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2009 Poultry Science 88 :2549–2554doi: 10.3382/ps.2009-00404

Key words: spleen , thymus , bursa of Fabricius , cold stress , peroxisome proliferator-activated receptor-γ

ABSTRACT This study was to investigate the ex-pression trait of the peroxisome proliferator-activated receptor-γ (PPAR-γ) gene and the effect of cold stress on the mRNA levels of PPAR-γ in spleen, thymus, and bursa of Fabricius of chickens. Eighty-four 1-d-old male chickens were randomly allocated to 12 groups (7 chick-ens per group). There was 1 control group and 5 treat-ment groups for acute cold stress and 3 control groups and 3 treatment groups for chronic cold stress. Chick-ens were maintained in our animal facility, kept under a 16L:8D cycle and temperature (30 ± 2°C), and given free access to standard chow and water. The cold stress was initiated when the birds were 15 d of age, with the duration of the acute cold stress being 1, 3, 6, 12, and 24 h, and the chronic cold stress was 5, 10, and 20 d, respectively. Cold stress temperature was 12 ± 1°C. Spleen, thymus, and bursa of Fabricius were collected for the assessment of the mRNA levels by real-time PCR after stress termination. The results showed that

the PPAR-γ gene is expressed in spleen, thymus, and bursa of Fabricius, and its expression level is different in different tissues and at different ages. Acute cold stress significantly decreased (P < 0.05) the mRNA levels of the PPAR-γ gene of spleen and thymus in all treatment groups and significantly increased (P < 0.05) the mRNA levels of the PPAR-γ gene of bursa of Fab-ricius in all treatment groups. Compared with the cor-responding control groups, chronic cold stress resulted in a significant increase (P < 0.05) of the mRNA levels of the PPAR-γ gene in spleen and a significant decrease (P < 0.05) of the mRNA levels of the PPAR-γ gene in thymus and bursa of Fabricius. The results indicate that the PPAR-γ gene is expressed in all 3 immune or-gans and has different expression traits. The magnitude and direction of change in PPAR-γ gene expression dif-fers with the type of cold stress applied and also varies by tissue.

Effects of cold stress on the messenger ribonucleic acid levels of peroxisome proliferator-activated receptor-γ in spleen, thymus,

and bursa of Fabricius of chickens

J. T. Wang ,*† S. Li ,* J. L. Li ,* J. W. Zhang ,† and S. W. Xu *1

* College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, P. R. China; and † Institute of Animal Science and Veterinary Medicine, Hei Longjiang Academy of Land-Reclamation Sciences,

Harbin 150038, P. R. China

INTRODUCTION Animals will face various kinds of environmental stres-

sors every day, and cold stress, as one of the stressors, commonly exists in the cold region. Researchers have demonstrated that cold stress dramatically affected the health and welfare of animals in the cold region (Yang and Li, 1999; Li et al., 2006). Moreover, studies also proved that cold stress could significantly influence the immune system in mice, human, and chicken (Janský et al., 1996; Banerjee et al., 1999; Brenner et al., 1999; Cichoń et al., 2002; Hangalapura et al., 2004, 2006).

Peroxisome proliferator-activated receptors (PPAR), composed of 3 proteins: PPAR-α, PPAR-δ (also known as PPAR-β), and PPAR-γ, are members of the nuclear receptor superfamily of the ligand-activated transcrip-tional factors, and the 3 subtypes have different tissue distributions and seem to have distinct but overlapping biological functions (Berger and Moller, 2002; Castrillo and Tontonoz, 2004). In mammals, PPAR-γ is promi-nently expressed in brown and white adipose tissue, the colon, differentiated myeloid cells, and the placenta and has a crucial role in controlling adipocyte differentia-tion (Tontonoz et al., 1994; Barak et al., 1999; Will-son et al., 2001), but other studies also indicated that PPAR-γ had a dramatic effect on the immune system, such as affecting the phagocytosis, cytokine production, antigen presentation, and the monocyte phenotypic dif-ferentiation (Clark et al., 2000; Faveeuw et al., 2000; Szatmari et al., 2004; Bouhlel et al., 2007). Therefore,

Received August 16, 2009. Accepted September 11, 2009. 1 Corresponding author: xushiwen101@sohu.com

© 2009 Poultry Science Association Inc.

PPAR-γ signals are important factors in the homeosta-sis of lipid metabolism and immune responses.

Although some studies have been done on the dis-tribution and function of the PPAR-γ gene of chickens (Sato et al., 2004; Meng et al., 2005a; Hojo et al., 2006; Wang et al., 2008), it was very little compared with that in mammals. Moreover, these studies were not involved in the immune organs. Therefore, there were 2 aims in this present study. The first goal was to investigate the distribution trait of the PPAR-γ gene in the im-mune organs (spleen, thymus, and bursa of Fabricius) of chickens, and the second was to evaluate the effect of cold stress on the mRNA levels of the PPAR-γ gene in spleen, thymus, and bursa of Fabricius of chickens.

