General and Comparative Endocrinology 160 (2009) 216–222

Contents lists available at ScienceDirect

General and Comparative Endocrinology

journal homepage: www.elsevier .com/locate /ygcen

Osmotic stress induced alteration in the expression of arginine vasotocinreceptor VT2 in the pituitary gland and adrenal function of domestic fowl

Dharmendra Sharma a, Lawrence E. Cornett b, Chandra Mohini Chaturvedi a,*

a Department of Zoology, Banaras Hindu University, Varanasi-22105, Indiab Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA

a r t i c l e i n f o

Article history:Received 23 May 2008Revised 17 November 2008Accepted 17 November 2008Available online 6 December 2008

Keywords:Osmotic stressAVTVT2 receptorAdrenalPOMC3b HSDCortico-medullary ratio

0016-6480/$ - see front matter � 2008 Elsevier Inc. Adoi:10.1016/j.ygcen.2008.11.025

* Corresponding author. Fax: +91 542 2368323.E-mail address: cmcbhu@yahoo.com (C.M. Chaturv

a b s t r a c t

The role of arginine vasotocin in the regulation of the pituitary-adrenal axis of domestic fowl was ana-lyzed by studying the expression of its recently cloned pituitary receptor VT2 and adrenal activity follow-ing osmotic stress. Four days of water deprivation induced an increase in plasma osmolality—a knownstimulator of AVT synthesis and release from hypothalamic magnocellular neurons. Water deprivationalso 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 pituitaryincreased, suggesting a possible role for post-transcriptional mechanisms in the regulation of this recep-tor. Further, adrenal activity (as judged by adrenal weight, cholesterol content, 3b hydroxysteroid dehy-drogenase, cortical cord width and cortico-medullary ratio) showed stimulation in water-deprivedchicken as compared to control. On the basis of present findings, it is concluded that water deprivationdown regulates the mRNA expression of AVT receptor VT2 as well as POMC but stimulates adrenal func-tion. It is also suggested that in addition to the release of magnocellular AVT into the neurohypophysis toact as antidiuretic hormone following water deprivation, AVT may also modulate HPA axis to cope withthe osmotic stress.

� 2008 Elsevier Inc. All rights reserved.

1. Introduction

Exposure of an organism to any kind of stimulus/stressor thatdisturbs its homeostasis often results in the activation of hypothal-amo-pituitary-adrenal (HPA) axis and the consequent release ofglucocorticoids into the blood. Similar to mammals, birds also havetwo hypothalamic peptides: corticotrophin-releasing hormone(CRH), and arginine vasotocin (AVT) are considered to be the regu-latory factors for the synthesis and release of adrenocorticotrophichormone (ACTH) from the anterior pituitary gland (Castro et al.,1986; Romero, 2006). Avian neurohypophyseal peptide AVT, theantidiuretic hormone synthesized in the magnocellular neuronsof supraoptic (SON) and paraventicular (PVN) nuclei, is reportedto increase during water deprivation/salt loading (Arad et al.,1985; Chaturvedi et al., 1994,1997; Seth et al., 2004). In view ofantidiuretic as well as ACTH regulating role of AVP/AVT, it is obvi-ous that this peptide is released through neurohypophysis intoperipheral circulation and from the median eminence into thepituitary portal circulation, respectively (Holmes et al., 1986). Inmammals two different populations of vasopressin secreting neu-rons have been reported. Magnocellular neurons of PVN releaseAVP into the neural lobe of the pituitary, which is subsequently se-

ll rights reserved.

edi).

creted into the peripheral circulation and is responsible for its anti-diuretic action; whereas parvocellular neurons of PVN secrete AVPinto the pituitary portal circulation and is reported to be responsi-ble for the ACTH release (Antoni, 1993). However, no data is avail-able in birds regarding such differentiation between magno andparvocellular population of vasotocin neurons. On the basis ofmammalian reports that magnocellular nerve fibers also releasevasopressin in the inner zone of the median eminence, it has alsobeen postulated that in addition to the parvocellular vasopressin-ergic system, magnocellular AVP also plays a role in the regulationof ACTH secretion (Wotzak et al., 2002). Several physiological andpathophysiological conditions inducing high circulating AVP levelsare reported to influence ACTH secretion from pituitary cortico-trophs (Rittmaster et al., 1987). Moreover, inhibition of endoge-nous magnocellular vasopressin following hyponatremia causes adecrease in the pituitary ACTH response to stress (Dohanicset al., 1991). On the other hand, hypertonic saline drink reducedthe pituitary ACTH response to both acute stress and to exogenousCRF in rats despite a marked increase in magnocellular AVP expres-sion, suggesting that magnocellular AVP does not stimulate corti-cotroph function (Chowdry et al., 1991).

