Effects of the thyroid hormone responsive spot 14α gene on chicken growth and fat traits

Effects of the thyroid hormone responsive spot 14α gene on chicken growth and fat traits C. d’André Hirwa, W. Yan, P. Wallace, Q. Nie, C. Luo, H. Li, X. Shen, L. Sun, J. Tang, W. Li, X. Zhu, G. Yang, and X. Zhang1 Department of Animal Genetics, Breeding and Reproduction, College of Animal Science, South China Agricultural University, Guangzhou 510642, Guangdong Province, China significantly associated with abdominal fat weight (P = 0.0445). The above new THRSPα polymorphisms were also significantly associated with the total cholesterol and low-density lipoprotein, in which the THRSPα GA/AG genotype was associated with lipid and lipoprotein and the THRSPα BB indel genotype was significantly associated with liver weight in chicken breeds. The mRNA expression analysis in vivo and in vitro culture studies suggested that the THRSPα gene is more responsive to glucose than triiodothyronine. In conclusion, the 3 variations of the chicken THRSPα gene were associated with both growth and fat traits in this study. Such effects of the THRSPα gene were further supported from the data of observations in association analysis of the gene with phenotypic records and plasma lipid profiles, in the THRSPα gene expression in chicken development, and in vivo and in vitro cell culture observation of liver and abdominal fat tissues.

Key words: thyroid hormone responsive spot 14α, growth, fat deposition, chicken 2010 Poultry Science 89:1981–1991 doi:10.3382/ps.2009-00582

INTRODUCTION Thyroid hormone responsive spot 14 (THRSP) is an important transcription factor that controls the expression of several lipogenic genes (Towle et al., 1997). In addition to sensing triiodothyronine (T3) and glucose levels, the expression of the THRSP gene is regulated by liver X receptor, a transcription factor that is activated by cholesterol. The known target genes of THRSP are specific to lipogenesis, indicating that it is a control point in the accumulation of lipids, although its biochemical function is elusive. The THRSPα gene was found on chromosome 1 at location 197836316– 197838040 in chicken (GenBank, http://www.ncbi.nlm. ©2010 Poultry Science Association Inc. Received November 29, 2009. Accepted May 25, 2010. 1 Corresponding author: [email protected]

nih.gov and http://www.genome.ucsc.edu/cgi-bin/hgBlat); the chromosome domain where the genes exist is related with the adiposity. Previous research showed that the THRSP gene was identified to duplicate into 2 paralogs, THRSPα and THRSPβ (Wang et al., 2004). An insertion-deletion (indel) polymorphism was identified in the chicken THRSPα gene and was found to be associated with abdominal fatness (Cogburn et al., 2004; Wang et al., 2004) and to be involved in lipid metabolism as a BW indicator in chickens (Cao et al., 2007). However, association of the THRSPα gene with other fat traits like i.m. fat has not been reported. Besides the indel, other variations of the THRSPα gene were also associated with chicken growth (Cao et al., 2007). The alignment of the THRSPα cDNA and genomic sequences showed that the THRSPα gene contains 2 exons and 1 intron. This gene is also detectable in chicken adipose tissue involved in lipid metabolism (Wang et

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ABSTRACT The thyroid hormone responsive spot 14α (THRSPα) gene plays important roles in chicken growth and fat deposition. The aim of this study was to identify new variations in the gene to determine their effects on growth and fat traits in chicken and to observe the effects of the THRSPα gene on chicken lipid profile and lipoprotein and glucose and triiodothyronine effects on the THRSPα expression in liver and fat cells. Two new variations, namely A197835978G and G197836086A, and a reported 9-bp insertion-deletion (indel) of the THRSPα gene were genotyped by singlestranded conformational polymorphism in a Xinghua × White Recessive Rock F2 full-sib resource population. The results showed that the A197835978G was significantly associated with hatch weight and BW at 28 d of age and breast muscle weight at 90 d of age in chickens (P < 0.05). The G197836086A was significantly associated with cingular fat width (P = 0.0349) and breast muscle crude fat content (P = 0.0349). The indel was

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MATERIALS AND METHODS Experimental Populations and Their Management Red Jungle Fowls, XH chicken, White Recessive Rock (WRR) chicken, Leghorn layers, and Beijing fatty chicken, each with 10 individuals, were used to identify the SNP and other variations in the 5′ flanking region of the THRSPα gene. An F2-designed full-sib resource population from the cross of WRR and XH (Lei et al., 2005) was used to analyze the association of the THRSPα variations with growth and fat traits. The cross of WRR and XH produced 17 F1 families and 454 F2 full-sib individuals. The F2 chicks were raised in floor pens and fed with commercial corn-soybean-based diets that met the NRC requirements (NRC, 1994). Hatch weight (HW) and BW at 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, and 90 d of age were recorded. All birds were slaughtered at 90 d of age. The fat traits such s.c. fat thickness, cingular fat width (CFW, the fat measured between the leg and breast muscles; when the CFW becomes abnormally wide, it means the bird is too fat), and abdominal fat pad weight (AFW) were recorded in all F2 full-sib individuals, and crude fat content of breast muscle (CFCBM) and crude fat content of leg muscle were measured and recorded in 213 of 454 F2 full-sib individuals.

