Expression of hypothalamic arginine vasotocin gene in response to water deprivation and sex steroid administration in female Japanese quail
1 Department of Zoology, Banaras Hindu University, Varanasi - 221 005 (UP),
India
2 Department Functional Genomics and Bioregulation, Institute for Animal
Science (FAL), Mariensee - 31535, Neustadt am Rübenberg,
Germany
* Author for correspondence (e-mail: cmcbhu{at}indiatimes.com)
Accepted 20 May 2004
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Summary |
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Key words: arginine vasotocin gene, sex steroid, Japanese quail, Coturnix coturnix japonica, dehydration, paraventricular nuclei
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Introduction |
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Water deprivation causes an increase in hypothalamic AVT mRNA by increasing
the amount of transcript per neuron and by recruitment of clusters of
magnocellular AVT neuron that are not identifiable in the basal condition
(Chaturvedi et al., 1994). In
chickens, a similar effect was observed, by in situ hybridization,
following saline drinking, hypertonic saline administration or hemorrhage
(Chaturvedi et al., 1997
;
Jaccoby et al.,
1997
). In view of the effect of osmotic stress and sex steroids on
the AVT system, the present experiment was undertaken to study the
simultaneous effects of water deprivation and sex steroid administration on
the expression of the hypothalamic AVT gene and on the magnocellular
neurons of paraventricular nuclei (PVN) synthesizing AVT.
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Materials and methods |
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Northern blot analysis
Brains were quickly removed, and hypothalami were isolated, snap frozen in
dry ice and stored at -70°C until used for northern blot analysis. Total
RNA was isolated using Trizol reagent (Invitrogen, Karlsruhe, Germany)
according to the method described by Chomczynski and Sacchi
(1987). 20 µg of total RNA
was separated on 1.4% w/v agarose denaturing formaldehyde gel in
morpholino-propane sulfonic acid (MOPS) buffer, pH 8.0, and subsequently
blotted overnight by capillary transfer onto nylon membranes (Hybond
N+; Amersham, Braunschweig, Germany), followed by UV crosslinking
(150 mJ; gene linker, Bio-Rad, München, Germany). The AVT-specific probe
(a 260 bp cDNA directed towards the distal 3' glycopeptide part of the
chicken AVT gene; Hamann et al.,
1992
) was labeled with [32P]dCTP by the random priming
method (megaprime DNA labeling system; Amersham) according to Feinberg and
Vogelstein (1983
) and
separated from unincorporated nucleotides by using Sephadex G50 columns (Nick
columns; Pharmacia, Freiburg, Germany). Hybridization proceeded overnight at
42°C in 50% formaldehyde-containing buffer according to Wahl et al.
(1979
). Approximately
5x106 c.p.m. were used per filter in 6 ml of hybridization
buffer without dextran sulfate. Exposure time was 48 h at -70°C using one
intensifying screen. After stripping of the AVT cDNA, the filter was
rehybridized with the housekeeping gene GAPDH to show the loading of
RNA samples in each lane. Autoradiographs showing AVT (700 bp) and
GAPDH (1.4 kb) gene expression were sequentially exposed to storage
phosphor screens (Bio-Rad), and densitometric bands corresponding to AVT and
GAPDH were calculated. The volume of the AVT band was divided by the volume of
the GAPDH band and multiplied by 100 to determine the % hypothalamic AVT
mRNA.
Immunohistochemistry (IHC)
Quail were anaesthetized using pentobarbital sodium (3-4 mg per 100 g body
mass), and a blood sample (1.5 ml) was obtained from the wing vein in a
heparinized syringe. Whole-body perfusion was done through the heart using
0.02 mol l-1 phosphate-buffered saline (PBS) and Zamboni fixative
(Stefanini et al., 1967
) by
perfusion pump at a speed of 2-3 ml min-1. Fixed brain tissue was
processed for cryostat sectioning and subsequent IHC. For cryoprotection,
brains were transferred to 25% sucrose solution in PBS at 4°C until the
brain sank to the bottom (
24 h). Brains were frozen using tissue-freezing
medium and cut at 18 µm in a cryostat. For IHC, sections were washed
several times in 0.02 mol l-1 PBS until fixative was washed out
completely, treated with 0.6% hydrogen peroxide (Sigma) in PBS for 30 min,
incubated in 5% normal goat serum (DAKO, Hamburg, Germany) containing 0.2%
Triton X-100 for 30 min, followed by incubation for 36 h at 4°C in a
1:5000 solution of rabbit anti-AVT serum in PBS containing 0.2% Triton X-100,
1% normal goat serum and 0.1% sodium azide. The AVT antiserum was kindly
provided by Dr D. Gray (Max Planck Institute for Physiological and Clinical
Research, Bad Nauheim, Germany). Sections were thoroughly washed and incubated
in goat anti-rabbit biotinylated IgG (DAKO; 1:500 in PBS containing 0.2%
Triton X-100) for 90 min at room temperature, rinsed in PBS (four times for 15
min each) and incubated for 90 min with avidin biotin peroxidase conjugate
(ABC-HRP; DAKO; 1:1000) solution in PBS containing 0.2% Triton X-100 and 1%
crystalline bovine serum albumen (Sigma). For immunodetection, 3,3'
diaminobenzidene (DAB; Sigma) in 0.05 mol l-1 Tris buffer (pH 7.6)
with 0.005% hydrogen peroxide was used. Sections were subsequently rinsed in
PBS and distilled water, air-dried and coverslipped using entellan (Merck,
Darmstadt, Germany). To control the specificity of the immune reaction,
sections were incubated with 5% normal goat serum instead of AVT antiserum. In
control sections, no immune-positive signal was detected
(Fig. 7).