In mammals, AVP stimulated ACTH release is mediated by AVPreceptor sub type V1b present on the corticotrophs in adenohy-pophysis (Dupasquier et al., 1991; Antoni, 1993). But in birds, theregulation of HPA axis by vasotocin at the anterior pituitary level

Table 1Sequence of primers used for RT-PCR.

Gene Primer sequence Amplicon size

VT2 F 50-GCG AGA TCT GCA AGA ACC-30 565 bpR 50-GGA AGC AGT GAC TGA ATC-30

POMC F 50-GAG AGC ATC CGC AAG TAC GTG-30 511 bpR 50-CTG ATG ACT CTG TTC AAA ACG-30

GAPDH F 50-AGT CAT CCC TGA GCT GAA TG-30 330 bpR 50-ACC ATC AAG TCC ACA ACA CG-30

D. Sharma et al. / General and Comparative Endocrinology 160 (2009) 216–222 217

is not very clear except in the one study of Mikami and Yamada(1984), suggesting its possible role in the ACTH secretion on thebasis of the presence of immunoreactive AVT in the median emi-nence. It has been also demonstrated that in addition to behavioraland cardiovascular effects, AVT also influences corticosterone re-sponses of starlings to crowding (Nephew et al., 2005). However,it has been found that vasotocin does stimulate the secretion ofACTH from cultured avian pituitary cells (Castro et al., 1986). Re-cently cloned vasotocin receptor VT2 is reported to be expressedin anterior pituitary gland only, unlike its mammalian counterpartV1b that is expressed in the anterior pituitary as well as in thebrain (Cornett et al., 2003; Jurkevich et al., 2005).

In addition to acting as an osmotic stimulus, water deprivationalso serves as a potent stress that stimulates the adrenal gland toincrease the plasma concentration of corticosterone (Freemanet al., 1983). In view of the conflicting information regarding roleof magnocellular vasopressin in the regulation of ACTH secretionand complete lack of such reports in avian system, the presentstudy was undertaken. This experiment was designed to studythe expression of vasotocin receptor VT2 in the pituitary glandand to assess the adrenal responses of chicken subjected to waterdeprivation, which is reported to stimulate vasotocin synthesis andrelease from the hypothalamic magnocellular neurons (Chaturvediet al., 1994). Adrenal activity was assessed by measuring the adre-nal cholesterol content (precursor molecule) and 3b HSD enzymeactivity (responsible for the conversion of pregnenolone to proges-terone) as an indicator of HPA activity. Depletion of adrenal choles-terol content and increase in the activity of 3b HSD reflectsstimulation of the adrenal steroidogenesis. Moreover, changes inthe cortical cord width as well as cortico-medullary ratio were alsonoted. These cytometric methods have been used widely as param-eters of adrenocortical activity, which is generally stimulated byenhanced secretion of pituitary ACTH.

2. Materials and methods

Day old white leg horn chicks were procured from local hatch-ery (Shreeji Hatchery, Shivpur, Varanasi, India) and reared underthe laboratory conditions in 12L:12D. Chicks were provided withhigh protein diet: chicken starter ration up to the age of 6 weeksand thereafter broiler finisher diet. All experiments were per-formed in accordance with institutional practice and within theframework of revised animals (scientific procedures) Act of 2002of Government of India on animal welfare.