Ninety-five XH (50 males and 45 females) and 81 QYP (37 males and 44 females) from Guangdong Wen’s Co. (Guangdong, China) and Qingyuan, Guangdong, China, respectively, were reared under the same environmental and management practices at the South China Agricultural University Poultry Farm, Guangzhou, China. Both were used for phenotypic record and plasma lipid level analysis. Live BW at 1, 2, 3, 4, 5, 6, 7, 8, and 9 wk of age were recorded and blood samples were collected. Live weight, fat weight (abdominal fat + gizzard fat), liver fat content, and abdominal fat content at 5, 6, 7, 8, and 9 wk of age were determined. Serum samples to determine triglyceride (TG), total cholesterol (TC), high-density lipoprotein (HDL) cholesterol, and low-density lipoprotein (LDL) cholesterol were taken at 5, 6, 7, 8, and 9 wk of age. The XH and QYP chickens were also used to analyze the association of the THRSPα variations with phenotypic traits, lipid profile, and lipoprotein.

Diets All birds were fed a nutritionally adequate corn-soybean diet (NRC, 1994) and had free access to water. In the feeding experiment that entailed the use of glucose and T3, 4 individuals were randomly selected every week from 3 groups with 3 replicates each. Between the ages of 1 and 4 wk, liver samples were excised for the THRSPa gene mRNA expression and afterward the 4-wk-old chickens received diets containing 1 mg of T3/ kg of BW and 4 mg of glucose/kg of BW for 5 wk (i.e., wk 5, 6, 7, 8, and 9).

Blood Sample Collection and DNA Extraction The 1- to 1.5-mL blood samples were collected from the wing vein with a 2.5-mL syringe, containing EDTA as an anticoagulant. The extraction of the DNA was conducted with the phenol method from the fresh blood or the blood that had been stored at −20°C.

Serum Samples Serum was harvested by centrifugation at 870 × g for 15 min. Total serum analysis (free cholesterol + cholesterol esters) and TG were calculated using a commercial enzymatic kit supplied by Shanghai Shenergy-Diasys Diagnostic Technology Co. Ltd (Shanghai, China). The concentrations of TG, TC, and HDL in the serum were determined using a Beckman Coulter machine (Beckman Coulter, Fullerton, CA), from the Institute of Animal Science, Guangdong Academy of Agricultural Sciences (Guangzhou, China). Low-density lipoprotein cholesterol was calculated using the following TG (Friedewald et al., equation: LDL = (TC - HDL) + 5 1972; Gazi et al., 2006).

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al., 2007). It exists at high level in the liver of birds that grow rapidly and accumulate excess fat. Generally in birds that grow slowly and do not accumulate fat, there are no THRSP gene expressions (Cogburn et al., 2003). Liver and adipose tissues synthesize fatty acids for use as fuel (Brown et al., 1997). In those tissues, lipogenesis is regulated by availability of dietary substrates and circulating hormones that control fuel metabolism. If the intake of fuel is in excess of immediate needs, the carbohydrate calories can be stored as glycogen for immediate glucose release or converted to fat for subsequent energy release. Conversion of carbohydrate to fat occurs in several tissues, such as liver and adipose tissue, by a process called de novo lipogenesis (Anderson et al., 2009). The spot 14 gene is rapidly upregulated by signals that induce lipogenesis such as enhanced glucose metabolism and thyroid hormone administration (Tsatsos et al., 2008). The objective of the present study was to identify new polymorphisms of the chicken THRSPα gene and to evaluate their associations with growth and fat traits using a F2 resource population. Such associations were further confirmed by the correlation analysis between the THRSPα gene polymorphisms, the phenotypic records, and their plasma lipid profiles using 2 breeds, Xinghua (XH) and Qingyuan partridge (QYP) chickens. In addition, the influences of glucose and T3 on the THRSPα mRNA gene expression were also studied.

