GENERAL AND COMPARATIVE

ENDOCRINOLOGY

www.elsevier.com/locate/ygcen

General and Comparative Endocrinology 137 (2004) 177–186

Changes in expression of AVT and AVT receptor (VT1) genein hypothalamus and shell gland in relation to egg laying in

white leghorn hen

R. Seth,a,b Y.X. Xu,b,c R. Grossmann,b and C.M. Chaturvedia,*

a Molecular Endocrinology Lab, Department of Zoology, Banaras Hindu University, Varanasi 221005, UP, Indiab Department Functional Genomics and Bioregulation, Institute for Animal Science and Animal Behaviour (FAL), Mariensee,

31535 Neustadt am R€ubenberg, Germanyc Nanjing Agricultural University, Nanjing, PR China

Received 6 June 2003; revised 1 March 2004; accepted 4 March 2004

Available online 13 April 2004

Abstract

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-poly-

merase 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 (Naþ, Kþ, Ca2þ, 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.

� 2004 Elsevier Inc. All rights reserved.

1. Introduction

Oviposition or egg laying occurs almost daily after

puberty during the reproductive phase of the life cycle in

poultry birds. Oviposition is a complex phenomenon

involving various regulatory mechanisms, mainly at the

level of hypothalamus, hypophysis, ovary, and oviduct.

Regulatory factors include follicular growth, ovulation,

egg transport through the oviduct, and neuropeptidearginine vasotocin (AVT) and prostaglandin(s) (PG).

Oviposition involves contraction of the smooth muscles

* Corresponding author. Fax: +91-542-2368323.

E-mail addresses: cmcbhu@indiatimes.com, sethrohit@uams.edu

(C.M. Chaturvedi).

0016-6480/$ - see front matter � 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.ygcen.2004.03.003

of uterus (shell gland) and relaxation of the utero-vag-inal sphincter leading to egg expulsion from the oviduct.

After the rupture of mature graffian follicle, the ovum

travels down the infundibulum, magnum, isthmus, and

finally reaches to the shell gland in albumen deposited

form where it stays for about 20 h. During its stay in the

shell gland the egg acquires shell before being laid.

AVT has both oxytocic and antidiuretic properties

and is selectively released during oviposition or hyper-tonic saline infusion in chickens (Koike et al., 1988;

Nouwen et al., 1984; Sawyer, 1977; Shimada et al.,

1986). Both, the sensitivity of uterine muscle to AVT

and receptor density is maximal at oviposition (Koike

et al., 1988; Saito et al., 1987). The ovary in laying hen

contains large amount of AVT which varies in different

178 R. Seth et al. / General and Comparative Endocrinology 137 (2004) 177–186

locations in relation to the oviposition cycle (Saito andGrossmann, 1999). For example, AVT levels in follicu-

lar venous plasma are lower than in brachial venous

plasma, suggesting that the ovarian peptides are not

released into the systemic circulation (Shimada et al.,

1987).

Under the influence of the hormones argine vasotocin

(AVT) and prostaglandins (PG), uterine muscles con-

tract leading to expulsion of the egg. Administration ofPG (PGE1, PGE2, and PGF2a), and free fatty acids

(which act as precursors to prostaglandins) induces

premature oviposition in hen and Japanese quail

(Hertelendy et al., 1975; Rzasa, 1984; Saito et al., 1987).

Acetylcholine administration also induces premature

oviposition in birds. However, administration of indo-

methacin before the predicted time of oviposition delays

egg lay by 8–14 h. It is reported that, AVT plays a keyrole in releasing PG from the uterus (Rzasa, 1984).

[3H]AVT binding sites in the uterine membrane repre-

sent physiological receptors that interact with AVT

during oviposition. The concentration of AVT and MT

(mesotocin) are very high at the time of oviposition, but

fall significantly after egg laying (Koike et al., 1988).

Therefore, the movement of the egg into the cloaca, as

well as its extrusion into the nest (oviposition) is con-trolled by AVT and PGs. High doses of AVT (up to

1 lg) increases oviposition upto 100%. However, bearing

down of oviposition is observed with higher doses (10

and 20 lg) of injected AVT; this could be caused by the

hormonal stimulus of AVT receptors present in vaginal

tissues (Takahashi and Kawashima, 2003).

