Arginine Vasotocin Gene Expression Research Articles

Testosterone modulates pituitary vasotocin receptor expression and adrenal activity in osmotically stressed chicken

Regulation of arginine vasotocin (AVT), avian neurohypophyseal hormone, is an important component of the hypothalamo-pituitary–adrenal axis. Changes in plasma osmolality levels and sex steroids are known to affect AVT gene expression. The present study reports the effect of water deprivation and testosterone treatment independently, as well as simultaneously, on the pituitary vasotocin receptor VT2R expression and adrenal steroidogenic activity in sexually immature male chicken ( Gallus gallus). Birds were divided into four groups— control, water deprived (WD), testosterone injected (TE) and TE treated water deprived (TE + WD). WD decreased and TE treatment alone or in combination with WD (TE + WD) increased VT2R expression compared to the control. Expression of pro-opiomelanocortin (POMC) was also studied since this gene is a polypeptide precursor of ACTH and is under the negative feedback of adrenal corticoids. TE treatment as well as WD separately or when coupled together decreased the POMC mRNA expression in the pituitary but stimulated adrenal steroidogenic activity. Further, VT2R expression decreased in TE + WD compared to TE group, but it was not different from the vehicle treated control group suggesting that the suppressive effect of WD on VT2R expression was inhibited by the stimulatory effect of testosterone. Similarly, although both TE and WD decreased POMC expression and increased steroidogenic activity, no further decrease or increase in these parameters was observed when these two (WD and TE) treatments were combined together. Although, the exact mechanism is not clear, data indicate a stimulatory action of testosterone on VT2R expression and adrenal function despite a decreased expression of POMC mRNA. Results also suggest that testosterone treatment to sexually immature birds, in addition to its effect on hypothalamic AVT neurons (earlier study) and pituitary VT2R expression (present study), masks or inhibits osmotic stress-induced alterations in pituitary–adrenal activity.

TonEBP regulates hyperosmolality-induced arginine vasotocin gene expression in the chick ( Gallus domesticus)

Arginine vasotocin (AVT) is expressed mainly in the paraventircular and supraoptic nuclei of the hypothalamus in chicken. This peptide is known to act as an antidiuretic hormone and its gene expression is stimulated by hyperosmolality. However, the transcription factors that regulate the AVT gene expression induced by hyperosmolality are still unknown. In this study, we examined the role of hyper-tonicity enhancer binding protein (TonEBP) in the transcriptional regulation of AVT gene in chicken. TonEBP mRNA expression levels increased at 1 h after salt-loading treatment in the hypothalamus. This increase preceded that in AVT and c-fos mRNA expression. Intracerebroventricular injections of TonEBP antisense oligonucleotides, before the salt-loading treatment, prevented the increase in AVT gene expression. These results, all together, suggest that the transcription factor TonEBP may be involved in the regulation of AVT genes expression in response to a hyperosmotic environment in chicken.

Changes in expression of AVT and AVT receptor (VT1) gene in hypothalamus and shell gland in relation to egg laying in white leghorn hen

Oviposition is a complex phenomenon involving various regulatory mechanisms at the neuroendocrine levels. Present study was designed to access the changes in arginine vasotocin (AVT) and its receptor (VT1) gene expression in relation to the time of egg laying of white leghorn hen. The expression of AVT gene (Northern blot analysis and in situ hybridization) in the hypothalamus and localization of ir-AVT in the magnocellular neurons of paraventricular nuclei was studied 2 h before (−2 h), immediately after (0 h) and 2 h after (+2 h) egg laying. Simultaneous changes in the AVT and VT1 receptor gene in the shell gland, which finally responds to AVT for smooth muscle contraction and expulsion of egg, were also determined by semi-quantitative reverse transcriptase-polymerase chain reaction. The findings indicated increased hypothalamic AVT gene expression immediately after egg laying (0 h) when compared to 2 h before and 2 h after egg laying. AVT receptor gene expression in the shell gland also followed the same pattern. However, AVT gene expression in the shell gland, unlike that of hypothalamus was higher at −2 h compared to 0 and +2 h of oviposition. While highly significant increase was noted in plasma AVT concentration at the time of egg laying, other parameters such as plasma osmolality and ionic concentration (Na+, K+, Ca2+, and Cl−) did not show any change. It is suggested that in addition to increased hypothalamic AVT transcript and peripheral release, local synthesis of AVT in the shell gland (paracrine release) may contribute to the contraction of shell gland smooth muscles during egg laying. Moreover, these findings clearly indicate temporal correlation of AVT and its receptor gene expression in different tissues during oviposition.