THE CHICKEN THYROID HORMONE RESPONSIVE SPOT 14α GENE

Primer Design and PCR Amplification for SNP Identification The primers (Table 1) were designed referring to the published cDNA sequences of the THRSPα gene, AY568628 and AY568629 (http://www.ncbi.nlm.nih. gov), and using GENETOOL software (BioTools, Alberta, Canada). The PCR products with different mutations were purified and sequenced using the protocol described by BioAsia Biotechnology Co. Ltd. (Shanghai, China). The sequences were analyzed to identify the SNP and other variations in the chicken THRSPα gene using DNASTAR software (http://www.biologysoft.com/).

Based on SNP locations in the chicken THRSPα gene, 2 SNP of A197835978G and G197836086A were selected to be genotyped. A reported 9-bp indel (Wang et al., 2004) was also genotyped in the present study. Twenty microliters of PCR reaction mixtures containing 40 ng of chicken genomic DNA, 10 × PCR buffer, 12.5 pmol of primer, 10 mM deoxynucleoside triphosphate, 25 mM MgCl2, and 0.2 U of Taq DNA polymerase (Sangon Biological Engineering Technology Co., Shanghai, China) were prepared. Polymerase chain reaction was performed with 33 cycles of 30 s at 94°C, 30 s at 59.5°C, and 1 min at 72°C. After denaturation, the setting was at 94°C for 2 min. The final extension was carried out at 72°C for 10 min. The amplified DNA fragments were identified on 1% agarose gel and stained with ethidium bromide. The amplified pattern was visualized on a UV transilluminator and photographed. The PCR products were separated on 12% PAGE for 24 h at 180 V, 8°C.

Liver Cell Culture Liver cells were isolated from chicken based on a modified method of Fraslin et al. (1992). The birds were first washed with 75% ethanol and anesthetized using i.m. mebumal sodium injection (75 mg/kg of

BW) followed by an intravenal heparin (1,750 IU/kg of BW) injection. The liver was first digested with solution A (10 mM HEPES, 137 mM NaCl, 3 mM KCl, 3 mM Na2HPO4·2H2O, 5 mM EDTA, pH 7.4) and then by the same buffer supplemented with collagenase (0.2 mg/mL) and CaCl2 (0.6 mg/mL). The liver was excised and put into a Petri dish. The outer membrane was removed and liver cells were collected in Williams’ medium E supplemented with 0.2% bovine serum. The cells were filtered through stainless metal mesh; washed 3 times by centrifugation (109 × g for 2 min); washed twice in HEPES buffer to eliminate cell fragments, erythrocytes, and nonparenchymal cells; and finally washed in Williams’ medium E. Cell viability was estimated by the trypan blue exclusion test. After counting the liver cells, the cells were concentrated to 3 × 105 cells/cm2 and plated on a 96-well plate with 100 μL of suspended cells per well (the cell density was 1 × 105 cells/cm2). The Williams’ medium was supplemented with penicillin-streptomycin, 5.5 mM glucose, and 10% fetal bovine serum. The cell attachment was achieved between 4 and 5 h. After 5 h, the cells were incubated under low (5.5 mM) and high (25 mM) glucose conditions in the presence or absence of 100 and 200 nM T3, then the cells were incubated under 100 nM T3 in the absence of glucose for a period of 48 h. There was a medium renewal every 24 h. The cells were cultured at 37°C, in an atmosphere of 5% CO2. The RNA extraction was conducted after 24, 48, and 96 h.

Abdominal Fat Cell Culture The birds were washed with 75% ethanol, and the abdomen was opened and fat was excised and put into a Petri dish. The blood vessels and fascia were removed and abdominal fat cells were collected in Williams’ medium E supplemented with 10% bovine serum. The abdominal fat cells were digested with collagenase. The cells were filtered through stainless metal mesh, washed 2 times by centrifugation (435 × g for 10 min), and finally washed in Williams’ medium E. Cell viability was estimated by the trypan blue exclusion test. After counting the liver cells, the cells were concentrated to 3 × 105 cells/cm2 and plated on a 96-well plate with

Table 1. Primers used for amplifying the 5′ flanking region of the chicken thyroid hormone responsive spot 14α (THRSPα) gene sequence Primer

Sequence (5′ to 3′)1

I

F: cct gat ggt gtt ggg aga R: tgc gtg ccg aga agt ac F: agg gtc tcga agt ggg ttg c R: gca atc cac gcc aca gca ct F: gca ggct ggg gaa cat cac c R: ccc tcc tca cca ccc tgc tc F: gga aga ccc ctc gca gca g R: acg tga gga gag gca agg cg F: gcc tcc gtc acc gat cag R: cgg tca gaa cct gct gca a

II III IV V

1F

= forward; R = reverse.