The major intracellular cation is potassium and its

concentration in intracellular water is nearly 35 timesgreater than in extracellular water. After potassium,

sodium is the next major intracellular cation and it is

nearly absent in the extracellular water. As sodium is a

major contributor to osmolality of blood, changes in

sodium balance can change both the distribution of

body water and its volume. Physiological levels of ir-

AVT (immunoreactive-AVT) are related to appropriate

changes in free-water clearance and other measures ofrenal osmoregulatory function in ducks (Gerstberger

et al., 1985; Mohring et al., 1980). An increase in plasma

AVT is observed after intracarotid infusion of 1.0M

NaCl (Simon-Oppermann and Simon, 1982). In ducks

maintained on 2% NaCl for 5–10 months, plasma os-

molality is elevated and AVT levels become three times

higher than in water adapted birds (Szczepanska-Sado-

wska et al., 1985). Thus, any change in plasma osmo-lality leads to an increase in plasma AVT concentration,

establishing a relationship between plasma AVT and

concentration of ionic variants.

Availability of the AVT mRNA sequence of chicken

(Hamann et al., 1992) and recent cloning of vasotocin

receptors designated as VT1 (Tan et al., 2000) and VT2

(Cornett et al., 2003) has revolutionized the study of

involvement of AVT in regulating various physiologicalevents at the genetic level. VT1 is expressed in brain and

shell gland and VT2 in pituitary gland. Studies have

suggested that in birds, as for vasopressin neurons in

mammals, some of the hypothalamic vasotocin neurons

project to the median eminence relative to the pars

nervosa to target the corticotrophs of the pars distalis,

thereby producing hypothalamic–pituitary–adrenal

(HPA) axis activation. This concept has been strength-ened in birds by the expression of VT2 receptor in the

pituitary gland of chicken, the avian homologue of the

mammalian V1b receptor (Cornett et al., 2003). In ad-

dition to its expression in hypothalamus, AVT gene was

identified by RT-PCR in ovary and shell gland (Cha-

turvedi et al., 1994a); however, precise timing of ovi-

position was not taken into consideration.

In view of the interaction of AVT and VT1 receptorsexpressed in the shell gland during oviposition, present

study was designed to access simultaneous changes in

the (a) AVT gene expression in the hypothalamus and

shell gland, (b) AVT receptor (VT1) gene expression in

the shell gland, and (c) concentration of plasma AVT, in

relation to the time of oviposition.

2. Materials and methods

Seven to eight months old white leghorn hens were

kept in individual cages with food and water ad libitum.

The hens were maintained under long days (16L:8D)

and personally monitored for 2–3 weeks to determine

the egg laying pattern and exact time of oviposition.

Only those hens which have a regular laying cycle of 6–7days were used in the following experiments.

Three groups of chicken (n ¼ 5 per group) were sac-

rificed by cervical dislocation 2 h before ()2 h), imme-

diately after egg laying (0 h) and 2 h after egg laying

(+2 h). Birds were dissected to separate out hypothala-

mus from the brain and shell gland from the oviduct.

These tissues were immediately frozen on dry ice and

kept in )70 �C until the extraction of total RNA. Thiswas followed by Northern blot analysis of hypothalamic

RNA and RT-PCR of shell gland RNA for the detec-

tion of AVT and AVT receptor gene. For another set of

studies, 5 chickens from each of the three groups ()2, 0,and +2 h) were anaesthetized by pentobarbital sodium

(30–40mg/kg body wt) and blood sample (�1.5ml) was

obtained from the wing vein in a heparinized syringe.

Plasma separated and stored at )20 �C for radioimmu-noassay of AVT. Thereafter, whole body perfusion was

done through carotids using 0.02M phosphate-buffered

solution (PBS) and Zamboni fixative given via perfusion

pump at the speed of 2–3ml/min. Brain was removed

and postfixed in fresh Zamboni fixative. For cryopro-

tection, brain was transferred to 25% sucrose solution in

PBS at 4 �C until it sank to the bottom (�24 h). Brains

R. Seth et al. / General and Comparative Endocrinology 137 (2004) 177–186 179

were frozen using tissue freezing medium and cut at18 lm in a cryostat. These sections were processed for in

situ hybridization and immunohistochemistry.

