Comparative Biochemistry and Physiology, Part A 158 (2011) 87–93

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Comparative Biochemistry and Physiology, Part A

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Testosterone modulates pituitary vasotocin receptor expression and adrenal activityin osmotically stressed chicken

Dharmendra Sharma, Chandra Mohini Chaturvedi ⁎Department of Zoology, Banaras Hindu University, Varanasi 22105, India

⁎ Corresponding author. Tel./fax: +91 542 2368323.E-mail address: cmcbhu@yahoo.com (C.M. Chaturve

1095-6433/$ – see front matter © 2010 Elsevier Inc. Aldoi:10.1016/j.cbpa.2010.09.008

a b s t r a c t

a r t i c l e i n f o

Article history:Received 6 August 2010Received in revised form 10 September 2010Accepted 12 September 2010Available online 17 September 2010

Keywords:Vasotocin receptorTestosteroneHPA axisPOMCAdrenalChicken

Regulation of arginine vasotocin (AVT), avian neurohypophyseal hormone, is an important component of thehypothalamo-pituitary–adrenal axis. Changes in plasma osmolality levels and sex steroids are known to affectAVT gene expression. The present study reports the effect of water deprivation and testosterone treatmentindependently, as well as simultaneously, on the pituitary vasotocin receptor VT2R expression and adrenalsteroidogenic activity in sexually immaturemale chicken (Gallus gallus). Birds were divided into four groups—control, water deprived (WD), testosterone injected (TE) and TE treated water deprived (TE+WD). WDdecreased and TE treatment alone or in combination with WD (TE+WD) increased VT2R expressioncompared to the control. Expression of pro-opiomelanocortin (POMC) was also studied since this gene is apolypeptide precursor of ACTH and is under the negative feedback of adrenal corticoids. TE treatment as wellas WD separately or when coupled together decreased the POMC mRNA expression in the pituitary butstimulated adrenal steroidogenic activity. Further, VT2R expression decreased in TE+WD compared to TEgroup, but it was not different from the vehicle treated control group suggesting that the suppressive effect ofWD on VT2R expression was inhibited by the stimulatory effect of testosterone. Similarly, although both TEand WD decreased POMC expression and increased steroidogenic activity, no further decrease or increase inthese 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 andadrenal function despite a decreased expression of POMC mRNA. Results also suggest that testosteronetreatment 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 inpituitary–adrenal activity.

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1. Introduction

The presence of a relationship between hypothalamic–pituitary–adrenal (HPA) and hypothalamic–pituitary–gonadal axis has beendocumented in mammalian literature. Stimulation of the HPA axisfollowing stress is known to affect reproductive function (McGrady,1984; Rabin et al., 1988; Rivier and Revest, 1991). Components of thegonadal axis have also been shown to modulate the functioning ofnormal and stress-induced HPA activity. Demonstrations of anincreased or decreased corticosterone secretion following gonadec-tomy imply an inhibitory and excitatory action of testosterone andestrogen respectively (Handa et al., 1994; Viau and Meany, 1996;Suzuki et al., 2001).

The HPA axis in birds is similar to the HPA axis in mammals andtwo neuropeptides; corticotrophic releasing hormone (CRF) andarginine vasotocin (AVT) are known to regulate adrenocorticotrophic

(ACTH) hormone synthesis and corticosterone production (Castroet al., 1986; Hazard et al., 2007). These hypothalamic peptides interactwith their corresponding membranous receptors localized on theanterior pituitary corticotrophs; CRF with CRF-R1, and AVT with VT2R(Yu et al., 1996; Cornett et al., 2003; Jurkevich et al., 2005). Studies onthe stress response in birds are mainly limited to seasonal changes inadrenocortical responses (Romero, 2002, 2006; Romero et al., 1998).An exception is the study by Madison et al. (2008) showing a genderrelated difference in plasma corticosterone release in response tointracerebroventricular injections of AVT and CRF. However, role ofgonadal steroids in the regulation of VT2R receptor and avian HPA axisfunction is not well understood.

