Intramuscular fat (IMF) plays an important role in meat quality. However, the molecular mechanisms underlying IMF deposition in skeletal muscle have not been addressed for the sex-linked dwarf (SLD) chicken. In this study, potential candidate genes and signaling pathways related to IMF deposition in chicken leg muscle tissue were characterized using gene expression profiling of both 7-week-old SLD and normal chickens. A total of 173 differentially expressed genes (DEGs) were identified between the two breeds. Subsequently, 6 DEGs related to lipid metabolism or muscle development were verified in each breed based on gene ontology (GO) analysis. In addition, KEGG pathway analysis of DEGs indicated that some of them (GHR, SOCS3, and IGF2BP3) participate in adipocytokine and insulin signaling pathways. To investigate the role of the above signaling pathways in IMF deposition, the gene expression of pathway factors and other downstream genes were measured by using qRT-PCR and Western blot analyses. Collectively, the results identified potential candidate genes related to IMF deposition and suggested that IMF deposition in skeletal muscle of SLD chicken is regulated partially by pathways of adipocytokine and insulin and other downstream signaling pathways (TGF-
In the past decades, poultry breeding predominantly focused on increasing growth rate and meat yield and improving body composition by producers. Poultry production has dramatically increased, meeting both consumer demand and commercial profit requirements desired by producers; however, the impressive progress made in these traits has been accompanied by deterioration of taste quality of the meat which leads consumers to seek better tasting chicken meat [
IMF is mainly distributed in the epimysium, perimysium, and endomysium and accumulated between muscle fibers or within muscle cells. Previous studies demonstrated that a certain amount of IMF can enhance meat quality, such as the flavor, juiciness, water holding capacity, and tenderness [
For the molecular mechanism of IMF deposition, numerous studies have been performed on livestock [
In the present study, characterization of mRNA expression profiles in skeletal muscles of 7-week SLD and normal chickens was performed using Affymetrix chicken gene chips and 6 candidate genes that may affect IMF deposition were selected out. Then, the expression changes of 6 candidate genes and other adipogenesis-related signaling pathway genes were measured by using qRT-PCR and/or Western blot analysis to examine the association of those genes with IMF deposition in skeletal muscle of SLD chicken.
SLD and normal recessive White Rock chickens, both bred for nearly 10 generations, were used. Dwarf chickens had a 1773-bp deletion mutation at the end of exon 10 and in the 3′UTR of GHR. The weight of dwarf chickens was about 30% less than that of normal chickens. The two strains were fed under the same conditions (ad libitum feeding, the same food stuff) to 7 weeks of age. All animal experiments involved in this study were approved by the Animal Care Committee of South China Agricultural University (Guangzhou, China). Chickens were euthanized as necessary to ameliorate suffering.
Nine birds of similar weight from each breed were sacrificed for tissue collection. Samples of the left leg gastrocnemius muscles were excised, divided into three parts, placed into cryopreservation tubes, and quickly snap-frozen in liquid nitrogen (−196°C) for preservation. The entire right leg muscle was collected and stored at −20°C for IMF content measurements.
IMF content of leg muscle was determined by the Soxhlet method according to previous studies [
Total RNA was isolated from skeletal muscle tissues with TRIzol (Takara Biotech Co. Ltd., Dalian, China) according to the manufacturer’s instructions. The purity and yield of RNA were determined using optical density at 260 and 280 nm. RNA integrity was examined by electrophoresis on a 1.2% denaturing formaldehyde gel.
Three pools of RNA were prepared for each chicken strain, with each pool containing RNA from three individuals. Microarray hybridization was carried out by Affymetrix Inc. (Beijing, China) using Agilent chicken gene chips with 38535 probes. The DEGs were selected out by using Significance Analysis of Microarrays (SAM) software, and the screening criteria were as follows:
To validate the microarray hybridization results, 6 genes were selected from the DEG list for qRT-PCR assays. In addition, 16 adipogenesis-related signaling pathway genes were analysed in the RNA samples by qRT-PCR. Using published genome sequences, the Primer Premier 5 software was used for primer design (Supplemental file 1 in the Supplementary Material available online at
The Western blot analysis was performed as described previously [
Results are presented as mean ± SEM, and qRT-PCR experiments included at least eight biological replicates per group and three technological replicates. Data were evaluated using a two-tailed Student’s
IMF content in the dwarf chickens is 1.2 times than that in the normal chickens (Figure
Body weight and IMF percentages of the dwarf and normal chickens. Significant differences in body weight between the two lines were apparent from the 3rd week; the IMF contents in dwarf chickens are significantly higher than those in normal chickens. Note:
Recessive sex-linkage dwarf gene (dw) is the only recessive mutant gene known to be benefit to human and has no harm to chicken health. SLD is perhaps the best characterized model of dw phenotype. Studies indicated that the SLD phenotype is caused by a mutation in the GHR gene that can result in a significant reduction in body weight, insulin-like growth factor 1(IGF1) levels, and increased adiposity and IMF content [
In our study, a total of 38,535 probes were used to detect mRNA expression profiles in chicken skeletal muscles, of those the probes displaying hybridization signals represented approximately 42.62–45.6% of the total; approximately 52.8–55.7% of probes lacked hybridization signals and about 1.5–1.7% of probes showed ambiguous hybridization signals (Table
Summary of gene expression in skeletal muscles of dwarf and normal chickens determined by microarray analysis.