In the third set of study chickens (n ¼ 10) were an-

aesthetized by sodium pentobarbital (30–40mg/kg body

wt) and were surgically cannulated in the wing vein us-

ing a silicone catheter (Dow Corning, Meckenheim,

Germany) for repeated withdrawal of blood samples at

hourly intervals over a period of 11 h (i.e., 5 h before egglaying, at the time of egg laying, and 5 h after egg lay-

ing). Blood was centrifuged, plasma separated, and

stored at )20 �C until used for radioimmunoassay (RIA)

of AVT, osmolality, and ion analysis.

2.1. In situ hybridization

Sections were washed in PBS, dehydrated in gradedethanol series, and air-dried. The 270 bp AVT probe

complementary to the glycoprotein coding 30 terminus

of the chicken AVT cDNA (Hamann et al., 1992) was

labeled with [a-33P]dCTP (Dupont, NEN, Bad Hom-

burg, Germany) by the random priming method

(megaprime DNA labeling system; Amersham, Braun-

shweig, Germany) according to Feinberg and Vogelstein

(1983) and separated from unincorporated nucleotidesby using Sephadex G-50 columns (Nick columns,

Pharmacia, Frieburg, Germany). The probe was diluted

with hybridization buffer (50% formamide, 5� Den-

hardt�s solution, 10% dextran sulfate, 0.75M NaCl,

25mM Pipes (1,4-piperazine bis-2-ethanosulfonic acid),

25mM EDTA, 0.2% (w/v) sodium dodecyl sulfate

(SDS), and 250 lg/ml herring sperm DNA) to give ap-

proximately 2500 cpm/ll. The sections were prehybrid-ized with hybridization buffer (mentioned above) for 1 h

at 52 �C in a moist chamber followed by hybridization

with labeled probe. Forty microliters of AVT probe was

applied on each section and covered by plastic cover-

slips. Hybridization was performed for about 16 h. at

52 �C in a moist chamber. The sections were washed at

room temperature in 4� sodium chloride–sodium citrate

buffer (SSC) for three times, 10min each and in 2� SSCfor three times, 10min each. Final washing was done in

70% ethanol and then sections were dried in vacuum for

2 h. Sections were covered with photographic emulsion

(LM-1, Amersham, Braunschweig, Germany) diluted

1:1 with distilled water. After exposure under total

darkness, for 7–10 days at 4 �C, slides were developed

using Ilford Phenisol, lightly counterstained with tolui-

dine blue, and, after air drying, were coverslipped withEntellan (Merk). Sections were viewed with a Nikon

Epiphot microscope equipped with a dark field con-

denser. To check the specificity of hybridization signals

subsequent controls were treated with RNase A (50 lg/ml, 0.5M NaCl, 0.01M Tris–HCl, 1mM EDTA, pH

7.5; Boehringer, Mannheim, Germany) for 10min at

37 �C before prehybridization.

2.2. Immunohistochemistry (IHC)

The brain sections were rinsed in 0.02M PBS until

fixative is washed out completely. Thereafter, sections

were treated with 0.6% hydrogen peroxide (Merk) in

PBS for 30min, incubated in 5% normal goat serum

(Sigma, USA) containing 0.2% Triton X-100 (Sigma,

USA) for 30min, followed by incubation for 36 h at 4 �Cin a 1:2000 solution of rabbit anti-AVT serum (gifted byDr. D.A. Fisher, USA; Rosenbloom and Fisher, 1974)

in PBS containing 0.2% Triton X-100, 1% normal goat

serum, and 0.1% sodium azide. Sections were thor-

oughly washed and incubated in goat-anti-rabbit bioti-

nylated IgG (Vector, USA) 1:500 in PBS containing

0.2% Triton X-100 for 90min at room temperature,

rinsed in PBS (4� 15min), and incubated for 90min

with avidin biotin peroxidase conjugate (ABC–HRP,1:1000; Vector) solution in PBS containing 0.2% Triton

X-100 and 1% crystalline bovine serum albumin (Sigma,

USA). For immunodetection, 3,30-diaminobenzidene

(DAB; Sigma) in 0.05M Tris buffer (pH 7.6) with

0.005% hydrogen peroxide was used. Sections were

subsequently rinsed in PBS and distilled water, air-dried,

dehydrated, and coverslipped using DPX mountant

(Qualigens; Glaxo, India). To control the specificity ofimmune reaction, sections were incubated with 5%

normal goat serum in place of primary antibody (Cha-

turvedi et al., 1994b). In control sections no immune

positive signal was detected. The ant-AVT serum used in

this study was validated previously by Gray and Simon

(1983). The specificity of the revealed immunolabeling

was tested with the same primary antiserum presorbed

with synthetic AVT or nonimmune rabbit IgG by Jur-kevich et al. (1997). These reactions resulted in no de-

tectable staining.