Water deprivation up-regulates the synthesis and secretion of AVTfrom hypothalamic paraventricular (PVN) and supraoptic nuclei intothe neurohypophysis from where it is released into the peripheralcirculation as an antidiuretic hormone (Mühlbauer et al., 1992;Chaturvedi et al., 1994, 1996, 1997). AVT has been shown to beinvolved in various physiological functions. Its role in the regulation ofwater balance is well documented. However, its role in stressfulconditions is not well understood. AVT has been found to regulate

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an integrated stress response by simultaneously modulating thebehavioral, cardiovascular, and corticosterone responses during acutecrowding (Nephew et al., 2005).

Male gonadal steroids have been shown to stimulate hypothalamicAVT gene expression (Seth et al., 2004). Moreover, seasonality andreproductive state of the bird is also reported to affect the hypo-thalamic vasotocinergic system in parallel with adrenal activity inJapanese quail (Singh and Chaturvedi, 2006, 2008), suggesting a rolefor sex steroid hormones in the regulation of AVT function and adrenalactivity. Keeping in mind the dual role of water deprivation (as anosmotic stimulus and as a stressor), and the knowledge that gonadalsteroids affect hypothalamic AVT gene expression as well as HPAfunction, the experiment carried out in the present study aimed toreveal the effect of testosterone on pituitary VT2R expression and theadrenal function in sexually immature and osmotically stressedchickens.

2. Materials and methods

Day-old white leghorn chicks were procured from a local hatchery(Shreeji hatchery, Shivpur, Varanasi, India) and reared underlaboratory conditions in a 12L:12D photoregime. Chicks were pro-vided with a high protein diet: starter chicken ration up to the age ofsix weeks. Thereafter, young ones were provided with a broilerfinisher diet. Except where noted, all of the birds were provided withwater ad libitum. The experiment was performed in accordance withthe institutional practice andwithin the framework of revised animals(scientific procedures) Act of 2002 of Govt. of India on animal welfare.

Five to 6 week old (300–400 g) sexually immature birds weredivided into two groups (n=16, each). The first group of birds wasinjected with testosterone enanthate (TE, German Remedies, 1 mg/100 g body weight, diluted in olive oil; s.c.) daily for fourteen days,while the second group served as a control and treated with thevehicle (oil) only. After ten days of injections, eight birds from boththe groups were subjected to osmotic stress by four days of waterdeprivation (WD; TE+WD), while the remaining birds wereprovided with water ad libitum. On the fourteenth day birds wereweighed and four individuals from all the subgroups were sacrificedby decapitation. Subsequently, trunk blood was collected in heparin-ized tubes and centrifuged at 1000 g, plasma separated and storedat −20 °C for hormonal (testosterone) analysis and plasma osmolal-ity measurements (Freezing point Osmometer). Pituitaries wereimmediately dissected out, snap frozen in liquid nitrogen and keptat −80 °C until RNA isolation and RT-PCR analysis of VT2R and pro-opiomelanocortin (POMC) transcript was performed. Adrenal glandswere removed and stored for the biochemical analysis of cholesterolcontent (Rosenthal et al., 1960) and 3β-hydroxysteroid dehydroge-nase (3β-HSD) enzyme activity (Shivanandappa and Venkatesh,1997). The remaining four birds from all the subgroups wereanaesthetized by injection with sodium pentobarbital (25–30 mg/kgbody weight), and then perfused intracardially with 0.02 M PBScontaining 0.01% heparin, followed by Zamboni Fixative (4% PFA inPBS+15% picric acid). After whole body perfusion, pituitaries andadrenals were removed and post fixed in a fresh fixative (4 h forpituitary and 48 h for adrenal). After fixation, tissues were dehydratedin a graded series of alcohol, cleared in xylene and embedded inparaffin wax. 8 μm thick sections of the pituitary and 6 μm thicksections for the adrenal were cut with a rotary microtome, attached tothe gelatin-coated slides and processed for the histochemical studieson pituitary sections (in situ hybridization and immunohistochemis-try) and routine haematoxylin–eosin staining for the adrenal sections.