Changes in expression of AVT and AVT receptor (VT1) gene in hypothalamus and shell gland in relation to egg laying in white leghorn hen

Oviposition is a complex phenomenon involving various regulatory mechanisms at the neuroendocrine levels. Present study was designed to access the changes in arginine vasotocin (AVT) and its receptor (VT1) gene expression in relation to the time of egg laying of white leghorn hen. The expression of AVT gene (Northern blot analysis and in situ hybridization) in the hypothalamus and localization of ir-AVT in the magnocellular neurons of paraventricular nuclei was studied 2 h before (−2 h), immediately after (0 h) and 2 h after (+2 h) egg laying. Simultaneous changes in the AVT and VT1 receptor gene in the shell gland, which finally responds to AVT for smooth muscle contraction and expulsion of egg, were also determined by semi-quantitative reverse transcriptase-polymerase chain reaction. The findings indicated increased hypothalamic AVT gene expression immediately after egg laying (0 h) when compared to 2 h before and 2 h after egg laying. AVT receptor gene expression in the shell gland also followed the same pattern. However, AVT gene expression in the shell gland, unlike that of hypothalamus was higher at −2 h compared to 0 and +2 h of oviposition. While highly significant increase was noted in plasma AVT concentration at the time of egg laying, other parameters such as plasma osmolality and ionic concentration (Na +, K +, Ca 2+, and Cl −) did not show any change. It is suggested that in addition to increased hypothalamic AVT transcript and peripheral release, local synthesis of AVT in the shell gland (paracrine release) may contribute to the contraction of shell gland smooth muscles during egg laying. Moreover, these findings clearly indicate temporal correlation of AVT and its receptor gene expression in different tissues during oviposition.

Sex dimorphism in the avian arginine vasotocin system with special emphasis to the bed nucleus of the stria terminalis

The avian neuropeptide arginine vasotocin (AVT) originally characterized as the antidiuretic hormone ( Munsick et al., 1960, Endocrinol. 66, 860–871) is produced by neurosecretory cells within the brain. Numerous neuroanatomical studies that employed immunocytochemical and in situ hybridization techniques revealed such cells in the following anatomical brain locations: (a) preoptic area including supraoptic nucleus; (b) paraventricular nucleus; (c) the bed nucleus of the stria terminalis (BnST) ( Viglietti-Panzica, 1986, J. Hirnforsch. 27, 559–566; Mühlbauer et al., 1993, J. Neuroendcrinol. 5, 281–288; Jurkevich et al., 1997, Cell Tiss. Res. 287, 69–77; Aste et al., 1998, J. Comp. Neurol. 369, 141–157). The BnST which influences reproduction and sexual behavior shows sex differences in morphology, steroid responsiveness and synthesis of neuropeptides including AVT ( Viglietti-Panzica et al., 1994, Brain Res. 657, 171–184). AVT is the main endocrine regulator of fluid balance in avian species and, in addition, is involved in oviposition in these species. Our recent studies clearly demonstrated that AVT secretion after osmotic stimulation is sexually dimorphic. In order to investigate whether AVT is expressed and synthesized in the BnST in a sexually dimorphic manner we have used in situ hybridization technique and immunocytochemistry to analyze AVT gene expressing neurons in the parvocellular (small-celled nulei) BnST of adult male and female chickens. In cocks, AVT peptide-containing neurons were detected in the parvocellular BnST and the lateral septal area, whereas no AVT immunoreactive neurons were detected in the corresponding regions of the hen. Even after osmotic stimulation AVT gene expression in neurons of the parvocellular BnST of hens was not upregulated ( Jurkevich et al., 1996, Cell Tiss. Res. 287, 69–77). These results demonstrate: (a) AVT gene expression in the BnST of chickens; and (b) a strong sexual dimorphism in this region. Furthermore, AVT synthesis is regulated on the transcriptional level independent from osmotic stimuli. Thus, sex steroids might be the main regulator of AVT gene expression in the BnST. In this paper we not only review the sexual dimorphic vasotocinergic system in the BnST, we also focus on the ontogeny of sex differences and the role of gonadal hormones in organization and retention of these differences.