To determine the effect of osmotic stress on the expression ofvasotocin receptor VT2, 8 weeks old male young chickens (n = 10per group) were deprived of water for 4 days without any foodrestriction. Control birds received both food and water ad libitum.At the end of four days, five birds from both the groups wereweighed and sacrificed by decapitation, blood was collected inheparinized tubes and centrifuged at 1000g for 20 min, plasmaseparated and stored in �20 �C for the measurement of plasmaosmolality. Pituitaries were immediately dissected out, snap frozenin liquid nitrogen and kept at �70 �C until used for RNA isolation,followed by RT-PCR. Adrenal glands were also removed, weighedand stored in �20 �C for the biochemical analysis of ascorbic acid(Schaffert and Kingsley, 1955), cholesterol content (Rosenthalet al., 1960) and 3b-hydroxysteroid dehydrogenase (3b HSD) activ-ity (Shivanandappa and Venkatesh, 1997). The remaining five birdsfrom each of the water-deprived and control groups were anaes-thetized by injecting sodium pentobarbitol (25–30 mg/kg bodyweight) and then perfused intracardially with 0.02 M PBS contain-ing 0.01% heparin, followed by Zamboni Fixative (4% PFA inPBS + Picric acid). After whole body perfusion, pituitaries and adre-nals were dissected out and post fixed in fresh Zamboni fixative(4–6 h for pituitary and 24–48 h for adrenal). After fixation, tissues

were dehydrated in graded series of alcohol, cleared in xylene andembedded in paraffin wax. Eight micrometers thick sagittal sec-tions of the pituitary were used for in situ hybridization and immu-nohistochemistry. Six micrometers thick transverse sections of theadrenal were processed for routine hematoxylin-eosin staining.

2.1. Semi quantitative RT-PCR

Reverse transcriptase polymerase chain reaction (RT-PCR) wasused to analyze VT2 and POMC mRNA level in the pituitary. TotalRNA was isolated from pituitaries using TRI reagent (Sigma,USA). The integrity of the RNA was verified by 1.5% denaturing aga-rose gel electrophoresis. To rule out the possibility that PCR prod-ucts would result from the amplification of genomic DNA, RNAsamples were treated with DNAse. One microgram of total RNAwas used to synthesize cDNA by reverse transcription, using firststrand cDNA synthesis kit (Fermentas, USA) according to the man-ufacturer’s guidance. For PCR reactions 2 ll of the RT product wastaken and PCR was carried out using 2� PCR Master Mix kit (Fer-mentas, USA). After initial denaturation by incubating at 94 �C for5 min, 35 cycles were performed, including denaturation for1 min at 94 �C, annealing of the primers for 1 min at 55 �C (forVT2), 52 �C (for POMC) and 58 �C for GAPDH, and extension for1 min at 72 �C, followed by a final elongation step at 72 �C for5 min Primer sequence for each gene and the size of amplicon isprovided in Table 1. PCR products were analyzed on 2% agarosegel containing ethidium bromide (0.5 lg/ml). Stained bands werecaptured digitally and densitometric analysis was conducted usingimage analysis software Alpha Imager (Alpha Innotech Corpora-tion, USA).

2.2. Immunohistochemistry

After initial dewaxing in xylene and rehydration in graded ser-ies of alcohol, slides were processed for immunohistochemistryusing rabbit-VT2 antisera (1:6000) for 24 h (Jurkevich et al.,2005), followed by incubation in HRP conjugated secondary anti-body. Diaminobenzidine hydrochloride (DAB) was used as a chro-mogen molecule for the immunological detection. For negativecontrol, primary antibody was omitted and the sections were incu-bated in normal goat serum only.

2.3. In situ hybridization

In situ hybridization of VT2 mRNA was performed using digox-igenin (DIG) labeled 250 bp fragment of the VT2 cDNA. The probesequence comprises of the fragment from exon 1 (703–953 bp) ofVT2 gene (GenBank Accession No. AY008272, Cornett et al.,2003). Hybridization was carried out in hybridization solution(50% DI formamide, 10% dextran sulfate, 1� Denhart’s solution,4� SSC, 10 mM DTT, 1 mg/ml ssDNA) containing 300–400 ng/mlof labeled probe at 42 �C for 16 h. Post hybridization washingsand immunological detection was performed as described earlier(Srivastava et al., 2007). For negative control, sections were incu-bated in a hybridization buffer lacking DIG labeled probe.

218 D. Sharma et al. / General and Comparative Endocrinology 160 (2009) 216–222

2.4. Quantitative analysis

For image analysis of in situ hybridization and immunohisto-chemical signals in the sections of the pituitary gland, relativequantification method was employed as described by Sakharkaret al. (2005). The image analysis system consisting of a Leitz Labor-Lux S microscope and CCD video camera (JVC, Japan) connected toa computer was used. The microscope images were digitized andanalyzed using Leica Qwin standard software (version 3). Ran-domly selected 5 square areas (21,389 lm2 each) in each sagittalsection of the pituitary (5 sections from each pituitary and total4 pituitaries) from the region of cephalic lobe were subjected toanalysis.