2.3. Morphometric measurements

Two to three best matched sections containing ir-

AVT neurons were selected from the corresponding

slides used for in situ hybridizaton in the PVN region oneither side of the third ventricle, from each group of

hens. The cells were counted manually under ordinary

light microscope (Weswox Optik, Ambala Cantt, India,

model-TR Hl 66). The nomenclature of the brain

structures and stereotaxic planes of the sections were

adjusted in reference to the chicken brain atlas of Ku-

enzel and Masson (1988).

2.4. RNA isolation

Total RNA was obtained from the hypothalamus and

the shell gland using the method described by

Chomczynski and Sacchi (1987). RNA yield and pu-

rity was assessed by absorbance at 260 and 280 nm in a

Gene Quant spectrophotometer (Pharmacia Biotech,

180 R. Seth et al. / General and Comparative Endocrinology 137 (2004) 177–186

Uppsala, Sweden). Ratios of the absorption (A260=280) ofall preparations were between 1.8 and 2.

2.5. Northern blot analysis

Twenty micrograms of total RNA was separated on a

1.4% w/v (weight/volume) agarose denaturing formal-

dehyde gel in morpholino-propane sulfonic acid (Mops)

buffer (pH 8.0). The RNA was subsequently blottedovernight by capillary transfer onto nylon membranes

(Hybond Nþ, Amersham), followed by UV (ultraviolet)

crosslinking (150mj; Gene Linker, Bio-Rad, Munich,

Germany). The AVT specific probe (a 260 bp cDNA

directed towards the distal 30 glycopeptide part of the

chicken AVT gene (Hamann et al., 1992)) was labeled

with [32p]dCTP by random priming method (Megaprime

DNA labeling system; Amersham, Braunschweig, Ger-many) according to Feinberg and Vogelstein (1983), and

separated from unicorporated neucleotides using Se-

phadex G-50 columns (Nick Columns, Pharmacia,

Freiburg, Germany). Hybridization proceeded over-

night at 42 �C in a 50% formaldehyde containing buffer

according to Wahl et al. (1979). Approximately

5� 106 cpm (counts per minute) were used per filter in

6ml of hybridization buffer without dextran sulfate.Exposure time was 48 h at )70 �C using one intensifying

screen. The filter hybridized with the AVT probe was

dehybridized and reprobed with 18S cDNA and ana-

lyzed for normalization of the AVT gene. For estimating

Northern blot data/densitometry of autoradiographs,

filters sequentially hybridized with the AVT and 18S

cDNAs were exposed to storage phosphor screens (Bio-

Rad Laboratories, Munich, Germany), and the densityof bands corresponding to AVT and 18S were calcu-

lated. The volume of the AVT band was divided by the

volume of the 18S band to determine the amount of

hypothalamic AVT mRNA in relative units.

2.6. Semi-quantitative RT-PCR

Duplex RT-PCR was performed to study the ex-pression of two genes, i.e, gene of interest (AVT or VT1)

and the house keeping gene 18S. Duplex RT-PCR is a

powerful tool for getting reliable results since both, the

house keeping gene (internal control) and the gene to be

analyzed are amplified simultaneously in the same re-

action mix using two sets of primers. This procedure is

much simple and less prone to contamination than the

Table 1

Sequence of forward and reverse primers used for RT-PCR

Gene Primer sequence

AVT 50-CAG CCT TCC CCG AAC GCA TAG C-30

50-CTG CCG CAA GGC TGT ATC ACC C-30

AVT R (VT1) 50-CGT GCC AAG TTA AGA TTT GCA A-30

50-AAT ACT TCC TGT GAC CAC CGA A-30

traditional two-step procedure. Aliquots of RNA sam-ples were electrophoresed to verify their integrity.