A Sexual Dimorphism in Hypothalamic Arginine Vasotocin (AVT) Gene Expression and AVT Plasma Levels in the Japanese Quail (Coturnix coturnix japonica) in Response to Water Deprivation

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 (P < 0.05) 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.

Development of sexually dimorphic vasotocinergic system in the bed nucleus of stria terminalis in chickens.

The bed nucleus of stria terminalis (BnST) of the domestic fowl contains two groups of parvicellular vasotocinergic neurons that are sexually dimorphic. In adult cockerels, arginine vasotocin (AVT) synthesis is well expressed in the dorsolateral and ventromedial portions of the BnST, whereas in corresponding brain areas of hens, AVT synthesis is completely lacking. In the present study, in situ hybridization and immunocytochemical methods were used to compare the ontogeny of sexually dimorphic AVT gene expression in the BnST of male and female chickens from day 12 of embryonic development (E12) until the onset of sexual maturation. By E12, both parvicellular groups of AVT-immunoreactive (AVT-ir) perikarya in the developing BnST can be distinguished in some males, whereas in females their presence is questionable. A quantitative analysis, beginning at E14, showed that the parvicellular dorsolateral portion of the BnST of male embryos had more AVT perikarya compared with females. In contrast, no evident sex difference in distribution pattern and number of AVT mRNA containing neurons in this BnST portion was observable by in situ hybridization at E15. At E18, as well as on the first and second days posthatch (D1 and D2), no differences in the number of AVT synthesizing cells and intensity of immunoreactive staining in male versus female chickens were found. Between D2 and D7, the number of AVT-ir cells in the BnST declined rapidly in both sexes until it disappeared completely in females before D35. In males, another increase in sexually dimorphic AVT-ir cells and innervation of the lateral septum was associated with the onset of puberty and fully matched a pattern observed in adult fowls. These results demonstrate that the sexually dimorphic part of the AVT system undergoes sexual differentiation during early stages of ontogeny.

Arginine Vasotocin Gene Expression and Secretion during Osmotic Stimulation and Hemorrhagic Hypotension in Hens

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 3MNaCl (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 byin situhybridization, both osmotic stimulation and hypovolemia resulted in an increase in the number of AVT mRNA-containing neurons in the supraoptic 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 gene expression in hypothalamic neurons is up-regulated in chickens drinking hypertonic saline: an in situ hybridization study.

Osmotic stress stimulates the release of the avian hypothalamic neuropeptide arginine vasotocin (AVT) into the peripheral circulation. We conducted the present study to investigate the effects of salt-loading on AVT secretion and AVT gene expression in specific hypothalamic nuclei in chickens. White Leghorn chickens were provided food ad lib and either water or 2% NaCl to drink. Both plasma osmolality and plasma AVT levels were significantly increased in chickens that drank 2% NaCl for either two or four days compared to that in chickens that drank water. Results from in situ hybridization analysis demonstrated an increase in the number of neurons expressing AVT mRNA in the supraoptic (SON) and paraventricular nuclei (PVN) in chickens provided 2% NaCl to drink compared to chickens that were provided water to drink. The number of grains per neuron increased in the PVN, but not in the SON of osmotically stimulated birds. Thus, increased osmolality resulting from ingestion of hypertonic saline is an effective stimulus to increase hypothalamic AVT mRNA content in chickens.

Distribution of AVT gene expressing neurons in the prosencephalon of Japanese quail and chicken

Detailed anatomical studies about the distribution of arginine-vasotocin (AVT) gene expressing neurons in the avian brain are so far non-existent. Most of the investigations are limited to discrete brain nuclei in the chicken and concern particular experimental conditions. Since no data on AVT gene expression are up to now available in Japanese quail, we report here the distribution of AVT mRNA containing neurons within the prosencephalon of this bird and a comparison with the same system of the chicken. The AVT mRNA containing perikarya were detected with a specific AVT cDNA probe directed toward the distal 3' glycopeptide encoding part of the chicken AVT gene. The specificity of the probe was tested by Northern and Southern blot analysis. The AVT gene expressing neurons showed the same pattern of distribution in both species; however, a larger number of labelled neurons was observed in the chicken by visual inspection of the sections. AVT gene expressing neurons were distributed within the hypothalamus (in the medial and ventral aspects of the rostral preoptic area, in the lateral forebrain bundle, close to the ependyma of the third ventricle and in several periventricular hypothalamic nuclei) and in some extrahypothalamic regions (around the nucleus rotundus, close to the nucleus commissurae pallii and in a region probably corresponding to the nucleus strial terminalis). AVT gene expressing neurons were observed close to the organum septi laterale of the chicken. Taken together, these results confirm the specificity of the employed probe and its suitability for further studies on the modulation of AVT gene expression in quail.