For adrenal morphometry, 5 randomly selected sections fromthe mid region of the adrenal (4 adrenals per group) were chosen.The width of 5 cortical cords as well as the area occupied by lightcolored steroidogenic cords (cortical) and dark patches of chromaf-fin tissue (medulla) from each section were measured to calculatecortico-medullary (C:M) ratio. These cytometric methods havebeen used widely as parameters of adrenocortical (steroidogenic)activity (Siller et al., 1975; Chaturvedi and Kumar, 2007).

2.5. Statistical analysis

Data are presented as the mean ± SEM. Statistical significance ofthe difference between the control and experimental water-de-prived group was determined by student t test. P < 0.05 was con-sidered statistically significant.

3. Results

Osmotic stress of 4 days water deprivation resulted in a signif-icant decrease in body weight and increase in plasma osmolality as

Fig. 1. Effect of water deprivation (WD) on the body weight and plasma osmolalityin chicken. Values are the mean ± SEM of the data obtained from 5 birds per group.**P < 0.01; ***P < 0.001, significance of difference from control.

compared to control (Fig. 1). Densitometric analysis of the RT-PCRproducts, after normalization with the values of housekeepinggene GAPDH, showed a significant decrease in the expression ofpituitary VT2 and pro-opiomelanocortin (POMC) mRNA in water-deprived birds compared to chickens provided water ad libitum(Fig. 2).

Non-radioactive in situ hybridization and immunohistochemi-cal study showed positively hybridized as well as immunoreactivesignals in the cephalic lobe of the pars distalis, along with very fewscattered cells observed in the caudal lobe as well. Results of therelative quantitative analysis of in situ hybridization of VT2showed a significant decrease in the area showing positive hybrid-ization signals in the pituitary, following 4 days of water depriva-tion. Image analysis of the ir-VT2 did not show a correlation withthe RT-PCR as well as ISH data. A significant increase in the areashowing positive immunoreactive signals was observed in thepituitary of water-deprived birds compared to control (Fig. 3).

Adrenal weight and 3b HSD enzyme activity increased. Adrenalcholesterol decreased, but the adrenal ascorbic acid values did notshow any change in water-deprived chicken compared to control(Fig. 4). Histologically, the adrenal gland showed a typical avianpattern i.e., steroidogenic/interrenal cords (cortical tissue) andthe patches of chromaffin cells (medullary tissue) intermingled

Fig. 2. RT-PCR analysis of VT2, POMC and GAPDH in water-deprived chicken. Upperpanel shows the representative agarose gels. Lower panel shows the relativedensitometric values of amplified product of VT2 and POMC normalized with thatof the GAPDH. Values are the mean ± SEM (n = 4). *P < 0.05; ***P < 0.001, significanceof difference from control.

Fig. 3. In situ hybridization (upper row) and immunohistochemical (lower row) localization of vasotocin receptor VT2 in the anterior pituitary gland of control and water-deprived chickens. Histograms represent relative quantitative analysis of the hybridization and immunoreactive signals. Values are the mean ± SEM of the data obtained byrelative quantitative analysis of signals in the pituitaries of 4 birds per group. ***P < 0.001, significance of difference from control.

D. Sharma et al. / General and Comparative Endocrinology 160 (2009) 216–222 219

with each other. In hematoxylin-eosin stained sections, interrenaltissue appears lighter (pinkish) whereas chromaffin tissues are dis-tinguished by dark (blue) color. Histological observations of adre-nal gland in water-deprived chicken revealed an activeappearance showing broader cortical cords with prominent nucleiarranged in double rows and less area of medullary patches ascompared to control. Cortico-medullary ratio (i.e., area occupiedby the cortical cords per unit of the area occupied by medullary tis-sue) also showed a significant increase in the water-deprived birds(Fig. 5).

4. Discussion

The objective of the present experiment was to study whetherosmotic stress following water deprivation affects vasotocin recep-tors VT2 that have been earlier reported to be co-localized (immu-nocytochemically) in the pituitary corticotrophs with ACTH(Jurkevich et al., 2005). It has already been established that waterdeprivation up-regulates AVT mRNA in the magnocellular neurons

of hypothalamic paraventricular (PVN) and supraoptic nuclei(SON) to stimulate the synthesis and secretion of AVT in theperipheral circulation (Chaturvedi et al., 1994, 1997; Seth et al.,2004). Further, the present study reports that pituitary expressionof VT2 receptor mRNA decreases concurrently with the downregu-lation of POMC transcript in water-deprived chickens.