2.1. Semi-quantitative RT-PCR

Reverse transcriptase polymerase chain (RT-PCR) reactions wereused to analyze VT2R and POMC mRNA levels in the pituitary. Total

RNA was isolated from a pool of two pituitaries using TRI reagent(Sigma, USA) and the integrity of the RNA was verified by 1.5%denaturing agarose gel electrophoresis. To rule out the possibility thatPCR products would result from the amplification of genomic DNA,RNA samples were treated with DNA-free (Ambion, USA). Onemicrogram of total RNA was used and reverse transcription wasperformed using the first strand cDNA synthesis kit (Fermentas, USA)according to the manufacturer's protocol. For cDNA amplifications, 1/10 of the RT product was used and PCR was carried out using the 2×PCR Master Mix kit (Fermentas, USA) as described earlier (Sharmaet al., 2009). Primer sequences to amplify VT2R, POMC and GAPDH areas follows: VT2R forward, 5′-GCG AGA TCT GCA AGA ACC-3′, andreverse, 5′-GGA AGC AGT GAC TGA ATC-3′; POMC forward, 5′-GAGAGC ATC CGC AAG TAC GTG-3′, and reverse, 5′-CTG ATG ACT CTG TTCAAA ACG-3′; and GAPDH forward, 5′-AGT CAT CCC TGA GCT GAA TG-3′, and reverse, 5′-ACC ATC AAG TCC ACA ACA CG-3′. Followingamplification, PCR products were analyzed on 2% agarose gelcontaining ethidium bromide (0.5 g/mL). Stained bands were cap-tured digitally and densitometric analysis was conducted using imageanalysis software Alpha Imager (Alpha Innotech Corporation, USA).

2.2. In situ hybridization

In situ hybridization of VT2R mRNA was performed using adigoxigenin (DIG)-labeled 250 bp fragment of VT2R cDNA. The probesequences comprise the fragment from Exon 1 (703–953 bp) of theVT2R gene (GenBank Accession No. AY008272, Cornett et al., 2003).Hybridization was carried out in hybridization solution (50% DIformamide, 10% dextran sulfate, 1× Denhart's solution, 4× SSC,10 mM DTT, 1 mg/mL ssDNA) containing 300–400 ng/mL of labeledprobe at 42 °C for 16 h. Post hybridization washings and immuno-logical detection were performed as described earlier ( Srivastava etal., 2007). For the negative control, sections were incubated inhybridization buffer lacking the DIG-labeled probe.

2.3. Immunohistochemistry

After initial dewaxing in xylene and rehydration in graded series ofalcohol, slides were processed for immunoreactive (ir-) localization ofVT2R. Rabbit-VT2R antiserum was obtained as a kind gift from Dr. L.E.Cornett, University of Arkansas for Medical Sciences, Little Rock, AR.The specificity of the antibody used in the present study has beenconfirmed in an earlier study (Jurkevich et al., 2005). Pituitarysections were incubated in primary antisera for 24 h followed byincubation in horseradish peroxidase conjugated secondary antibody.Diaminobenzidine hydrochloride (DAB) was used as a chromogenmolecule for the immunological detection. For the negative control,primary antiserum was omitted, and sections were incubated innormal goat serum only.

2.4. Radioimmunoassay and plasma osmolality

Plasma levels of testosterone were determined with the help of acommercially available RIA kit (Immunotech, Marseille, France).Briefly, 50 μl of the plasma sample was added in an antibody coatedtube and assay was performed according to the instruction manual.All of the samples were processed in duplicates. Analytical sensitivityof the kit was 0.025 ng/mL.

Plasma osmolality was measured with Fiske Freezing point microsample osmometer.

2.5. Quantitative and cytometric analysis

For image analysis of in situ hybridization and immunoreactivesignals, a relative quantification method (Sakharkar et al., 2005) wasemployed. The image analysis system consisting of a Leitz LaborLux S

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microscope and CCD video camera (JVC, Japan) connected to acomputer was used. The microscope images were digitized andanalyzed using Leica Qwin standard software (version 3). Randomlyselected 5 square areas (21,389 μm2 each) in each sagittal section ofthe pituitary (5 sections from each pituitary and total 4 pituitaries)from the region of cephalic lobe were subjected to analysis.