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Morphometric measurements
Brain sections containing ir-AVT neurons in the PVN were selected from each
experimental and control group of quail. Immunostained cells/neurons were
counted manually under an ordinary light microscope (Weswox Optik model TR Hl
66; Ambala Cantt, India). The nomenclature of the brain structures and
stereotaxic planes of the sections were adjusted in reference to the chicken
brain atlas of Kuenzel and Masson
(1988). Area/size of these
neurons was measured by occulomicrometer (length x width). Student's
t-tests were used to assess comparisons between group means.
In situ hybridization (ISH)
Fixed brain tissue was processed for cryostat sectioning and subsequent
ISH. Brain sections were washed in PBS, dehydrated in a graded ethanol series
and air-dried. The probe was labeled by the random priming method as described
for northern analysis but using [33P]dCTP. The probe was diluted
with hybridization buffer [50% formamide, 5x Denhardt's solution, 10%
dextran sulfate, 0.75 mol l-1 NaCl, 25 mmol l-1 PIPES,
25 mmol l-1 EDTA, 0.2% (w/v) sodium dodecyl sulfate (SDS) and 250
µg ml-1 herring sperm DNA] to give final counts of 2500
c.p.m. µl-1. 40 µl of AVT probe was applied to each section,
and hybridization was carried out for
16 h at 52°C in a moist
chamber. Washing was carried out in 4x sodium chloride-sodium citrate
buffer (SSC) for 3x10 min and 2x SSC for 3x10 min at room
temperature. Final washing was done in 70% ethanol and then sections were
dried under vacuum for 2 h. Sections were covered with photographic emulsion
(LM-1; Amersham) diluted 1:1 with distilled water. After exposure for 7-10
days at 4°C, slides with coated emulsion were developed using Ilford
Phenisol, lightly counterstained with toluidine blue, and after air drying
were coverslipped with Entellan (Merck). Sections were viewed with a Nikon
Epiphot microscope equipped with a dark-field condenser. Hybridization
signals, in the form of silver grains observed over the neurons under dark
field, represent AVT mRNA. To check the specificity of hybridization signals,
subsequent controls were treated with RNase A (50 µg ml-1;
Boehringer, Mannheim, Germany; 0.5 mol l-1 NaCl, 0.01 mol
l-1 Tris-HCl, pH 7.5, 1 mmol l-1 EDTA) for 10 min at
37°C before the prehybridization (Fig.
7).
Plasma analysis
Plasma samples were extracted and processed for radioimmunoassay (RIA) for
ir-AVT by the method of Gray and Simon
(1983). AVT was extracted from
plasma with two volumes of acetone and two volumes of petroleum ether. The
extract was dried under vacuum in a speed vac concentrator (Savant Instruments
Inc., New York, USA). The dried extract was dissolved in assay buffer (0.1 mol
l-1 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 standard (Sigma). Plasma osmolality was measured by vapor pressure osmometry
(Wescor, model 5500; Logan, UT, USA). Plasma levels of sodium and potassium
were analyzed using a sodium/potassium analyzer (Ciba Corning Diagnostics,
Sudbury, UK).
Statistics
For statistical analysis of data, analysis of variance (ANOVA) followed by
Newman-Keul's multiple range test was applied. For estimating northern blot
data, Student's t-test was employed to compare between control and
experimental groups. Student's t-test was also employed to make
individual comparisons between sex steroid and sex steroid coupled with water
deprivation groups.
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Results |
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Northern blot analysis
An AVT mRNA transcript corresponding to 700 bp was detected in the
hypothalamus of quail. After normalization with the housekeeping gene
GAPDH, % increase in AVT gene expression was calculated.
Levels were lower in controls when compared with water-deprived and
steroid-treated animals. Although increased hypothalamic AVT transcript was
detected in all the treated groups, it was highly significant
(P<0.001) in animals treated with sex steroids coupled with water
deprivation (Fig. 3).