It has been reported that under normal circumstances plasmaAVT level shows a circadian variation in chicken with peak andtrough values occurring at 08.00 and 20.00 h, respectively. Thiscyclicity gets abolished following four days of water deprivation,and AVT concentration is maintained at a higher level (Chaturvediet al., 2001). In view of reports that the membrane receptors forpeptide hormones can be downregulated by their own ligands(Catt et al., 1979), it is possible that increased AVT secretion inosmotically stressed chickens led to a downregulation of VT2receptor mRNA expression in the pituitary. Further, this studyalthough indirectly, supports the view that magnocellular vasoto-cinergic system takes part in the regulation of HPA axis via itsreceptor VT2 localized on pituitary corticotrophs as in mammals

Fig. 4. Effect of water deprivation (WD) on the adrenal responses of chicken. Values are the mean ± SEM of the data obtained from 4 birds per group. *P < 0.05; **P < 0.01;***P < 0.001, significance of difference from control.

Fig. 5. Transverse section of the adrenal gland of control and water-deprived (WD) chicken showing intermingled light colored steroidogenic (cortical) cords and darkchromaffin (medullary) patches. Histograms represent changes in the cortical cord width and corticomedullay (C:M) ratio. Values are the mean ± SEM of the data obtained byrandomly selected 5 sections from the mid region of the adrenal of bird (4 birds per group). ***P < 0.001, significance of difference from control.

220 D. Sharma et al. / General and Comparative Endocrinology 160 (2009) 216–222

D. Sharma et al. / General and Comparative Endocrinology 160 (2009) 216–222 221

(Engelmann et al., 2004). In mammals, hypothalamic vasopressin isreported to reach the anterior pituitary through hypothalamo-hypophyseal portal blood via median eminence (Holmes et al.,1986; Wotzak et al., 1996, 2002) and/or through short portal vesselthat carry the blood from posterior pituitary to anterior pituitary(Sawchenko et al., 1992). But no such direct evidence is reportedin birds. Further, no change in AVP mRNA has been reported inthe parvocellular neurons of water-deprived rats (Grinevichet al., 2001). Again, comparable information is lacking in avianspecies.

Results of the RT-PCR analysis correlate with the in situhybridization study. However, despite decreased expression ofVT2 mRNA, immunoreactivity for VT2 increased in the pituitaryof water-deprived chickens. The reason for this discrepancy isnot fully clear, but may indicate a role for post-transcriptionalmechanisms. Osmotic stress may have triggered increased trans-lation of VT2 mRNAs but not the increased transcription of VT2mRNAs, thereby shifting the balance towards elevated levels ofVT2 protein but less mRNA. Moreover, in co-localization studyusing the same antisera, it has been suggested that pituitary lac-totrophs may also be the site expressing VT2 receptor (Jurkevichet al., 2005). Therefore any apparent role of prolactin in waterbalance cannot be ruled out. In view of this, there may be apossibility that the dehydration-induced secretion of vasotocinmay also regulate lactotroph function. But again the role of lac-totrophs in the regulation of pituitary-adrenal axis remainsunexplained.

Consistent reports from the mammalian as well as avian litera-ture indicate an increase in plasma osmolality following dehydra-tion/osmotic stress, which in turn stimulates the secretion ofarginine vasotocin from the hypothalamic magnocellular neurons.However, there are conflicting reports in the avian species relatingto the dehydration induced changes in plasma corticosterone con-tent. The adrenal steroids are reported to increase following dehy-dration in turkey (Brown, 1961), chicken (Freeman et al., 1983) andduck (Harvey et al., 1981); while in quail (Kobayashi et al., 1980)and fowl (Arad et al., 1985), water deprivation failed to alter theplasma corticosterone level. It has been found that the level ofthe plasma corticosterone does not show the actual state of theadrenal since even handling of the animals itself causes an increasein the corticosteroid level (Place and Kenegy, 2000; Vleck, 2001).Hence we have chosen other parameters to study the adrenalactivity. The level of adrenal cholesterol and ascorbic acid has beenfound to be inversely related to steroid synthesis. In the presentstudy, increased adrenal weight, cortical cord width, cortico-med-ullary ratio, and 3b HSD (a rate limiting enzyme of steroid biosyn-thesis) indicate increased adrenal activity and steroidogenesis.However, adrenal ascorbic acid unlike cholesterol content did notshow inverse relation with adrenal activity. This is in accordancewith the earlier reports in chicken, where stimulation of the adre-nal gland by stressful conditions fails to deplete adrenal ascorbicacid content (Breitenbach, 1962) unlike other avian species includ-ing Japanese quail (Chaturvedi and Thapaliyal, 1978; Singh andChaturvedi, 2008).