For adrenal cytometry, 5 randomly selected sections from the midregion of the adrenal (4 adrenals per group) were chosen. The width of 5cortical cords as well as the area occupied by light colored steroidogeniccords (cortical) and dark patches of chromaffin tissue (medulla) fromeach section were measured to calculate cortico-medullary (C:M) ratio.

2.6. Statistical analysis

Data are presented as the mean±SEM of values obtained from thenumber of birds indicated in the figure legends. Statistical significanceof difference between control and experimental groups was deter-mined by one-way ANOVA followed by Student Newman KeulsMultiple Comparison test. A “p” value less than 0.05was considered asstatistically significant.

3. Results

3.1. Body weight, plasma osmolality and testosterone content

No significant differences either in the body weight or plasmaosmolality levels were observed after androgen administration

Fig. 1. Effect of four-day water deprivation (WD), testosterone enanthate (TE) and testoosmolality (B), testosterone levels (C) and adrenal activity (D,E,F) in chicken. Data are presegroup (*, Pb0.05). Double cross denotes a significant difference from the WD group (#, Pb

compared to the vehicle treated control animals. WD alone as wellas when coupled with TE (TE+WD group) resulted in a decrease inthe body weight and an increase in the plasma osmolality valuescompared to controls as well as TE treated birds (Fig. 1). Plasmatestosterone levels were almost negligible in sexually immature birds.However, daily administration of the androgen resulted in asignificant increase in testosterone level. When these androgentreated birds were water deprived (TE+WD), a decrease in theplasma testosterone values was noted (Fig. 1). Since in the presentstudy, we have administered TE to the animals, therefore the decreasein the level following WD may involve a dramatic change inmetabolism.

3.2. Assessment of the adrenal steroidogenic activity

In order to assess the adrenal function, we measured variousparameters like weight, changes in cholesterol content 3β-HSDenzyme levels, and histomorphological alterations in the adrenalgland. These methods have been earlier used as a significant indicatorof adrenocortical activity (Bhattacharyya and Ghosh, 1965; Siller et al.,1975; Sharma et al., 2009). Except TE+WD group, no significantchange was observed in the adrenal weight following water depri-vation or TE administration. On the other hand, a significant decreasein the adrenal cholesterol contents and an increase in the 3β-HSDenzyme levels were observed in WD, TE as well as in the TE+WDbirds in comparison to controls indicating increased adrenal steroido-genic activity in all the three groups. However, when compared with

sterone coupled with water deprivation (TE+WD) on the body weight (A), plasmanted as mean±SEM (n=4). Asterisk denotes a significant difference from the control0.05). Lower case letter denotes a significant difference from the TE group (a, Pb0.05).

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TE treated group, osmotic stress did not change these values signifi-cantly in TE+WD birds (Fig. 1D,E,F).

In stained sections of the adrenal, interrenal tissues appear lighter(pinkish), typically composed of a double row of columnar or cuboidalcells with their long axes in the transverse plane of the strandwhereaschromaffin tissues are distinguished by dark (blue) color patches(Fig. 2). To support the biochemical parameters, histomorphologicalchanges (cortical cord width as well as C:M ratio) in the adrenal glandwere also noted (Fig. 2). Cytometric analysis revealed a stimulatedappearance of adrenal having broader cortical cords and increasedC:M ratio in WD, TE and TE+WD birds as compared to saline treatedcontrols. However, compared to TE groups these values were notdifferent in TE+WD group (Fig. 2).

3.3. RT-PCR analysis of the VT2R and POMC transcripts

To study the effect of WD and TE on the VT2R and POMC mRNAexpression, RT-PCR was performed and products were resolved onagarose gel. Densitometric analysis of the PCR amplified products,after normalization with the values of housekeeping gene GAPDHshows a decrease in VT2R expression following water deprivation incomparison to the vehicle treated control birds. However, a significantincrease in VT2R expression was observed following testosteronetreated birds as compared to saline treated controls as well as WDbirds (Fig. 3). Although osmotic stress resulted in a decrease in VT2Rtranscript levels in TE+WD birds in comparison to TE group, thevalues were not different from the saline treated control birds (Fig. 3).On the other hand, expression of POMC mRNA decreased in all thethree groups as compared to the control. However, in comparison toTE treated group, the values were not significantly different in TE+WD group (Fig. 3).