MATERIALS AND METHODS

Birds and Tissue Collection

Eighty-four 1-d-old male chickens were purchased from Weiwei Co. Ltd. (Harbin, China) and were ran-domly allocated to 12 groups (7 chickens in each group). There was 1 control group and 5 treatment groups for the acute cold stress and 3 control groups and 3 treat-ment groups for the chronic cold stress. Therefore, there were 4 control groups and 8 treatment groups in all. The chickens were maintained in our animal facil-ity, kept under a 16L:8D cycle and temperature (30 ± 2°C), and given free access to standard chow and water. The cold stress was initiated when the birds were 15 d of age, with the duration of the acute cold stress being 1, 3, 6, 12, and 24 h, and the chronic cold stress was 5, 10, and 20 d, respectively. The cold stress temperature was 12 ± 1°C. The chickens were killed by decapitation after the corresponding duration of cold stress, and the killing time was 16 d of age (1-h treatment group, 3-h treatment group, 6-h treatment group, 12-h treatment group, 0-h control group, 24-h treatment group), 20 d of age (5-d treatment group and 5-d control group), 25 d of age (10-d treatment group and 10-d control group), and 35 d of age (20-d treatment group and 20-d control group), respectively. Five chickens were killed in each group, and the spleen, thymus, and bursa of Fabricius from each bird were collected, immediately frozen on dry ice, and then stored at −70°C for RNA isolation.

Primer DesignTo design primers, we used the chicken PPAR-γ

mRNA GenBank sequence with an accession number of AF470456. Chicken glyceraldehyde phosphate dehydro-genase (GAPDH, GenBank accession number K01458) as a housekeeping gene was used as an internal refer-ence. Primers (Table 1) were designed using the Oligo 6.0 Software (Molecular Biology Insights, Cascade, CO) and were synthesized by Invitrogen Biotechnology Co. Ltd. in Shanghai, China.

Total RNA Isolation and Reverse Transcription

Total RNA was isolated from each of the organs of each bird using Trizol reagent according to the instruc-tions of the manufacturer (Invitrogen). Reverse tran-scription reaction (40 μL) consisted of the following: 10 μg of total RNA, 1 μL of Moloney murine leukemia virus reverse transcriptase, 1 μL of RNase inhibitor, 4 μL of deoxynucleoside triphosphate, 2 μL of Oligo dT, 4 μL of dithiothreitol, and 8 μL of 5× reverse tran-scriptase buffer. The procedure of the reverse transcrip-tion was according to the instructions of the manufac-turer (Invitrogen).

Real-Time Quantitative Reverse Transcription PCR

Real-time quantitative reverse transcription PCR was used to detect the expression of the PPAR-γ gene in different tissues by using SYBR Premix Ex Taq (Ta-kara, Shiga, Japan), and real-time PCR work was done on each of the organs. Reaction mixtures were incubat-ed in the ABI PRISM 7500 real-time PCR system (Ap-plied Biosystems, Foster City, CA). The program was 1 cycle at 95°C for 30 s and 40 cycles at 95°C for 5 s and at 60°C for 34 s. Dissociation curves were analyzed by Dissociation Curve 1.0 Software (Applied Biosystems) for each PCR reaction to detect and eliminate the pos-sible primer-dimer and nonspecific amplification. Re-sults (fold changes) were expressed as 2−ΔΔCt in which

ΔΔCt = (CtPPAR-γ − CtGAPDH)t

− (CtPPAR-γ − CtGAPDH)c,

Table 1. Gene-specific primers for glyceraldehyde phosphate dehydrogenase (GAPDH) and peroxi-some proliferator-activated receptor-γ (PPAR-γ) used in the real-time quantitative reverse transcrip-tion PCR

Gene Accession

number1 Primer (5′→3′) Product size, bp

GAPDH K01458 Forward: AGA ACA TCA TCC CAG CGT 182Reverse: AGC CTT CAC TAC CCT CTT G

PPAR-γ AF470456 Forward: TGG AGG CAG TGC AGG AGA TTA C 100Reverse: TGG ACA CCA TAT TTC AGG AGG G

1GenBank accession number for sequence from which primers were designed.

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where CtPPAR-γ and CtGAPDH are the cycle thresholds for chicken PPAR-γ and GAPDH genes in the different treated groups, respectively, t is the treatment group, and c is the control group.