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In situ hybridization
Following in situ hybridization studies under dark field, silver
grains representing steady-state levels of AVT mRNA/hybridization signals were
localized over neurons that are immediately lateral to the third ventricle
(3V). Compared with controls, an increase in the intensity and density of
silver grains over AVT neurons can be visualized (not quantified) in the PVN
region of steroid-treated animals. But, a highly remarkable increase in the
density of silver grains representing AVT transcript is apparent in
water-deprived quail and in quail subjected to sex steroid administration and
water deprivation simultaneously (Fig.
4).
|
Immunohistochemistry
A significant increase in the number and area of ir-AVT neurons as well as
in the intensity of immunostaining was seen in the PVN of WD, EB, EB+WD and
TP+WD quail compared with controls (Fig.
5). Although TP-treated quail appeared to have more ir-AVT neurons
compared with the control (Fig.
6), the difference was not statistically significant
(Fig. 5).
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Discussion |
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Physiologically, steroid hormones produce three different effect periods.
Either these hormones produce long-term effects (for many days or weeks or for
life), short-term effects (for a lesser time) or rapid effects, which are also
called immediate effects (for seconds or minutes). Sex steroids can act at the
genetic level by lowering or overexpressing genes and can alter the sequence
of biochemical events. The sex steroids can act on neuronal cells in several
ways, causing an increase or change in cell body size or shape alteration,
changes in nuclear size, changes in neuronal enzyme content, neuropeptide and
neurotransmitter production, growth of dendrite processes, modification and
alteration of efferent and afferent circuits, etc
(Panzica et al., 1997). The
variation in cell number generally occurs on the basis of three different
mechanisms: (1) the steroid hormone stimulates neurogenesis in one sex, (2) it
could regulate processes influencing the differentiation of neurons or (3)
could prevent neuronal death (Arnold and
Schlinger, 1993
). Gonadal steroid implants are reported to restore
the behavioral effect of AVT injection. One hypothesis also suggests that
gonadal steroids affect the AVT target neuron by altering AVT receptor
concentration or binding affinity (Moore,
1992
).
The present study suggests a significant upregulation of the AVT gene in the magnocellular neurons of PVN of Japanese quail following 2 days of water deprivation. Although sex steroid administration over a period of 16 days also induced some increases in number of ir-AVT neurons and an increase in the steady-state level of AVT mRNA transcript in these neurons as well as in total hypothalamic RNA, the effect was remarkably augmented when the two conditions (water deprivation and sex steroid administration) were applied simultaneously. Increased localization of the steady-state level of AVT mRNA transcript and ir-AVT in the neurons of PVN and a simultaneous increase in the concentration of plasma AVT following water deprivation suggest that the osmotic stress stimulates transcription of the AVT gene in hypothalamic neurons and causes release of AVT in the peripheral circulation, thus affecting synthesis (transcription and translation) in the hypothalamus as well as secretion of AVT from neurohypophysis. Furthermore, since the number of neurons expressing ir-AVT in the TP-treated group was not statistically different from the control group, but the amount of AVT transcript in the hypothalamus and the intensity of hybridization signals (not quantified) in individual neurons was higher in these quail, it appears that testosterone treatment upregulates AVT gene expression (transcription only) in the existing neurons. However, estradiol administration induced significant increase in the number of neurons expressing ir-AVT in the PVN region, suggesting a differential response of sex steroids on the AVT system in birds.
Comparing the two effects, i.e. water deprivation and TP administration, on AVT neurons, it is obvious that, although both conditions upregulate AVT gene expression in the hypothalamus (northern analysis) and also in existing (basal) numbers of ir-AVT neurons of PVN, an additional number of neurons (which were dormant in basal condition) is recruited for AVT gene expression in only the osmotically stimulated condition (but not in TP-treated quail). Another point of difference is that, unlike in WD quail, localization of ir-AVT/intensity of immunostaining and plasma AVT concentration was not increased in the TP-treated quail. Taken together, these observations suggest that TP upregulates the transcription of AVT in PVN neurons (as well as in other vasotocinergic regions), an effect that appears to be mediated through testosterone receptors present on these AVT neurons, but possibly has no role at the translational level. On the other hand, the water-deprivation-induced effect on the AVT system (at both a transcriptional and translational level) is due to/mediated through an increase in plasma osmolality (not observed in the TP-treated group). A direct relationship exists between increase in osmolality and AVT secretion following upregulation of AVT gene expression. On the other hand, when comparing estradiol-treated and control group birds, a highly significant increase in AVT gene expression as well as in the number of ir-AVT neurons and intensity of immunostaining is seen in the same region. These findings further suggest the differential role of sex steroid/breeding status in upregulating transcription of the AVT gene in the hypothalamus/PVN neurons and hence appears to show a stimulatory role on the activity of the hypothalamo-neurohypophyseal axis (AVT system) of birds in response to osmotic stress.
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Acknowledgments |
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