cDNAs were synthesized using 1.0 lg of total RNA

from each sample. RNA samples were denatured at

65 �C for 15min and placed on ice for 5min before re-

action. The final reaction volume was 25 ll, which

contained 1� reaction buffer, 5mM MgCl2, 1mM each

of dNTP, 3.2 ll of random primers, 50U of RNase in-

hibitor, 0.01mg/ml gelatin, and 20U of AMV reversetranscriptase (First strand cDNA synthesis kit, Boerh-

inger–Mannheim, IN, USA). Cycles used in reaction

conditions were 25 �C for 10min, 42 �C for 60min, and

99 �C for 5min for enzyme heat inactivation and 4 �Cfor 5min. Samples were either stored at )20 �C or pro-

ceeded to the amplification step. Prior to the sample�sevaluation for RT reaction, 25U of DNase 1 (Roche,

Cat. No. 2222, Germany) were added and the samplewas incubated at 30 �C for 30min, followed by heat

inactivation of the enzyme. Contamination of genomic

DNA amplification was periodically checked using

control experiments in which reverse transcriptase (po-

sitive control) or RNA (negative control) were omitted

during the RT step.

PCR was performed in 50 ll reaction volume con-

taining 3 lg cDNA, 10mM KCl, 10mM (NH4)2SO4,20mM Tris–HCl (pH 8.8, 25 �C), 2mM MgSO4, 0.1%

Triton X-100, 0.32 lM upstream primer, 0.32 lMdownstream primer, and 0.04 lM 18S primer pairs. The

ratio of 18S primer pairs to the competimer was 1:9.

Competimers are specially modified primers that are

identical in sequence to the normal primers but cannot

be extended. By adjusting the ratio of competimer to

normal 18S rRNA primers, the signal for 18S rRNA canbe attenuated to the level of even rare messages by

modulating the efficiency of amplification of 18S rRNA

PCR product. The annealing temperature was 58 �C for

60 s, primer extension was done at 72 �C for 30 s, and

denaturing at 94 �C for 40 s. The reaction was set for 32

cycles (linear range 22–35 cycles) for AVT receptor and

36 cycles (linear range 30–40 cycle) for the AVT gene.

Two units of Vent R DNA polymerase (Biolabs),0.2mM of each dNTP, and 0.4 lM of each of the

primers was used in each cup.

Primer sequences and the length of amplified frag-

ments are given in Table 1. To obtain optimal condi-

tions for amplification (i.e., the exponential phase PCR),

the PCR cycle number was tested first for each target

gene. The Quantum RNA 18S primer and competimer

Forward Reverse Size (bp)

1 176 176

63 443 381

R. Seth et al. / General and Comparative Endocrinology 137 (2004) 177–186 181

were used as internal controls of amplification; thisprimer pair (Cat. No. 1716 Ambion, Austin, TX) am-

plifies a 489 bp fragment. Amplification was performed

in a personal cycler (Biometra, Tampa, FL). Duplex

RT-PCR end point products were subjected to electro-

phoresis on 1% agarose gel. Data were analyzed by

reading densitometric bands corresponding to AVT or

VT1 and 18S on computer using software that comes

with Bio-Rad phosphoimager (Munchen, Germany).

2.7. Radioimmunoassay

Plasma samples were extracted and processed for

RIA of AVT using anti-rabbit AVT serum (gift by Dr.

D. Gray, Max Plank Institute of Physical and Clinical

Research, Baad Nauheim, Germany) using the method

of Gray and Simon (1983). AVT was extracted fromplasma with 2 volumes of acetone and 2 volumes of

petroleum ether. The extract was dried under vacuum in

speed Vac concentrator (Savant Instruments, Holbrook,

NY, USA). The dried extract was dissolved in assay

buffer (0.1M Tris–HCl, pH 7.4, 2% BSA, and 0.2%

neomycin) and stored at )20 �C until assayed. RIA was

performed in duplicate using synthetic AVT as a stan-

dard (Sigma-chemie GmbH, Deisenhofen, Germany).Plasma osmolality was measured by vapor pressure

osmometry (Wescor, Logan, UT, model 5500). Plasma

levels of ionic concentration was analyzed using sodium/

potassium analyzer (Ciba Corning Diagnostics, Hal-

stead, UK).

2.8. Statistics

For the statistical analysis of the data, ANOVA fol-

lowed by Newman–Keul�s multiple range test was em-

ployed to compare results at various time points.