Length (bp)

Temperature (°C)

18 18 20 20 20 20 19 20 18 19

59 58.5 60 63.5 59.5

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Genotyping with PCR-Single-Strand Conformational Polymorphism Approach

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100 μL of suspended cells per well (the cell density was 1 × 105 cells/cm2). The Williams’ medium was supplemented with penicillin-streptomycin, 5.5 mM glucose, and 10% fetal bovine serum. The cell attachment was achieved between 4 and 7 h. After 7 h, the cells were incubated under low (5.5 mM) and high (25 mM) glucose conditions in the presence or absence of 100 and 200 nM T3, then the cells were incubated under 100 nM T3 in the absence of glucose. There was a medium renewal every 24 h. The cells were cultured at 37°C, in an atmosphere of 5% CO2. The RNA extraction was conducted after 4 d.

Total RNA Isolation and Reverse Transcription

Determination of mRNA Levels by Real-Time Reverse Transcription PCR After reverse transcription, the cDNA of the THRSPα gene was amplified by real-time reverse transcription PCR. This was performed in 96-well plates of 25-μL capacity containing 2.5 μL of diluted cDNA, 10 μM concentrations of the forward and reverse primers, SYBR

Statistical Analysis Data were analyzed by ANOVA (SAS Institute, 1989). The association between the polymorphism and traits was subjected to GLM procedures with the genotypes and sexes as fixed effects and hatch, family, and sex as random effects. The following model was used for association analysis using the F2 population: Y = µ + G + S + h + f + e, where Y = the dependent variable; µ = population mean; G = genotype; S = sex; h = hatch; f = family; and e = the random error.

Table 2. Identified SNP in the chicken thyroid hormone responsive spot 14α (THRSPα) gene1 No.

SNP location in the THRSPα

SNP location in the whole genome

Region

SNP sequence

Allelic frequencies

1 2 3 4 5 6 7 8 9 10 11 12 13

A164G G181A C215A A234G T250C G271A C275G A281C T311A G330A C571T G583C T663G

A197835978G A197835998G C197836030A A197836049G —2 G197836086A — — — — 197836567G-C — —

5′ 5′ 5′ 5′ 5′ 5′ 5′ 5′ 5′ 5′ 5′ 5′ 5′

G-A A-G C-A A-G T-C G-A C-G A-C T-A G-A T+ G-C T-G

FA = 0.10 FG = 0.10 FA = 0.33 FG = 0.10 FC = 0.33 FA = 0.43 FG = 0.05 FC = 0.33 FA = 0.14 FA = 0.10 FA = 0.05 FC = 0.33 FG = 0.14

Flanking Flanking Flanking Flanking Flanking Flanking Flanking Flanking Flanking Flanking Flanking Flanking Flanking

1Single nucleotide polymorphism position was determined based on sequences of GenBank accession no. AY568628, AY568629, and chromosome 1 (http://www.ncbi.nlm.nih.gov) and extended by BLAT Searche Genome (http://www.genome.ucsc.edu/cgi-bin/hgBlat). The A197835978G caused an activator protein 1 change, in which the activator protein 1 exists in allele A. 2Indicates that an SNP is not reported in GenBank.

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Total RNA in chicken was collected each week and tissues of interest were taken immediately after cervical dislocation, snap-frozen in liquid nitrogen, and stored at −80°C until extraction of RNA. Total RNA was extracted using the Redzol kit (SBS Genetech Co., Beijing, China) and its concentration was determined by reading the optical density at 260 nm. Samples were diluted in RNase-free water. The quality of extracted total RNA was examined by agarose gel electrophoresis, in which the rRNA (28S/18S) was observed for integrity. When not in use, total RNA samples were stored at −80°C. The RNA was reverse-transcribed using the RevertAid Fist Strand cDNA Synthesis Kit (Fermentas, Shenzhen, China)

Green Master Mix, and diethylpyrocarbonate water (Sangon Biological Engineering Technology Co.). Each sample was assayed in triplicate and the reaction was performed with an Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems, Carlsbad, CA). The 18S rRNA chosen as reference was determined with the TaqMan Universal qPCR Master Mix kit (Applied Biosystems). A specific primer was designed from the chicken sequences to be intron-spanning to avoid amplification of genomic DNA. The cycling conditions consisted of a denaturation step at 95°C for 1 min followed by a 2-step amplification program (15 s at 95°C, followed by 15 s at 60°C) repeated 40 times. At the end, a dissociation program consisting of 1 min at 95°C, 15 s at 60°C, and 15 s at 60°C was performed. The calculation of absolute mRNA levels was based on the PCR efficiency and the threshold cycle (Ct) deviation of unknown cDNA versus the control cDNA, and then quantitative values were obtained from the Ct values, which were the inverse ratio relative to the starting PCR product. The relative quantification was obtained by 2ΔΔCt, in which ΔCt = Cttarget gene − Ctreference gene (18S). The following represented the primers used for the THRSPα gene expression: forward: 5′-gcc tcc gtc acc gat cag-3′ and reverse: 5′-cgg tca gaa cct gct gca a-3′.