Statistical AnalysisStatistical analysis of all data was performed using

SAS procedures (SAS Institute Inc., Cary, NC). The effect of cold stress on mRNA levels in chickens was as-sessed by 1-way ANOVA. All values were expressed as means ± SD, and P < 0.05 was considered a significant difference.

RESULTS

The Distribution of PPAR-γ in Spleen, Thymus, and Bursa of Fabricius of Chickens

The expression distribution of the PPAR-γ gene in spleen, thymus, and bursa of Fabricius was analyzed by real-time quantitative reverse transcription PCR. The results showed that the PPAR-γ gene was expressed in all 3 immune organs, and its expression level changed with increasing age. Compared with that in spleen, the expression level of the PPAR-γ gene in the bursa of Fabricius was significantly decreased (P < 0.05) at the same time point. In addition, the expression level of the PPAR-γ gene in thymus was significantly higher (P < 0.05) than that in spleen except the 15-d group (Figure 1).

Effects of Cold Stress on the mRNA Levels of PPAR-γ in Spleen of Chickens

Effects of acute cold stress on the mRNA levels of the PPAR-γ gene in spleen are presented in Figure 2A. Compared with the 0-h control group, acute cold stress in 1-, 3-, 6-, 12-, and 24-h treatment groups all signifi-cantly decreased (P < 0.05) the mRNA levels of the PPAR-γ gene in spleen. Except for the 3- and 24-h groups and the 6- and 12-h groups, there were signifi-cant differences (P < 0.05) in the mRNA levels of the PPAR-γ gene among different treatment groups.

Effects of chronic cold stress on the mRNA levels of the PPAR-γ gene in spleen are presented in Figure 3A. Compared with the corresponding control groups, chronic cold stress in 5-, 10-, and 20-d treatment groups significantly increased (P < 0.05) the mRNA levels of the PPAR-γ gene in spleen.

Effects of Cold Stress on the mRNA Levels of PPAR-γ in Thymus of Chickens

Effects of acute cold stress on the mRNA levels of the PPAR-γ gene in thymus are presented in Figure 2B. Compared with the 0-h control group, acute cold stress in 1-, 3-, 6-, 12-, and 24-h treatment groups all signifi-cantly decreased (P < 0.05) the mRNA levels of the PPAR-γ gene in thymus. Except the 6- and 12-h groups and the 12- and 24-h groups, there were significant dif-ferences (P < 0.05) in the mRNA levels of the PPAR-γ gene among different treatment groups.

Figure 1. The expression trait of the peroxisome proliferator-activated receptor-γ (PPAR-γ) gene in the immune organs of chickens by real-time PCR. Panels A to C are the expression traits of the PPAR-γ gene in the spleen, thymus, and bursa of Fabricius of chickens at different ages, respectively. The relative mRNA levels from the 15-d group are used as the reference values in panels A to C. Panel D is the comparative result of the expression trait of the PPAR-γ gene in spleen, thymus, and bursa of Fabricius. The relative mRNA levels from the spleen at different ages are used as the reference values in panel D. Each value represented the mean ± SD of 5 individuals. The different letters in panels A to C indicate that there are significant differences (P < 0.05) between any 2 groups. *Significant differences (P < 0.05) between the spleen and the thymus and bursa of Fabricius at the same time point. #Significant differences (P < 0.05) between the thymus and the bursa of Fabricius at the same time point.

PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR-γ EXPRESSION LEVELS 2551

Effects of chronic cold stress on the mRNA levels of the PPAR-γ gene in thymus are presented in Figure 3B. Compared with the corresponding control groups, chronic cold stress in 5-, 10-, and 20-d treatment groups all significantly decreased (P < 0.05) the mRNA levels of the PPAR-γ gene in thymus.

Effects of Cold Stress on the mRNA Levels of PPAR-γ in Bursa of Fabricius of Chickens

Effects of acute cold stress on the mRNA levels of the PPAR-γ gene in bursa of Fabricius are presented in Figure 2C. Compared with the 0-h control group, acute

Figure 2. Effects of acute cold stress on the mRNA levels of the peroxisome proliferator-activated receptor-γ (PPAR-γ) gene in spleen, thymus, and bursa of Fabricius of chickens. Relative mRNA levels of the PPAR-γ gene were detected by real-time PCR. Panels A to C are the results of spleen, thymus, and bursa of Fabricius, respectively. The relative mRNA levels from the 0-h control group were used as the reference values in panels A to C. Each value represented the mean ± SD of 5 individuals. The different letters in panels A to C indicate that there are significant differences (P < 0.05) between any 2 groups.