Fig. 1. Northern blot analysis of total cellular RNA isolated from the

hypothalamus of white leghorn hen 2 h before ()2 h), immediately after

egg laying (0 h), and 2 h after egg laying (+2 h). Each lane was loaded

with 20 lg total RNA. Autoradiograph showing (A) AVT (700 bp) and

(B) 18S (1.2 kb) gene expression. (C) % AVT mRNA levels in the

hypothalamus of white leghorn hen. Northern blots sequentially hy-

bridized to the AVT and 18S cDNAs were exposed to storage phos-

phor screens and densitometric bands corresponding to AVT and 18S

were calculated. The volume of AVT band was divided by the volume

of 18S band and multiplied by 100 to determine the amount of %

hypothalamic AVT mRNA. The data are presented as means�SEM

(n ¼ 5). **p < 0:01, ***p < 0:001, Significance of difference from 2h

before egg laying ()2 h).

3. Results

A highly significant increase in AVT transcript/geneexpression can be seen immediately after egg laying

(0 h), which had been low 2 h before ()2 h) and was

again low 2 h after egg laying (+2 h) (Fig. 1). In situ

hybridization of the brain section corresponding to PVN

region of hypothalamus, observed immediately after egg

laying, also showed a significant increase in the expres-

sion of AVT transcript compared to that of 2 h before

and 2 h after egg laying (Fig. 2). The amount of AVTmRNA, represented by silver grains, not only increased

in each neurons but many more neurons showed hy-

bridization signals at 0 h compared to )2 h of oviposi-

tion. When observed 2 h after oviposition, the amount

of AVT mRNA and the number of neurons having these

transcripts exhibited a decrease. Immunohistochemistry

on the brain sections corresponding to in situ hybrid-

ization in PVN region showed increased number of ir-AVT neurons and immunostaining at the time of egg

laying which was low 2 h before and 2 h after egg laying.

Distribution and localization of immunoreactive AVT

neurons was consistent with hybridization signals de-

termined by in situ hybridization (Figs. 2 and 3).

Plasma AVT increased significantly at the time of egg

laying (78.7� 12.4 pmol/L); however, it was low 2 h be-

fore (13.3� 1.6 pmol/L), and 2 h after (14.1� 2.9 pmol/L) (Table 2; Fig. 4). Plasma osmolality and other ionic

parameters like sodium, potassium, calcium, and chlo-

ride ion concentration did not show any difference in

relation to the time of oviposition (Table 2).

Semi-quantitative RT-PCR showed increased ex-

pression of the shell gland AVT gene, 2 h before egg

laying, followed by a decline. This low level was main-

tained until 2 h after the egg was laid (Fig. 5). RT- PCRalso showed an increase in AVT receptor (VT1) gene

expression in the shell gland immediately after egg lay-

ing, which remains low 2 h before and 2 h after egg

laying (Fig. 6).

Fig. 2. Photomicrographs of chicken brain section showing in situ hybridization (upper panel) and immunohistochemistry (lower panel) in the

paraventricular nuclei on either side of third ventricle. Arrows represent AVT mRNA (hybridization signals) over neurons of PVN region in

the upper panel and ir-AVT in the lower panel. In both the panels, photomicrographs Nos. 1 and 3 correspond to )2 h and +2h, respectively, and the

middle photomicrograph No. 2 represents 0 h in relation to oviposition. 3V¼ third ventricle. Scale bar is equivalent to 100lm.

Fig. 3. Number of neurons expressing ir-AVT in the PVN region of

hypothalamus in relation to egg laying in white leghorn hen. Data are

presented as means�SEM (n ¼ 5), ***p < 0:001, significance of dif-

ference from 2h before egg laying ()2 h).

182 R. Seth et al. / General and Comparative Endocrinology 137 (2004) 177–186

4. Discussion

The rationale of the present experiments was to study

temporal relation of central/hypothalamic (endocrine)

and peripheral/shell gland (paracrine) AVT gene ex-

pression in relation to the act/time of oviposition. AVT

gene expression was studied in chicken at different time

intervals, i.e., 2 h before ()2 h), immediately after (0 h),

and 2 h after (+2 h) egg laying. Results show low levels

of the hypothalamic AVT transcript (Northern blot

analysis and in situ hybridization) and ir-AVT in hy-

pothalamic neurons and in peripheral circulation at)2 h. These levels increased significantly at the time of

egg laying (0 h), but declined at +2 h. The increase in the

number of ir-AVT neurons and mRNA copies in mag-

nocellular neurons of paraventricular region following

oviposition, suggests a functional significance of AVT

during oviposition.