1985 in rows with different superscripts are significantly different. number shown in parentheses stands for the selected individuals. HW = hatch weight; BW28 = BW at 28 d of age; CFW = cingular fat width; AFW = abdominal fat pad weight; CFCBM = crude fat content of breast muscle. 1The

CFCBM (g)

AFW (g)

CFW (g)

BW28 (g)

a,bMeans

29.72 ± 0.13 (114) 309.0 ± 3.56 (115) 12.16 ± 0.29 (115)b 28.45 ± 1.29 (115)a 0.86 ± 0.09 (56) 29.87 ± 0.16 (33) 312.4 ± 4.23 (231) 11.46 ± 0.36 (235)ab 28.36 ± 1.65 (239)ab 0.97 ± 0.10 (106) 29.87 ± 0.14 (182)ab 305.1 ± 3.64 (182)ab 12.13 ± 0.30 (181)a 30 ± 1.34 (183)a 0.85 ± 0.10 (77)a

30.15 ± 0.17 (167)a 318.8 ± 4.45 (168)a 12.03 ± 0.37 (169)a 29.23 ± 1.63 (169)a 0.88 ± 0.10 (84)a

30.44 ± 0.29 (86)a 312.2 ± 5.30 (85)ab 11.73 ± 0.35 (85)a 27.87 ± 1.72 (87)a 0.913 ± 0.04 (49)b

30.08 ± 0.22 (139)a 315.2 ± 4.28 (137)a 11.74 ± 0.29 (140)a 27.42 ± 1.49 (140)a 0.973 ± 0.04 (68)ab

29.59 ± 0.18 (198)b 309.7 ± 3.52 (199)b 11.90 ± 0.27 (201)a 25.40 ± 1.26 (201)b 1.08 ± 0.04 (84)a

30.17 ± 0.34 (76) 297.1 ± 8.94 (75) 10.45 ± 0.754 (76) 20.91 ± 3.42 (76)b 0.761 ± 0.13 (39)

BB AB AA GG AG AA

29.37 ± 0.24 (81)b 316.0 ± 6.38 (78)b 11.62 ± 0.51 (83)a 24.89 ± 2.2 (83)b 0.90 ± 0.11 (42)a

The site G197836086A was significantly associated with live BW (P < 0.01) and liver weight (P < 0.05) in XH and QYP. There was also significant association between these parameters and serum cholesterol (P < 0.05) and LDL (P < 0.05) (Table 4). The site A197835978G was significantly associated with abdominal fat (P < 0.05) and cholesterol (P < 0.01). Abdominal fat pad weight in the heterozygotes of A197835978G

HW (g)

Association of the THRSPα Gene with Phenotypic Records and Plasma Lipid Profiles and THRSPα Gene Expression in Chicken Development

GG

The site A197835978G was significantly associated with HW (P = 0.0431), BW at 28 d of age (P = 0.0248), and ADG (P = 0.0454). Allele G was dominant in relation to growth (Table 3). No significant association of the SNP with s.c. fat thickness, CFW, AFW, CFCBM, and crude fat content of leg muscle was observed. The site G197836086A was significantly associated with both CFW (P = 0.0349) and CFCBM (P = 0.0349). Allele A was dominant in relation to abdominal fat deposition, whereas allele G was dominant in the case of fat deposition within breast muscles (Table 3). The indel was significantly associated with AFW (P = 0.0445), in which allele B was dominant both in abdominal fat deposition and general fat deposition (Table 3).

AG

Association of the THRSPα Gene SNP with Chicken Growth and Fat Traits

AA

Thirteen variation sites were identified in the 5′ flanking regions of chicken THRSPα gene by comparing amplified sequences using the DNASTAR software (Table 2). These SNP included 5 transitions (38.5%) and 8 transversions (61.5%), of which A197835978G and G197836086A were regarded as potential transcription sites.