Present findings reveal that osmotic stress given for 4 days de-creased the expression of pituitary VT2 and POMC mRNA whileadrenocortical activity increased. It is quite possible that waterdeprivation stress induced an initial but transient increase in theplasma ACTH that resulted in the stimulation of adrenal corticoids,which subsequently exerted a feedback effect on the pituitary cor-ticotrophs. There are reports in mammals too showing an increasein the plasma ACTH level after 12 h of water deprivation, which re-turns to the basal level after 48 h (Aguilera et al., 1993). Further,the apparent dissociation between pituitary corticotrophs andadrenal activity and stimulation of adrenal function despite de-creased ACTH activity (assessed on the basis of a decrease in POMC

level as well as in the VT2 mediated effect of AVT on corticotrophs)reflects increased sensitization of the adrenal to low ACTH levels. Itwill be worthwhile to study the pituitary-adrenal axis, especiallythe response of VT2 receptor expression during different kinds ofstressful conditions (other than osmotic stress) when AVT secre-tion is not stimulated from magnocellular neurons. Such studiesare required to understand the role of parvocellular AVT, if any,in the regulation of HPA axis in birds.

On the basis of multifarious response of various components ofthe HPA axis, the present study indicates that water deprivationis a stress that modulates HPA axis function involving AVT as anACTH secretagogue via the VT2 receptor expressed in corticotrophs.This osmotic stress reduces mRNA level of pituitary VT2 receptor aswell as the ACTH precursor POMC, but stimulates adrenal steroido-genesis. Further, in addition to the antidiuretic role of magnocellu-lar AVT, through neurohypophyseal system, this neuropeptide isalso an important component of the network controlling HPA axisby modulating pituitary ACTH synthesis via its receptor VT2.

Acknowledgments

This work was supported in part by a research Grant (F. No. 32-472/2006 (SR) to CMC, from University Grant Commission, NewDelhi. We also thank Council of Scientific and Industrial Research,for providing financial support in the form of Junior and Senior Re-search fellowships to DS. The authors wish to thank Ms. Sandie Ja-cobi, (USA) for the preparation of VT2 cDNA and Dr. N.K. Subhedar,Department of Pharmaceutical Sciences, University of Nagpur,Nagpur, India for providing the facility of image analysis.

References

Aguilera, G., Lightman, S.L., Kiss, A., 1993. Regulation of the hypothalamic-pituitary-adrenal axis during water deprivation. Endocrinology 132, 241–248.

Antoni, F.A., 1993. Vasopressinergic control of pituitary adrenocorticotropinsecretion comes of age. Front. Neuroendocrinol. 14, 76–122.

Arad, Z., Arnason, S.S., Chadwik, A., Skadhauge, E., 1985. Osmotic and hormonalresponses to heat and dehydration in the domestic fowl. J. Comp. Physiol. B 155,227–234.

Breitenbach, R.P., 1962. The effect of ACTH on adrenocortical secretion and ascorbicacid depletion in normal and testosterone treated cockerels. Poult. Sci. 41,1318–1324.

Brown, K.I., 1961. The validity of using plasma corticosterone as a measure of stressin the turkey. Proc. Soc. Exptl. Biol. Med. 107, 538–542.

Castro, M.G., Estivariz, F.E., Iturriza, F.C., 1986. The regulation of thecorticomelanotropic activity in Aves: II. Effect of various peptides on therelease of ACTH from dispersed, perfused duck pituitary cells. Comp. Biochem.Physiol. 3A, 71–75.

Catt, K.J., Harwood, J.P., Aguilera, G., Dufau, M.L., 1979. Hormonal regulations ofpeptide receptors and target cell responses. Nature 280 (5718), 109–116.

Chaturvedi, C.M., Thapaliyal, J.P., 1978. Effects of orchidectomy and male hormoneadministration on the adrenal of common myna, Acridotheres tristis. Ann.Endocrinol. 39, 417–420.