Hybridization |
Normal chickens | Dwarf chickens | ||||
---|---|---|---|---|---|---|
A1 | A2 | A3 | B1 | B2 | B3 | |
Present | ||||||
Probes | 17174 | 17124 | 17559 | 16506 | 17417 | 16425 |
% | 44.6 | 44.4 | 45.6 | 42.8 | 45.2 | 42.6 |
Absent | ||||||
Probes | 20764 | 20839 | 20335 | 21428 | 20513 | 21448 |
% | 53.9 | 54.1 | 52.8 | 55.6 | 53.2 | 55.7 |
Marginal | ||||||
Probes | 597 | 572 | 641 | 601 | 605 | 662 |
% | 1.5 | 1.5 | 1.7 | 1.6 | 1.6 | 1.7 |
|
||||||
Total probes | 38535 | 38535 | 38535 | 38535 | 38535 | 38535 |
From our results, 16782 and 17285 genes were detected as expressed genes in dwarf and normal chickens, respectively (Figure
Numbers of genes that were differentially expressed in skeletal muscles between dwarf and normal chickens.
Based on the known DEGs, GO analyses were performed in each breed, and the enriched GO-terms (
Through the gene enrichment analysis, BCL6 was found be to the most frequent gene involved in those biological functions, then were JMJD6 and KIT, and followed by CBFB, MB, HOXA3, PTN, GHR, ARNT, HLF, ICER, LOC417056, LOC417083, YFVI, MR1, AGTR1, CPZ, FGF1, FOXK2, LOC396260, NR1D2, OTOR, PON2, POSTN, MAFF, RORA, ST6GAL1, and TGIF1 genes (Supplemental file 4), suggesting that those genes may participate in the regulation of chicken skeletal muscle development with high frequency.
According to Cui et al. [
IGF2BP3, one of the important members of insulin-like growth factor RNA binding protein family (RNA-binding proteins, RBPs), has pro-growth functions by binding to IGFs [
To further validate the results of microarray testing, qRT-PCR was used to examine the relative expression of 6 DEGs selected in each breed. As shown in Table
Comparison of microarray and qRT-PCR fold changes for selected genes in skeletal muscles of dwarf and normal chickens.
Gene | Fold change (microarray) |
Fold change (qRT-PCR) |
Tendency |
---|---|---|---|
GHR | 5.263 | 1.727 | Consistency |
SOCS3 | 2.439 | 2.428 | Consistency |
THRSP | 2.273 | 2.580 | Consistency |
ROR |
0.433 | 0.533 | Consistency |
IGF2BP3 | 0.33 | 0.400 | Consistency |
NR1D2 | 2.381 | 6.525 | Consistency |
Next, the mRNA or protein levels of other downstream genes that related to adipogenesis (Supplemental file 1) were also measured by using qRT-PCR and Western blot analysis.
As shown from Figure
(a) The mRNA expression of some signaling pathway factors was measured by qRT-PCR. Data were normalized using
KEGG pathway analysis was performed on those 6 DEGs to explore the potential pathways that may relate to IMF deposition in dwarf and normal chicken. The results showed that IGF2BP3 is associated with the insulin signaling pathway; both SOCS3 and GHR are associated with adipocytokine and insulin signaling pathways and SOCS3 is also involved in the regulation network of GHR gene.
In adipocytokine signaling pathway, SOCS3 affects adipogenesis perhaps by regulating two downstream genes, lipophorin receptor (LEPR) and insulin receptor substrate 1(IRS1). On the one hand, SOCS3 increases PPAR-
Schematic illustrations for potential signaling pathway of IMF deposition in skeletal muscles regulated by GHR and SOCS3. GHR increases the expression of SOCS3. SOCS3 affected adipogenesis by inhibiting the expression of LERP and IRS1, participating in adipocytokine signaling pathway. IGF2BP3 affected insulin signaling pathway by regulating IGFs.
qRT-PCR analysis in our previous study [
In insulin signaling pathway, for dwarf chicken, upregulated SOCS3 inhibits the phosphorylation of IRS1 affecting insulin signaling. Previous studies have demonstrated that Wnt10a/catenin-
ROR
The present approach has used gene expression profiling to analyse the DEGs and used qRT-PCR and Western blot analyses to elucidate the molecular events of IMF deposition in chickens. Possibly regulated by multiply signaling pathways and modifications of circadian rhythms-related genes may also contribute.
In the present study, gene expression profiles of skeletal muscle sampled at 7 weeks old from dwarf and normal chickens were characterized and 173 DEGs were selected out between the two groups. Six DEGs, whose expressions were verified by qRT-PCR and Western blot analysis in both two groups, were tentatively revealed to play key roles in developmental processes of IMF since they participate in adipogenesis-associated signaling pathway. Therefore, the IMF deposition in chickens was proposed to be partially regulated by multiple signaling pathways and circadian rhythms-related genes. The findings obtained in the current study could provide meaningful information for the establishment of the groundwork to further explain the molecular mechanisms underlying IMF deposition in chicken.
The authors declare that there is no conflict of interests regarding the publication of this paper.
This work was financially supported by the Natural Scientific Foundation of China (no. 31272519).