paracrine role) in advance to facilitate the role of neurohypophyseal AVT during oviposition.

The avian neurohypophyseal hormone arginine vasotocin (AVT) is known to be involved in the regulation of adrenocorticotropin (ACTH) release by interacting with the VT2 receptor (VT2R), which is homologous to the mammalian vasopressin V1b receptor (V1bR). To study the role of glucocorticoid in the expression and regulation of the VT2R, corticosterone (1 or 5mg/bird/day) or metapyrone (10 or 50mg/kg body weight/day) were administered daily for 8 days to white leghorn chickens. While low doses were ineffective, a high dose of corticosterone upregulated, while metapyrone downregulated, pituitary VT2R mRNA expression and ir-VT2 in the cephalic lobe of the anterior pituitary. Further, although no change was observed in the expression of POMC mRNA, adrenal activity (as judged by the changes in total cholesterol, 3beta HSD, cortical cord width and cortico-medullary ratio) exhibited suppression or stimulation following treatment with corticosterone or metapyrone, respectively. In view of the classical negative feedback effect of glucocorticoids at the level of hypothalamic CRH neurons and pituitary corticotrophs, high corticosterone level-induced suppression of the CRH-ACTH axis may have been masked by VT2R-mediated stimulation of corticotrophs, and hence the POMC mRNA level did not change. The same argument could be used for metapyrone. It is concluded that expression of the VT2 receptor is regulated by glucocorticoids in chicken. These findings confirm a role for AVT, mediated by the VT2 receptor, in regulating ACTH secretion during stress and suggest that corticotroph VT2 receptor levels may be dynamically regulated depending on the HPA axis activity.

The role of arginine vasotocin in the regulation of the pituitary-adrenal axis of domestic fowl was analyzed by studying the expression of its recently cloned pituitary receptor VT2 and adrenal activity following osmotic stress. Four days of water deprivation induced an increase in plasma osmolality-a known stimulator of AVT synthesis and release from hypothalamic magnocellular neurons. Water deprivation also decreased pituitary mRNA levels for both the VT2 receptor and for pro-opiomelanocortin (POMC). Despite a decrease in the expression of VT2 mRNA, immunoreactive-VT2 receptor levels in the pituitary increased, suggesting a possible role for post-transcriptional mechanisms in the regulation of this receptor. Further, adrenal activity (as judged by adrenal weight, cholesterol content, 3beta hydroxysteroid dehydrogenase, cortical cord width and cortico-medullary ratio) showed stimulation in water-deprived chicken as compared to control. On the basis of present findings, it is concluded that water deprivation down regulates the mRNA expression of AVT receptor VT2 as well as POMC but stimulates adrenal function. It is also suggested that in addition to the release of magnocellular AVT into the neurohypophysis to act as antidiuretic hormone following water deprivation, AVT may also modulate HPA axis to cope with the osmotic stress.

Avian neurohypophysial hormone arginine vasotocin (AVT) is known to regulate shell gland contractility during oviposition. While studying the role of estrogen in the expression and regulation of AVT and its oxytocic-like receptor VT3, using in situ hybridization and immunohistochemistry, it was observed that the expression of AVT and its receptor was not detected in the shell gland of sexually immature Japanese quail. However, administration of estrogen to these birds not only stimulates the growth and activity (as assessed by increased mucosal fold length, total protein content and alkaline phosphatase level) of the shell gland but also upregulates the expression of AVT and VT3. Further, administration of estrogen antagonist tamoxifen to sexually mature bird shows opposite results. On the other hand, localization of ir-AVT, observed in the ovary of sexually mature bird, was not detected in the estrogen treated sexually immature quail. It is concluded that estrogen not only affects the growth and differentiation of avian oviduct, but also regulates the expression of shell gland AVT and its receptor VT3. Present findings suggest that the locally synthesized AVT acts in a paracrine way to upregulate VT3 receptor and thus facilitates the endocrine function of neurohypophysial AVT during oviposition.

Research in mammals has demonstrated a variety of regulatory effects of vasopressin and oxytocin on endocrine functions of the anterior pituitary gland. Less evidence is available regarding the hypophysiotropic action of arginine vasotocin (AVT) comprising vasopressic and oxytocic activities in birds. Some hypophysiotropic effects of AVT may result from its interactions with brain circuits controlling pituitary functions, whereas others are caused by its direct affect on pituitary cells. Use of an antiserum to the vasotocin receptor VT2 (VT2R) has revealed numerous immunoreactive cells in the anterior pituitary gland of the chicken. The objective of the present study has been to identify endocrine phenotypes of chicken pituitary cells containing VT2R by means of immunohistochemical labeling. VT2R immunoreactivity has been found in all cells immunoreactive for adrenocorticotropin and alpha-melanotropin. Approximately 10% of labeled lactotropes are also immunoreactive for VT2R and lie around the anatomical boundary dividing the cephalic and caudal lobes. In both corticotropes/melanotropes and lactotropes, immunoreactive VT2R is present in a narrow layer outlining cell bodies. Immunoreactive VT2R is not found in gonadotropes, thyrotropes, or somatotropes. These results provide evidence for the important role of VT2Rs in mediating effects of AVT on endocrine secretion from corticotropes and, partially, from lactotropes.