Figure 3. Effects of chronic cold stress on the mRNA levels of the peroxisome proliferator-activated receptor-γ (PPAR-γ) gene in spleen, thymus, and bursa of Fabricius of chickens. Relative mRNA levels of the PPAR-γ gene were detected by real-time PCR. Panels A to C are the results of spleen, thymus, and bursa of Fabricius, respectively. The relative mRNA levels from the 5-, 10-, and 20-d control group were used as the reference values in panels A to C. Each value represented the mean ± SD of 5 individuals. *Significant differences (P < 0.05) between the control group and the stress group at the same time point.

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cold stress in 1-, 3-, 6-, 12-, and 24-h treatment groups all significantly increased (P < 0.05) the mRNA levels of the PPAR-γ gene in bursa of Fabricius. Except for the 3- and 12-h groups, there were significant differ-ences (P < 0.05) in the mRNA levels of the PPAR-γ gene among different treatment groups.

Effects of chronic cold stress on the mRNA levels of the PPAR-γ gene in bursa of Fabricius are presented in Figure 3C. Compared with the corresponding control groups, chronic cold stress in 5-, 10-, and 20-d treat-ment groups all significantly decreased (P < 0.05) the mRNA levels of the PPAR-γ gene in bursa of Fabr-icius.

DISCUSSIONStudies indicate that the chicken PPAR-γ gene is ex-

pressed in almost all tissues, such as fat, brain, kidney, spleen, heart, lung, esophagus, stomach (glandular and muscular stomach), intestine (small and large intestine), liver, skeletal muscle, and ovaries, but results were not consistent. For example, the results from Meng et al. showed that the PPAR-γ gene was not expressed in liver and breast muscle by reverse transcription PCR and Northern blot, and the study from Sato et al. in-dicated that the PPAR-γ gene was expressed in liver and skeletal muscle by Northern blot (Sato et al., 2004; Meng et al., 2005a; Hojo et al., 2006). Our results indi-cated that the chicken PPAR-γ gene was also expressed in thymus and bursa of fabricius by real-time reverse transcription PCR besides the above tissues. This work makes a supplement to the tissue distributions of the PPAR-γ gene.

The reactive mechanism of cold stress is very compli-cated, and the results from different studies are not con-sistent. These may be attributed to the duration time of cold exposure, the temperature of cold exposure, the genetic background of experimental animal, and so on. But lots of studies have proved that cold stress could influence the energy metabolism and immune responses (Rybakina et al., 1997; Cichoń et al., 2002; Haman et al., 2002; Pereira-da-Silva, 2003). In recent years, more and more experimental evidence indicates that some factors not only affect metabolism but also play a role in the regulation of immune responses, such as cytok-ines, hormones, neuropeptides, and transcription fac-tors (Matarese and La Cava, 2004). As one of them, the role of PPAR-γ in fat metabolism and immunity has been well studied and illuminated in mammals (Bens-inger and Tontonoz, 2008). But in chickens, the role of PPAR-γ is just involved in adipocyte differentiation and fat deposition (Matsubara et al., 2005; Meng et al., 2005b; Wang et al., 2008). Therefore, this study did a preliminary investigation on the effect of cold stress on PPAR-γ expression in 3 immune organs of chickens.

In this study, acute cold stress significantly decreased (P < 0.05) the mRNA levels of the PPAR-γ gene of spleen and thymus in all treatment groups and sig-nificantly increased (P < 0.05) the mRNA levels of

the PPAR-γ gene of bursa of Fabricius in all treat-ment groups. Compared with the corresponding con-trol groups, chronic cold stress resulted in a significant increase (P < 0.05) of the mRNA levels of the PPAR-γ gene in spleen and a significant decrease (P < 0.05) of the mRNA levels of the PPAR-γ gene in thymus and bursa of Fabricius. These results indicated that PPAR-γ gene expression in immune organs could be influenced by cold stress, but because of little research, the effect of cold stress on the PPAR-γ gene needs to be further studied, especially in chickens.

In conclusion, this study indicated that the PPAR-γ gene was expressed in spleen, thymus, and bursa of Fabricius and took on different expression traits. This would provide some basic data for the research on the function of the PPAR-γ gene in chickens. Both acute and chronic cold stress could alter the mRNA levels of the PPAR-γ gene in 3 tissues, and it took on a differ-ent response to the different degrees of cold stress in the same or different tissues. The lipid metabolism and immune responses, which aspect of the PPAR-γ gene is more important in the immune organs during cold stress, still needs more studies.

ACKNOWLEDGMENTS

We thank Antonio La Cava in the Department of Medicine, University of California, Los Angeles, for help in the experimental design. We also thank the members of the veterinary internal medicine labora-tory, especially the members of the cold stress group, in the College of Veterinary Medicine, Northeast Agri-cultural University, for help in feeding the chickens and analyzing the data.

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