Interestingly, the amount of shell gland VT1 also fol-

lowed the same pattern, suggesting that an increased levelof shell gland AVT receptor at the time of oviposition

facilitated the sensitivity of avian uterus to AVT for its

oxytocic action during expulsion of the egg. An AVT

surge (78.7� 12.4 pmol/L) in the peripheral circulation at

the time of egg laying (0 h), strengthens oxytocic action of

AVT. This is independent and different from the increase

in plasma AVT following osmotic stimulation, as plasma

osmolality and ion contents did not change during ovi-position. AVT gene expression in shell gland decreased at

the time of egg laying. It is postulated that the synthesis as

well as secretion of hypothalamic AVT and an increase in

Table 2

Changes in plasma AVT, osmolality, and ionic concentrations in relation to egg laying in white leghorn hen

Parameters tested in plasma 2 h before egg lay ()2 h) Immediately after egg lay (0 h) 2 h after egg lay (+2 h)

AVT (pmol/L) 13.3� 1.6 78.7� 12.4��� 14.1� 2.9

Osmolality (mOsm/kg H2O) 293.8� 9.5 295.8� 6.9 301.0� 4.3

Sodium ion conc. (mmol/L) 152.3� 1.4 149.8� 0.5 149.1� 0.6

Potassium ion conc. (mmol/L) 3.77� 0.15 3.66� 0.19 3.40� 0.12

Calcium ion conc. (mmol/L) 0.73� 0.05 0.50� 0.05 0.55� 0.03

Chloride ion conc. (mmol/L) 114.8� 1.0 111.5� 1.2 113.8� 0.9

Values are means� SEM (n ¼ 5).*** p < 0:001; significance of difference from )2 h and +2h.

Fig. 4. Changes in plasma AVT concentration during a period of 10 h. Values are means� SEM. ***p < 0:001, significance of difference from 5h

before and after egg laying.

Fig. 5. (A) Duplex RT-PCR (semi-quantitative) reaction shown for the house keeping gene 18S (489 bp) and AVT gene (176 bp) in the shell gland. (B)

Histograms represent relative units of AVT transcript. *p < 0:05, ***p < 0:001, significance of difference from 2h before egg laying ()2 h).

R. Seth et al. / General and Comparative Endocrinology 137 (2004) 177–186 183

Fig. 6. (A) Duplex RT-PCR (semi-quantitative) reaction shown for the house keeping gene 18S (489 bp) and AVT receptor-VT1 gene (381 bp) in the

shell gland. (B) Histograms represent relative units of VT1 receptor transcript. ***p < 0:001, significance of difference from 2h before egg laying

()2 h).

184 R. Seth et al. / General and Comparative Endocrinology 137 (2004) 177–186

vasotocin receptors in shell gland, facilitate the contrac-

tion of smooth muscles. High AVT gene expression in the

shell gland at )2 h and low expression at 0 h, would sug-

gest paracrine role of AVT in the control of egg expulsion

from the shell gland. Though the present study describes a

correlation between the expression of central and pe-ripheral AVT gene with that of VT1 in the shell gland

during oviposition, but it does not suggest that AVT can

upregulate its own receptors.

The results support previously described changes of

the amount of plasma AVT concentration during egg

laying in chicken (Arad and Skadhauge, 1984; Nouwen

et al., 1984; Sasaki et al., 1998; Shimada et al., 1986).

Presence of mRNA encoding AVT in ovarian and uter-ine tissues have been reported following RT-PCR but

oviposition timings were not taken into consideration

(Chaturvedi et al., 1994a). Levels of vasotocin mRNA in

ovarian and uterine tissues vary from 1/50 to 1/1000

when compared with that of hypothalamus suggesting a

paracrine and/or autocrine role for AVT in these tissues

(Saito and Grossmann, 1999). An increase in plasma

AVT together with a concomitant decrease in neurohy-pophysial AVT coincides with oviposition (Koike et al.,