3.4. In situ hybridization and immunohistochemistry of VT2R in pituitary

To further assess the expression of VT2R, we performed the in situhybridization and immunohistochemical localization in pituitarysections. When stained slides were viewed under the microscope,

Fig. 2. Transverse sections of the adrenal gland from four-day water deprived (WD), testoHistograms represent changes in the cortical cord width and corticomedullary (C:M) ratio. Dadrenal from each bird. Asterisk denotes a significant difference from the control group (*,

VT2R mRNA as well as VT2R immunoreactivity was presentpredominantly in the cephalic lobe of the anterior pituitary (Fig. 4Aand C). Results of the quantitative analysis of the positive signals weresimilar to that of the RT-PCR, showing a decrease in positive signalarea following water deprivation; however, the same was signifi-cantly increased in androgen treated birds compared to control aswell as WD (Fig. 4B and D). In comparison to the TE group, VT2Rexpression decreased significantly in TE+WD birds (Fig. 4B and D).

4. Discussion

Disturbances in the functioning of HPA axis following gonadecto-my or sex steroid replacements have been studied in mammals. Malesand females differ in their response to stress and this sex difference inHPA function is principally due to the influence of sex steroids (Sealeet al., 2004). However, no such reports are available in birds whereseveral environmental factors affect the onset of reproductiveactivities. The present study discusses the effects of testosterone onthe expression of arginine vasotocin VT2R receptor and adrenalfunction of sexually immature chicken following osmotic stimulationby water deprivation. Results indicate that osmotic stimulation andtestosterone treatment increase the pituitary VT2R expression as wellas adrenal activity despite a decrease in the expression of POMCtranscript levels.

Availability of day light plays an important cue in timing the avianreproduction; long days are known as the photostimulatory whereasshort days are considered as gonadoinhibitory. Different studies fromour laboratory indicate that high plasma testosterone levels eitherduring breeding season or following long day treatment run parallelwith increased adrenal activity (Singh and Chaturvedi, 2006, 2008).Results of the present study together with the earlier reports showexistence of a parallel relationship between gonadal and adrenalcycle. A correlation between testosterone and corticosterone releaseas has been observed earlier during breeding season further supportsit (Chaturvedi and Thapaliyal, 1980; Ketterson et al., 1991; Schoechet al., 1999).

sterone enanthate treated (TE) and testosterone/water deprived (TE+WD) chicken.ata are presented asmean±SEM (n=4) from five randomly selected mid regions of thePb0.05).

Fig. 3. RT-PCR analysis of VT2R, POMC and GAPDH in four-day water deprived (WD), testosterone enanthate treated (TE) and testosterone/water deprived (TE+WD) chicken. Theleft panel shows the representative agarose gel. The right panel shows the relative densitometric values of amplified products of VT2R and POMC, normalized with the values ofGAPDH. Data are presented as mean±SEM (n=4). Asterisk denotes a significant difference from the control group (*, Pb0.05). Double cross denotes a significant difference fromthe WD group (#, Pb0.05). Lower case letter denotes a significant difference from the TE group (a, Pb0.05).

Fig. 4. (A) In situ hybridization, and (C) Immunohistochemical localization of VT2R in the pituitary gland of four-day water deprived (WD), testosterone enanthate treated (TE) andtestosterone/water deprived (TE+WD) chicken. Histograms (B and D) represent relative quantitative analysis of the ir-VT2R and hybridization signals. Data are presented as mean±SEM (n=4). Asterisk denotes a significant difference from the control group (*, Pb0.05). Double cross denotes a significant difference from theWD group (#, Pb0.05). Lower caseletter denotes a significant difference from the TE group (a, Pb0.05).