Corticotropin releasing hormone receptor (CRHR) and the VT2 arginine vasotocin receptor (VT2R) are vital links in the hypothalamic-pituitary-adrenal axis that enable a biological response to stressful stimuli in avian species. CRHR and VT2R are both G-protein coupled receptors (GPCRs), and have been shown by us to form a heterodimer via fluorescent resonance energy transfer (FRET) analysis in the presence of their respective ligands, corticotrophin releasing hormone (CRH) and arginine vasotocin (AVT). The dimerization interface of the heterodimer is unknown, but computational analyses predict transmembrane domains (TMs) as likely sites of the interaction. We constructed chimerical VT2Rs, tagged at the C-terminal ends with either cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP), by replacing the fourth transmembrane region (TM4) of VT2R with TM4 of the beta2-adrenergic receptor (beta2AR). The VT2R/beta2AR chimeras were expressed in HeLa cells and proper trafficking is confirmed by observing cell membrane localization using confocal microscopy. VT2R/beta2AR-YFP chimera functionality was confirmed with a Fura-2 acetoxymethyl ester (Fura-2AM) assay. FRET analysis was then performed on VT2/beta2AR-chimera/CRHR pairs, and the calculated distance was observed to be >10 nm apart, indicating that heterodimerization was partly disrupted by mutating TM4 of the VT2R. Therefore, TM4 may form one region of the possible dimerization interfaces between the VT2R and CRHR.

The avian neurohypophysial hormone arginine vasotocin (AVT) is an important regulatory hormone involved in many physiological processes including fluid balance, blood pressure regulation, stress responses and reproductive events including oviposition. The mechanisms by which AVT stimulates myometrial contraction during oviposition are not well established in birds. In the present study, immunohistochemistry and in situ hybridization were used to localize AVT and the oxytocin-like VT3 receptor in the shell gland of Japanese quail (Coturnix coturnix japonica). Using an AVT-specific antibody, immunoreactive AVT (ir-AVT) was observed in the myometrium of both photosensitive and photorefractory birds. Similarly, VT3 receptor gene transcripts were detected in the myometrial layer of the shell gland of both photosensitive and photorefractory birds. Body mass, shell gland mass and length of mucosal folds of the shell gland of photosensitive birds was higher compared to that of photorefractory birds. Treatment of photorefractory birds with estrogen increased shell gland activity (mass and length of mucosal folds) and levels of both AVT and VT3 receptor mRNA, whereas treatment of photosensitive birds with the estrogen antagonist tamoxifen decreased shell gland activity and levels of both AVT and VT3 receptor mRNA. Our results suggest that shell gland contractility in response to AVT may be regulated during the reproductive cycle of the Japanese quail and that, in part, this regulation is estrogen-dependent.

In birds, ACTH release from the anterior pituitary gland during stress is controlled by CRH and arginine vasotocin (AVT). Using 5-wk-old male chicks, simultaneous iv injections of CRH and AVT were found to result in a greater than additive increase in plasma corticosterone levels compared with that obtained with individual administration of either peptide hormone. In order to investigate molecular mechanisms underlying this observation, the chicken CRH receptor (CRHR) and vasotocin VT2 receptor (VT2R) were fused to cyan and yellow fluorescent proteins and expressed in HeLa cells. The resulting CRHR and VT2R fusion proteins were expressed appropriately in the plasma membrane and were found to couple to downstream signal transduction pathways. Quantitative fluorescence resonance energy transfer (FRET) analysis was used to determine whether the CRHR and VT2R formed heterodimers. In the absence of CRH and AVT, the FRET efficiency was 15-18%, and the distance between receptors was 5-6 nm. Treatment of the cells that expressed both cyan fluorescent protein-CRHR and yellow fluorescent protein-VT2R with CRH or AVT alone did not lead to a significant change in the FRET efficiency. However, simultaneous addition of these hormones increased the efficiency of the FRET signal and decreased the distance between the two receptors. In HeLa cells expressing both CRHR and VT2R, treatment with CRH and AVT resulted in a significant increase in cAMP production over that with CRH alone, indicating that heterodimer formation may enhance the ability of the CRHR to activate downstream signal transduction.

The neurohypophysial hormone arginine vasotocin (AVT) stimulates adrenocorticotropin hormone (ACTH) secretion from the avian anterior pituitary gland resulting in increased adrenal secretion of corticosterone in response to stress. Here, we report molecular cloning and functional characterization of a gene encoding an AVT receptor subtype, designated the VT2 receptor, that may mediate the stimulatory effect of AVT on ACTH secretion in birds. The open reading frame predicts a 425 amino acid polypeptide that includes seven segments of 19 to 24 hydrophobic amino acids, typical of guanine nucleotide-protein coupled receptors. Phylogenetic analysis revealed that the VT2 receptor shares highest identity with the mammalian V1b-vasopressin receptor subtype. Expressed VT2 receptors in COS7 cells mediate AVT-induced phosphatidylinositol turnover and Ca(2+) mobilization. In the domestic chicken, expression of VT2 receptor gene transcripts is limited to the pituitary gland. Based on similarities in sequence, site of expression and coupled signal transduction pathways, we conclude that the VT2 receptor is the avian homolog of the mammalian V1b-vasopressin receptor, and therefore may play an important role in the avian stress response.