1988). AVT levels in ovarian vein plasma is lower than

plasma AVT levels in peripheral circulation suggesting

that neurohypophysial AVT plays a critical role in egg

laying (Sasaki et al., 1998). Further, AVT concentration

is much higher at the time of oviposition than those

observed in the studies using prolonged dehydration

(Chaturvedi et al., 1996), hypertonic saline loading(Chaturvedi et al., 1997), and haemorrhage (Jaccoby

et al., 1997). To date, the exact mechanism that regulates

the release of AVT from the neurohypophysis at the time

of oviposition remains unknown. It is suggested that

stretching of the shell gland due to the increased size of

the egg may lead to increased release/surge of AVT for

shell gland contraction through reflex arc, as it occurs

during parturition in mammals. Elevated plasma sodiumlevel and osmolality during osmotic stress also leads to

an increase in plasma AVT level. But in the present

study, high concentrations of plasma AVT is related to

oviposition, and is not the result of osmotic stimulation

since no changes in plasma sodium and osmolality were

observed during the course of the study.

In the case of mammals, neurohypophysial hormones

have diverse actions including inhibition of diuresis,stimulation of glycogenolysis, contraction of smooth

muscles, and modulation of ACTH release from ante-

rior pituitary gland (Sawyer, 1977). To perform these

diverse functions at least three different vasopressin and

one oxytocin receptor has been reported from mammals.

Out of the three vasopressin receptors, V1a (vascular/

hepatic receptor) and V1b (anterior pituitary receptor)

act through phosphatidyl inositol hydrolysis to mobilizeintracellular Ca2þ and V2 (kidney receptor) act via

adenylate cyclase pathway (Burbach et al., 1995; Van

Kesteren and Geraerts, 1998). Just before the onset of

labour, uterine myometrium becomes extremely sensi-

tive to oxytocin due to presence of a G protein-coupled

oxytocin receptor. VT1 is the physiological receptor that

mediates AVT induced contractile response of chicken

shell gland during oviposition and this has been shownusing shell gland membrane preparations and ribonu-

clease protection studies (Tan et al., 2000).

R. Seth et al. / General and Comparative Endocrinology 137 (2004) 177–186 185

It is also reported that AVT-initiated PG synthesis/release in the shell gland/uterus causes the contraction of

the smooth muscles responsible for egg laying/parturi-

tion (Cornett et al., 2001; Hertelendy et al., 1975; Saito

et al., 1987). Similarly, in mammals, oxytocin has been

shown to have an indirect effect on myometrial con-

tractility mediated by prostaglandin PGF2a (Fuchs et

al., 1981). Further, the signal transduction properties of

VT1 receptor coupling to the phosphatidyl inositol/cal-cium pathway are similar to that of V1a and V1b

vasopressin receptors in mammals (Burbach et al.,

1995). It is suggested that the increased gene expression

of shell gland AVT, 2 h prior to egg laying and the re-

sultant paracrine AVT secretion initiates PG secretion

from surrounding tissues. This mechanism is further

enhanced by AVT surge (endocrine secretion from hy-

pothalamus and posterior pituitary) and AVT receptordensity in shell gland at the time of egg laying. It would

appear that the initiation/preparation of shell gland

contraction is mediated through local AVT synthesis/

release (proximate factor), and the endocrine synthesis

and secretion plays a final role in the expulsion of egg.

Present findings indicate an interrelation of AVT gene

expression at the central (hypothalamus) and peripheral

(shell gland) level and the expression of VT1 receptorduring oviposition. Increase in the shell gland AVT gene

expression at )2 h was followed by increased hypotha-

lamic AVT and shell gland receptor gene transcript at

0 h. We further suggest that local synthesis of shell gland

AVT and an increase in the amount of shell gland re-

ceptor (VT1) transcript may be responsible for the in-

creased sensitivity of shell gland during contraction of

its smooth muscles. Hence, our study proposes a si-multaneous increase in the expression of AVT gene in

the hypothalamus and its receptor gene expression in the

shell gland during oviposition.

Acknowledgments

The research work was supported by fellowships

from DAAD (German Academic Exchange Services),

Center of Advanced Study (University Grants Com-

mission, New Delhi, India) and Council for Scientific

and Industrial Research (New Delhi, India) to R.S. and

USDA research grant FG-IN- (IN-ARS-866) and De-

partment of Science and Technology, India, research

grant (SP/SO/C-44/99) to C.M.C. We thank the staff ofour Departments and the Experimental Station of the

Institute for Animal Science for their skillful assistance.

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