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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.

Arginine vasotocin is a neurohypophysial hormone in lower vertebrates including birds. Its actions are mediated through G-protein coupled receptors that belong to the vasopressin/oxytocin receptor family. Our laboratory recently cloned a vasotocin receptor, designated the VT2 receptor (VT2R), which shares high sequence identity at both the nucleotide and amino acid level with the mammalian V1b vasopressin receptor (V1bR). In the present study, we report development and use of an antibody to the VT2R to obtain anatomical evidence for testing the hypothesis that the VT2R is the avian homolog of the mammalian V1bR. Results verified the specificity of the antibody and demonstrated a receptor distribution occurring predominantly in the cephalic lobe of the pars distalis and co-localizing with adrenocorticotropin in corticotrophs. With respect to VT2R distribution and cell-type localization in pituitary gland, evidence presented support its similarity with the mammalian V1bR. In contrast to the mammalian V1bR, VT2R expression was not observed in chicken brain. Further research will be required to determine which receptor/s in the arginine vasotocin/mesotocin family are expressed in brain and mediate regulatory functions of vasotocin in the central nervous system.

To examine a possible sexual dimorphism in the osmotic control of arginine vasotocin (AVT) release in birds, age-matched male and female Japanese quail were subjected to water deprivation. The observed increased plasma osmolalities were accompanied by increased plasma AVT levels. Plasma mesotocin levels did not change with water deprivation. The sensitivity of the osmotic control of AVT release as determined by the slope of the relationship between plasma AVT levels and plasma osmolalities was significantly (P0.05) higher in males than in females. By Northern blot analysis, levels of hypothalamic AVT gene transcripts were increased 2.3 +/- 0.14- and 3.5 +/- 0.13-fold in water-deprived male and female Japanese quail, respectively, compared to normally hydrated birds. Our data suggest gender-related differences in the osmotic control of AVT release and in hypothalamic AVT gene expression in the Japanese quail.

In chickens, hyperosmolality stimulates the secretion of vasotocin (AVT) and up-regulates hypothalamic AVT gene expression. Hemorrhage, on the other hand, has not been considered an effective stimulus for AVT release in this species. The effects of acute osmotic stress and prolonged hemorrhagic hypotension on AVT gene expression and secretion were studied in White Leghorn hens. Conscious hens were osmotically stimulated by administering a single ip injection of 3 M NaCl (5 ml/kg). Urethane-anesthetized hens were bled to a mean arterial pressure of 80-90 mm Hg and the pressure was maintained within this range by additional bleeding. A total of about 30% of the estimated blood volume was removed. Both experiments were terminated after 1 hr of stimulation. Plasma AVT levels in the hyperosmotic and hypovolemic hens were 4- and 2-fold higher, respectively, compared to controls. Hypothalamic AVT mRNA levels, detected by Northern blot analysis, were 2.5- and 2-fold higher in the osmotically stimulated and hypotensive groups, respectively, compared to control groups. As determined by in situ hybridization, both osmotic stimulation and hypovolemia resulted in an increase in the number of AVT mRNA-containing neurons in the supra-optic and paraventricular nuclei. Our results indicate that, under the conditions used, hypotension and hyperosmolality are equally effective in stimulating AVT gene expression and secretion of AVT.

Arginine vasotocin (AVT) and corticotropin-releasing hormone (CRH) are two neuronal regulators in the hypothalamic–pituitary–adrenal (HPA) axis that modulate biological responses to stress in avian species. When AVT and CRH are administered together in vitro or in vivo , levels of adrenocorticotropic hormone (ACTH) or plasma corticosterone (CORT) are released, respectively, in a synergistic manner. The underlying mechanism of this greater than additive stress response was investigated by expressing the vasotocin receptor type 2 (VT2R) and CRH receptor type 1 (CRH-R1), both G-protein coupled receptors, in HeLa cells. Fluorescence resonance energy transfer (FRET) analysis provided the evidence for heterodimerization of the VT2R/CRH-R1 in the presence of their respective ligands, AVT and CRH. The VT2R and CRH-R1 were tagged at the C-terminal ends with either cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP), and a VT2R chimera was constructed by replacing the fourth transmembrane region (TM4) of the VT2R with TM-IV of the β 2 -adrenergic receptor (β 2 AR). When VT2R/β 2 AR chimera and CRH-R1 were expressed in HeLa cells, heterodimerization was partly disrupted. Taken together, these data indicate that TM-IV of the VT2R may provide an important interface for effective receptor dimerization, suggesting that direct molecular interaction between VT2R and CRH-R1 receptors plays a role in mediating an enhanced interaction between these two receptors. Their interaction at the anterior pituitary level may potentiate the endocrine output of the avian HPA system.

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.

The effects of water deprivation on the secretion of vasotocin (AVT) and expression of the AVT gene were studied in White Leghorn cockerels. Animals deprived of water for 4 days were compared with normally hydrated controls. Blood samples were obtained for measurements of plasma osmolality and AVT levels, and the hypothalamus was collected for extraction of total cellular RNA. A 519-bp AVT cDNA was prepared by reverse transcriptase–polymerase chain reaction and a 209-bpPstI/EcoRI restriction fragment from the 3′ region of the fowl AVT cDNA was used as a probe for Northern blot analysis. Plasma osmolality and AVT levels in dehydrated birds were about 30 and 350% greater, respectively, than those in normally hydrated controls. The quantity of hypothalamic AVT mRNA was 2.3-fold greater in water-deprived birds compared to controls. The size of the hypothalamic AVT transcript was about 100-bp longer in the water-deprived birds. As determined by RNase H treatment in the presence and absence of oligo(dT)12–18, the increase in mean size of the AVT mRNA in dehydrated animals was due to a longer poly(A) tract. Our results indicate that osmotic stress up-regulates expression of the AVT gene and increases the accumulation of AVT mRNA in the hypothalamus. This accumulation may, in part, be due to lengthening of the AVT mRNA poly(A) tail which is a general mechanism associated with stabilization of vertebrate mRNAs.