Chaturvedi, C.M., Kumar, P., 2007. Nitric oxide modulates gonadal and adrenalfunction in Japanese quail Coturnix coturnix japonica. Gen. Comp. Endocrinol.151 (3), 285–299.

Chaturvedi, C.M., Newton, B.W., Cornett, L.E., Koike, T.I., 1994. An in situhybridization and immunohistochemical study of vasotocin neurons in thehypothalamus of water deprived chickens. Peptides 15 (7), 1179–1187.

Chaturvedi, C.M., Cornett, L.E., Koike, T.E., 1997. Arginine vasotocin gene expressionin hypothalamic neurons is up-regulated in chickens drinking hypertonicsaline: an In situ hybridization study. Peptides 18, 1383–1388.

Chaturvedi, C.M., Choudhary, A., Cornett, L.E., 2001. Water deprivation and circadianchanges in plasma arginine vasotocin and mesotocin in the domestic hen(Gallus domesticus). Chronobiol. Int. 18 (6), 947–956.

Chowdry, H.S., Jessop, D.S., Patel, H., Lightman, S.L., 1991. Alteredadrenocorticotropin, corticosterone and oxytocin responses to stress duringchronic salt load. Neuroendocrinology 54, 635–638.

Cornett, L.E., Kirby, J.D., Viscarra, J.A., Ellison, J.C., Thrash, J., Mayeux, P.R., Crew,M.D., Jones, S.M., Ali, N., Baeyens, D.A., 2003. Molecular cloning and functionalcharacterization of a vasotocin receptor subtype expressed in the pituitarygland of the domestic chicken (Gallus gallus domesticus): avian homolog of themammalian V1b-vasopressin receptor. Regul. Pept. 110, 231–239.

Dohanics, J., Hoffman, G.E., Verbalis, J.G., 1991. Hyponatremia induced inhibition ofmagnocellular neurons causes stressor selective impairment of stimulatedadrenocorticotropin secretion in rats. Endocrinology 128, 331–340.

222 D. Sharma et al. / General and Comparative Endocrinology 160 (2009) 216–222

DuPasquier, D., Loup, F., Dubios-Dauphin, M., Dreifuss, J.J., Tribollet, E., 1991.Binding sites for vasopressin in the human pituitary are associated withcorticotrophs and may differ from other known vasopressin receptors. J.Neuroendocrinol. 3, 237–247.

Engelmann, M., Landgraf, R., Wotjak, C.T., 2004. The hypothalamic-neurohuypophysial system regulates the hypothalamic-pituitary-adrenal axisunder stress: an old concept revisited. Front. Neuroendocrinol. 25, 132–149.

Freeman, B.M., Manning, A.C.C., Falk, I.H., 1983. Adrenal cortical activity in thedomestic fowl, Gallus gallus, following withdrawal of water and food. Comp.Biochem. Physiol. 74A, 639–641.

Grinevich, V., Ma, X.M., Verbalis, J., Aguilera, G., 2001. Hypothalamic pituitaryadrenal axis and hypothalamic-neurohypophyseal responsiveness in water-deprived rats. Exp. Neurol. 171 (2), 329–341.

Harvey, S., Klandorf, H., Phillips, J.G., 1981. Effects of food or water deprivation oncirculating levels of pituitary, thyroid and adrenal hormones and on glucose andelectrolyte concentration in domestic ducks (Anas platyrhynchos). J. Zoo. 194,341–361.

Holmes, M.C., Antoni, F., Aguilera, G., Catt, K.J., 1986. Magnocellular neurons inpassage through the median eminence release vasopressin. Nature 319, 326–329.

Jurkevich, A., Berghman, L.R., Cornett, L.E., Kuenzel, W.J., 2005. Characterization andimmunohistochemical visualization of the vasotocin VT2 receptor in thepituitary gland of the chicken. Gen. Comp. Endocrinol. 143, 82–91.

Kobayashi, S., Umera, H., Takai, Y., 1980. Physiological role of the rennin-angiotensin system in dehydration. In: Epple, A., Stretson, M.H. (Eds.), AvianEndocrinology. Academic Press, New York, pp. 319–330.

Mikami, S., Yamada, S., 1984. Immunohistochemistry of the hypothalamicneuropeptides and anterior pituitary cells in the Japanese quail. J. Exp. Zool.232, 405–417.