Arginine vasotocin gene expression in neuroendocrine, reproductive and gastrointestinal tissues of the domestic fowl: detection by reverse transcriptase polymerase chain reaction

Arginine vasotocin gene transcripts in various tissues of the domestic fowl were detected by reverse transcriptase polymerase chain reaction followed by Southern blot analysis using a 209 bp fragment from the 3′-region of a cDNA encoding chicken arginine vasotocin as the probe. Relatively strong signals were observed with hypothalamic, adenohypophysial and proventricular RNA as the starting material. Lesser signals were obtained from RNA isolated from shell gland, adrenal gland, post-ovulatory follicles and ovarian thecal cells. Arginine vasotocin gene transcripts were undetectable in the posterior pituitary gland, small intestine and large intestine. These results suggest that in addition to its well-known antidiuretic and oxytocic actions, arginine vasotocin may act as a local neuromodulator or mediator and have other important autocrine or paracrine actions in non-hypothalamic tissues.

Arginine vasotocin gene expression and hormone synthesis during ontogeny of the chicken embryo and the newborn chick.

Chicken embryos at different developmental stages (embryonal day (E) 6 to 21) and chicks at posthatch day 1 (D1) were monitored for the development of their hypothalamo-neurohypophysial system as indicated by the kinetics of arginine vasotocin (AVT) gene expression via mRNA concentration and brain AVT content. Our data concerning the onset of gene expression support previous results from our laboratory and others about an early activation of the AVT gene transcriptional and translational activity around E6. We could detect measurable amounts of AVT in chicken embryo brains at E6 and an exponential increase during further development until D1. Dot blots of hypothalamic RNA extracts indicated that AVT gene transcript concentrations rose between E12 and E17 and slightly dropped thereafter. Northern hybridization showed that this drop was caused by a decrease of full length message and an increase of smaller transcripts during late embryonal and D1 stages, probably an AVT mRNA specific degradation phenomenon. The dissociation between the increase of AVT concentration and AVT mRNA concentration visible at the D1 stage might be due to accumulation and storage of AVT in the magnocellular neurons, preferentially in their axon terminals in the neurohypophysis. Blood samples taken from E14 onwards revealed a constant increase in plasma osmolality and plasma AVT concentration. Our data suggest that, in the chicken, AVT seems to be required early during embryonal development, either for osmoregulatory or further unknown functions.

Arginine Vasotocin Gene Expression During Osmotic Challenge in the Chicken

The avian hypothalamic nonapeptide arginine vasotocin (AVT) is released from axon terminals in the neural lobe upon the application of osmotic stimuli. We have investigated whether, and to what extent, hormone secretion from the neurohypophysis is related to gene expression in the hypothalamus. Results from hybridization experiments with an AVT-specific cDNA probe indicate that in adult chickens stimulated by water deprivation or by hypertonic saline (2% w/v) drinking water, an upregulation of the AVT mRNA pool takes place, since consistently higher AVT mRNA levels compared to controls were monitored in osmotically challenged birds. This stimulatory effect was even visible at the transcriptional level after 19 h of water deprivation when osmolality was still near the basal value. In hens osmotically challenged by hypertonic saline drinking water for 5 days, a dissociation between osmolality and AVT plasma concentration was visible: extremely high plasma osmolality was accompanied by only moderately increased plasma AVT concentration. This might be caused either by exhaustion of stored hormone, or by downregulation of the system after chronic challenge. The latter suggestion is supported by the fact that the AVT mRNA concentration after 5 days of hypertonic saline challenge was well below the AVT mRNA levels of the groups with the more short-term stimuli of water deprivation for 19 or 48 h. In 30-day-old chicks the hypothalamic AVT mRNA concentration hardly reached 70% of the adult value, although AVT plasma concentrations were similar to those in the mature bird. We conclude that osmotic challenge of the hypothalamo-neurohypophysial system not only causes secretion of AVT from stores in the neural lobe but is accompanied by upregulation of AVT gene expression. Upregulation already occurs after marginal increase in plasma osmolality, as seen after 19 h of water deprivation in hens. In 30-day-old chicks gene expression is only slightly upregulated after short-term water deprivation while increase in plasma AVT is even greater compared to hens.