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SNP Identification in the 5′ Flanking Region of the Chicken THRSPα Gene

Trait1

RESULTS

Indel

where Y = the dependent variable; µ = mean; G = genotype; S = sex; b = breed; and e = the random error. The statistical significance threshold was set at P < 0.05. Data were expressed as the mean ± SEM, and the differences in the means were compared using Duncan’s multiple range test at 5% level of significance.

G197836086A

Y = µ + G + S + b + e,

A197835978G

The following model was used to analyze the association of the THRSPα gene with phenotypic records and plasma lipid:

Table 3. Effect of the A197835978G, G197836086A, and the insertion-deletion (indel) in the thyroid hormone responsive spot 14α (THRSPα) gene on chicken growth and fat deposition

THE CHICKEN THYROID HORMONE RESPONSIVE SPOT 14α GENE

0.1054

0.2887

0.0146*

0.7439

0.0027**

0.4555

0.1603b

0.6439

BW (g)

LW (g)

AFW (g)

AFC (%)

CHOL (mmol/L)

TG (mmol/L)

HDL (mmol/L)

LDL (mmol/L

535.94 ± 19.4a 15.60 ± 0.5 9.49 ± 0.7ab 91.68 ± 1.68 7.6 ± 0.3a 4.62 ± 0.5 3.1 ± 0.1ab 2.2 ± 0.28

AA (52) 536.83 ± 23.8a 15.86 ± 0.7 9.79 ± 1.12a 92.1 ± 1.65 7.6 ± 0.4a 4.08 ± 0.3 3.24 ± 0.09a 2.1 ± 0.26

AG (30) 474.74 ± 18.45b 14.66 ± 0.4 6.46 ± 0.5b 92.43 ± 0.6 6.49 ± 0.2b 5.7 ± 0.7 2.89 ± 0.09b 2.28 ± 0.16

GG (94)

0.0317*

0.8369

0.4706

0.0123*

0.1642

0.2647

0.0122*

0.0059**

P-value 537.83 ± 57.2 13.97 ± 1.50 9.6 ± 2.4 95.92 ± 0.6 5.6 ± 0.8b 3.09 ± 0.8 2.89 ± 0.21 0.84 ± 0.16b

GG (7) 438.1 ± 13.84 13.93 ± 0.47 8.9 ± 0.6 90.65 ± 2.4 7.55 ± 0.4a 4.46 ± 0.3 3.01 ± 0.08 1,750.19ab

GA (137)

G197836086A

516.9 ± 15.01 15.49 ± 0.38 7.63 ± 0.53 92.26 ± 0.69 7± 0.2ab 5.3 ± 0.5 3.03 ± 0.07 2,470.15a

AA (32)

1The

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454.6 ± 36.84 13.29 ± 0.88b 6.34 ± 1.21b 93.37 ± 0.87 6.93 ± 0.45 6.84 ± 1.4 3.02 ± 0.22 2.69 ± 0.4

0.1175

0.4489

0.905

0.2027

0.7013

0.7094

0.7868

0.0402*

AA (28)

P-value

516.8 ± 14.96 15.58 ± 0.39a 8.64 ± 0.53a 92.08 ± 1.02 7.19 ± 0.21 4.73 ± 0.4 3± 0.08 1.99 ± 0.14

AB (94)

Indel2

505.4 ± 23.85 15.34 ± 0.62a 7.55 ± 0.91ab 91.56 ± 1.4 6.83 ± 0.37 4.8 ± 0.84 3.06 ± 0.1 2.56 ± 0.26

BB (54)

within a row with different superscripts are significantly different (P > 0.05). number shown in parentheses stands for the selected individuals. LW = liver weight; AFW = abdominal fat weight; AFC = abdominal fat content; CHOL = total cholesterol; TG = triglyceride; HDL = high-density lipoprotein; LDL = low-density lipoprotein. 2Indel = insertion-deletion. *P < 0.05; **P < 0.01.

a,bMeans

P-value

Trait

A197835978G

Table 4. Association of the thyroid hormone responsive spot 14α (THRSPα) gene with phenotypic traits, level of plasma, and lipoprotein concentration in chicken pure breeds1

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THE CHICKEN THYROID HORMONE RESPONSIVE SPOT 14α GENE

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was significantly different in XH and QYP, and allele A of A197835978G was dominant for cholesterol in XH and QYP. The site A197835978G and the indel were significantly associated with liver weight (P < 0.05). Allele B of the indel was dominant. The highest expression levels of the THRSPα gene in liver (Figure 1a) and abdominal fat (Figure 1b) were observed in XH females at 2 wk of age.