Nephew, B.C., Aaron, R.S., Romero, L.M., 2005. Effects of arginine vasotocin (AVT) onthe behavioral, cardiovascular, and corticosterone responses of starlings(Sturnus vulgaris) to crowding. Horm. Behav. 147 (3), 280–289.

Place, N.J., Kenegy, G.J., 2000. Seasonal changes in plasma testosterone andglucocorticoids in free living male yellow-pine chipmunks and the responseto capture and handling. J. Comp. Physiol. B 170, 245–251.

Rittmaster, R.S., Cutler, G.B., Gold, P.W., Brandon, D.D., Tomai, T., Loriaux, D.L.,Chrousos, G.P., 1987. The relationship of saline induced changes in vasopressinsecretion to basal and corticotrophin-releasing hormone stimulatedadrenocorticotropin and cortisol secretion in mammals. J. Clin. Endocrinol.Metab. 64, 371–376.

Romero, L.M., 2006. Seasonal changes in hypothalamic-pituitary-adrenal sensitivity infree living house sparrows (Passer domesticus). Gen. Comp. Endocrinol. 149, 66–71.

Rosenthal, H., Pfluke, M.L., Buscaglia, S., 1960. A stable iron reagent fordetermination of cholesterol. J. Lab Clin. Med. 50 (2), 318–322.

Sakharkar, A.J., Singuru, P.S., Sarkar, K., Subhedar, N.K., 2005. Neuropeptide Y in theforebrain of adult male cichlid fish Oreochromis mossambicus: distribution,effects of castration and testosterone replacement. J. Comp. Neurol. 489, 148–165.

Sawchenko, P.E., Cunningham Jr., E.T., Bittencourt, J.C., Chan, R.K.W., 1992.Aminergic and peptidergic pathways subserving the stress response. In:Kvetnansky, R., McCarty, R., Axelrod, J. (Eds.), Stress: Neuroendocrine andMolecular Approaches. Gordon and Beach Science Publishers, New York, pp. 15–27.

Schaffert, R.R., Kingsley, G.R., 1955. A rapid, simple method for the determination ofreduced, dehydro- and total ascorbic acid in biological material. J. Biol. Chem.212, 59–68.

Seth, R., Kohler, A., Grossmann, R., Chaturvedi, C.M., 2004. Expression ofhypothalamic arginine vasotocin gene in response to water deprivation andsex steroid administration in female Japanese quail. J. Exp. Biol. 207, 3025–3033.

Shivanandappa, T., Venkatesh, S., 1997. A colorimetric assay method for 3b-hydroxysteroid dehydrogenase. Anal. Biochem. 254, 57–61.

Siller, W.G., Teague, P.W., Mackenzie, G.M., 1975. The adrenal cortico-medullaryratio in the fowl. Br. Poult. Sci. 16 (4), 335–342.

Singh, S., Chaturvedi, C.M., 2008. Changes in vasotocin immunoreactivity ofparaventricular nuclei and adrenal function of Japanese quail in relation todifferent phases of sexual development. Domest. Anim. Endocrinol. 34 (3), 293–300.

Srivastava, R., Cornett, L.E., Chaturvedi, C.M., 2007. Effect of photoperiod andestrogen on expression of arginine vasotocin and its oxytocic like receptor inthe shell gland of the Japanese quail. Comp. Biochem. Physiol. A Mol. Integr.Physiol. 148 (2), 451–457.

Vleck, C.M., 2001. Comparison of corticosterone and heterophil to lymphocyteratios as indicators of stress in free-living birds. In: Dawson, A., Chaturvedi, C.M.(Eds.), Avian Endrocrinology. Narosa Publishing House, New Delhi, India, pp.402–411.

Wotzak, C.T., Kubota, M., Kohl, G., Landgraf, R., 1996. Release of vasopressin fromsupraoptic neurons within the median eminence in vivo. A combinedmicrodialysis and push–pull perfusion study in the rat. Brain Res. 726, 237–241.

Wotzak, C.T., Ludwig, M., Ebner, K., Russel, J.A., Singewald, N., Landgraf, R.,Engelmann, M., 2002. Vasopressin from hypothalamic magnocellular neuronshas opposite actions at the adenohypophysis and in the supraoptic nucleus onACTH secretion. Eur. J. Neurosci. 16, 477–485.