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Besides acting as an antidiuretic hormone, avian neurohypophy-seal hormone AVT is also known as an important component of theHPA axis, regulating corticotroph activity via VT2R receptors (Cornettet al., 2003; Sharma et al., 2009). Earlier reports from our laboratoryshow an up-regulation of hypothalamic AVT expression in birdsfollowing testosterone treatment (Seth et al., 2004). Not only that, aparallel relationship has been observed between AVT expression andadrenal function during different seasons as well as during differentphases of the reproductive cycle (Singh and Chaturvedi, 2006, 2008).In view of these earlier studies and an increase in VT2R expressionfollowing testosterone treatment in the present study, it may beassumed that in sexually immature chicken, increased synthesis ofhypothalamic AVT following androgen treatment may stimulateadrenal activity by interacting with VT2 receptor on pituitarycorticotrophs. However, despite an increase in VT2R expression andadrenal activity, POMC mRNA levels decrease in sexually immaturebirds. It would have been better to have plasma ACTH levels as mRNAexpression does not necessarily coincide with the protein expression.There may be unaltered plasma ACTH levels despite a decreasedPOMC mRNA expression. Furthermore, apart from the central ma-chinery (hypothalamic–pituitary)we cannot rule out a direct action ofthe testosterone at various enzymatic pathways leading to the adrenalsteroidogenesis. However, birds adapt various strategies to regulatetheir corticosterone production during different seasons and more-over, present study lacks the data on the CRF expression, we areunable to explain the exact mechanism responsible for adrenalstimulation.

Hyperosmolality following water deprivation not only up-regulates the AVT gene expression in existing magnocellular neu-rons but also recruits many more neurons to increase the synthesisand release of AVT (Chaturvedi et al., 1994, 1997) into the neuro-hypophysis. Besides acting as an osmotic stimulus, water depriva-tion acts as stress that modulates corticotroph function. Similar toour earlier studies involving normal uninjected birds (Sharma et al.,2009), results of the current study revealed that osmotic stressdecreased the expression of pituitary VT2R and POMC while itincreased adrenal steroidogenic activity in sexually immature salinetreated birds also. This apparent dissociation between stimulation ofadrenal function despite of decreased POMC levels may involveincreased sensitization of the adrenal to ACTH. However, it may alsobe due to an initial but transient increase in the plasma ACTH fol-lowing water deprivation that resulted in the stimulation of adrenalcorticoids. In rats, an increase in the plasma ACTH level has beenobserved following twelve hours of water deprivation which returnsto the basal level after 48 h (Aguilera et al., 1993). Further, results ofthe present study reveal that although, VT2R levels in TE treatedosmotically stressed birds were lower compared to TE treated birds,the values were not different from vehicle treated control birds. Thisdecrease in VT2R expression in TE+WD birds probably reflects amuch higher increase in the synthesis/secretion of AVT into themedian eminence. It has been observed that AVT expression wasremarkably augmented when the two conditions (water deprivationand testosterone administration) were applied simultaneously (Sethet al., 2004). Irrespective to the changes in AVT/VT2R system, POMCmRNA level and adrenal gland activity do not show furtherstimulation in TE+WD birds compared to TE birds. This unrespon-siveness of adrenal to osmotic stress following testosterone ad-ministration suggests that during breeding season (high plasmatestosterone level) birds can better cope up with osmotic stress. Ourstudy further suggests that increased synthesis and secretion ofhypothalamic AVT following osmotic stress and androgen treat-ment may act differentially with its pituitary receptor. Exactmechanism underlying increased adrenal activity following andro-gen treatment as well as water deprivation in spite of decreasedPOMC level cannot be explained at present. Further studies involvingcombined role of AVT and CRF are required to understand the

detailed pathway that regulates HPA axis during different seasons/breeding conditions in bird.

Acknowledgements

This work was supported in part by a research grant (F. No. 32-472/2006 (SR)) to CMC, from the University Grant Commission, NewDelhi. We also thank the Council of Scientific and Industrial Research,for providing financial support in the form of Junior and SeniorResearch fellowships to DS. The authors wish to thank Dr. L.E. Cornett(Department of Physiology and Biophysics, University of Arkansasfor Medical Sciences, Little Rock, AR, USA) for the gift of VT2R cDNA,and to Dr. N.K. Subhedar (Department of Pharmaceutical Sciences,University of Nagpur, Nagpur, India) for providing the facility of imageanalysis.

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