Influence of Glucose and T3 on THRSPα Gene Expression in Chicken Liver and Abdominal Fat

THRSPα Gene Expression in Chicken Hepatocytes and Adipocytes After 48 h, the THRSPα gene was highly expressed in hepatocytes maintained on 25 mM glucose without T3 (Figure 4). The THRSPα gene gave higher expression in the abdominal fat supplemented with 25 mM glucose but had lower expression compared with the liver culture (Figure 5).

DISCUSSION New variations of the chicken THRSPα gene have effects on both growth and fat traits. In this study, A197835978G in the 5′ flanking region of the chicken THRSPα gene is associated with growth traits like HW, BW at 28 d of age, and ADG. Cao et al. (2007) also observed that 2 variations in the first exon of the chicken THRSPα gene were associated with chicken growth. The site G197836086A in the 5′ flanking region of the chicken THRSPα gene was significantly associated with CFW and CFCBM. The reported indel of the chicken THRSPα gene was also associated with AFW in the F2 resource population used in this study, which supported the previous findings (Wang et al., 2004). From the observations in this study, the THRSPα gene has effects on chicken growth and fat traits through other variations beside the 9-bp indel. Development of chicken fat traits is linked to plasma lipid profiles, and THRSPα gene expression is correlated with plasma lipid profiles. The correlation analyses between the chicken THRSPα gene polymorphism and phenotypic records as well as their plasma lipid profiles in XH and QYP chickens reported that there are associations of the THRSPα gene with chicken lipoprotein metabolism. Variations in the chicken THRSPα gene

Figure 1. Thyroid hormone responsive spot 14α (THRSPα) expression in liver (a) and abdominal fat (b) from males and females. XH = Xinghua.

are also significantly associated with plasma lipid profiles. The G197836086A GA genotype of the THRSPα gene is significantly linked to TC and LDL levels, indicating that the above genotype is associated with lipid and lipoprotein metabolism. In addition, A197835978G in the 5′ flanking region of the chicken THRSPα gene is associated with TC level. Consequent to the human body, fat deposition is strongly associated with health problems such as diabetes mellitus, hypertension, and atherosclerosis. Therefore, the identification of the genes associated with fat deposition contributes to the efforts to fight against these health problems in human beings. Obesity is reportedly related to several disturbances in lipid and lipoprotein metabolism (Garrison et al., 1980). For instance, high concentrations of serum TG-rich lipoproteins and LDL cholesterol levels are usually much less elevated in obese conditions. In chickens, the expression of THRSP mRNA increases dramatically in the liver of newly hatched chicks as they begin to synthesize and deposit abdominal fat (Cogburn et al., 2003). The results mentioned that the THRSPα gene has an effect on lipid and lipoprotein processing at different stages of chicken development. In this study, further emphasis was put on the associative relationship based on the results of the correlation analysis between the THRSPα gene polymorphism and its expression levels in liver and abdominal fat.

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The highest expression of THRSPα gene in the liver of XH males fed with glucose was found during the fifth and eighth weeks (Figure 2). The highest expression of the THRSPα gene in the control group of female XH chicken was found during the eighth week (Figure 2). In terms of abdominal fat, during the sixth week, the THRSPα gene was highly expressed in XH females fed with T3 (Figure 3).

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Figure 3. Influence of glucose and triiodothyronine on the thyroid hormone responsive spot 14α (THRSPα) gene in vivo expression in chicken abdominal fat from 5- to 9-wk-old chicken. Relative mRNA levels of the THRSPα were measured by real-time PCR. XH = Xinghua.

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Figure 2. Influence of glucose and triiodothyronine on the thyroid hormone responsive spot 14α (THRSPα) gene in vivo expression in chicken liver from 5- to 8-wk-old chicken. Relative mRNA levels of the THRSPα were measured by real-time PCR. XH = Xinghua.

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The THRSPα gene is highly expressed in both liver and abdominal fat of female XH chicken at the second week. This testifies that THRSPα expression is experienced at the early stages of chicken development. A similar observation made by Cogburn et al. (2003) showed that the THRSP gene has high expression after

hatching (i.e., between 1 and 9 d of age). With respect to 5-wk-old broiler chicken, the THRSP gene was reported to be highly expressed in the liver other than in the adipose tissue (Wang et al., 2004). Yan (2006) also reported that the THRSPα gene in Yangshan chickens is highly expressed in abdominal and s.c. fat at 9 wk

Figure 5. Thyroid hormone responsive spot 14α (THRSPα) gene expression obtained from the culture of the abdominal fat at 4 d of age. The columns and error bars represent mean values and SE. T3 = triiodothyronine.

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Figure 4. Thyroid hormone responsive spot 14α (THRSPα) gene expression obtained from the culture of the liver cells in vitro for 48 h. The columns and error bars represent mean values and SE. T3 = triiodothyronine.

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dominal fat tissues. Other researchers are highly encouraged to conduct further studies on the role of spot 14α in chicken development and the physiological significance of its regulation by glucose and T3.

ACKNOWLEDGMENTS Funds for this work were partly provided by The Ministry of Higher Education Student Financing Agency of Rwanda, China Scholarship Council, and South China Agricultural University, Guangzhou, China. Further support was provided by the Major State Basic Research Development Program, China (project no. 2006CB102107), and the National High Technology Research and Development Program of China (863 Program, project no. 2007AA10Z163).

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of age, whereas the highest expression levels in Peking duck were found in fat tissues at 6 wk of age (Zhan et al., 2006). According to Wang et al. (2007), it was determined that at 7 wk of age, the THRSPα gene is highly expressed in the adipose tissue of chicken. Also, in the liver and abdominal fat in vivo and in vitro cell culture to study THRSPα mRNA gene expression, it was highly expressed in the liver during the fifth and eighth weeks in male XH chicken fed glucose. At the eighth week, the THRSPα mRNA level in the female XH chicken was high in the abdominal fat tissue. The elevation of the THRSPα mRNA levels in the liver of chicken by feeding a diet containing T3 was first reported by Wang et al. (2002). Cogburn et al. (2000) also reported that a large number of genes involved in lipogenesis, namely the THRSP gene, fatty acid desaturase, malic enzyme, and fatty acid-binding protein, were captured in the liver cDNA library. Due to the fact that the liver is the major site of lipogenesis (Goodridge and Ball, 1967), in vitro cell culture studies carried out in this work demonstrated that the THRSPα gene is highly expressed in liver cells maintained with 25 mM glucose without T3. Although glucose is the major substrate for de novo lipogenesis (Sanches-Rodriguez et al., 2005), the thyroid hormone is very important because it is involved in the growth and development of animals. Referring to the in vivo and in vitro cell culture, respectively, to liver and abdominal fat, THRSPα gene expression responds to both but more slightly to glucose than T3. Foufelle and Ferre (2002) reported the regulation of hepatic glucose metabolism as a key to the whole body energy metabolism because the liver is able to store and to produce glucose. It has been found that THRSP is involved in the mechanism that regulates lipogenic enzyme expression (Kinlaw et al., 1995; Zhu et al., 2001). For most genes involved in glucose carbon utilization, the induction of their mRNA expression by a carbohydrate-rich diet is powerful (4- to 25-fold), rapid (1- to 2-h range), and involves a transcriptional mechanism (Foufelle and Ferre, 2002). Indeed, the current study is in conformity with the known facts that THRSPα is controlled by age, sex, breed type, and nutritional factors. The findings in this study confirm that THRSPα gene expression plays a critical role in biological development. In conclusion, the current study revealed that A197835978G in the 5′ flanking region of the chicken THRSPα gene is significantly associated with the growth of chicken, whereas G197836086A in the 5′ flanking region and the indel in exon 1 of the chicken THRSPα gene are associated with fat deposition. Their association with fat traits was further supported by the correlation analysis between these chicken THRSPα gene variations and plasma lipid profiles such as TC and LDL. The effects of the chicken THRSPα gene on fat trait development were also confirmed by in vivo and in vitro cell culture observation of liver and ab-

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Wang, X., W. Carre, L. Rejto, and L. A. Cogburn. 2002. Global gene expression profiling in liver of thyroid manipulated and/or growth hormone injected broiler chickens. Poult. Sci. 81(Suppl. 1):63. (Abstr.) Wang, X., W. Carre, H. Zhou, S. J. Lamont, and L. A. Cogburn. 2004. Duplicated Spot 14 genes in the chicken: Characterization and identification of polymorphisms associated with abdominal fat traits. Gene 332:79–88. Yan, W. 2006. Effects of the chicken THRSPα gene on fat deposition. PhD Diss. South China Agric. Univ., Guangzhou. Zhan, K., Z. C. Hou, H. F. Li, G. Y. Xu, and N. Yang. 2006. Molecular cloning and expression of the duplicated thyroid hormone responsive spot 14 (THRSP) genes in ducks. Poult. Sci. 85:1746–1754. Zhu, Q., A. Mariash, M. R. Margosian, S. Gopinath, M. T. Fareed, G. W. Anderson, and C. N. Mariash. 2001. Spot 14 gene deletion increases hepatic de novo lipogenesis. Endocrinology 142:4363–4370.

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