INTRODUCTION
One important goal of farm animal industry is to produce high quality beef. Beef quality is normally defined by the compositional quality (lean to fat ratio) and the palatability factors such as visual appearance, smell, firmness, juiciness, tenderness, and flavor. Many studies have indicated that meat tenderness is not only affected by protein composition of muscle fibers, but also by handling and slaughtering conditions, genetic traits, and growth progress. In addition, there is some connection between tenderness and flavor through marbling of meat (Hughes et al., 2014).
Generally, marbling means the amount of intramuscular fat (Purslow, 2005; Nishimura, 2010), one of the main factors used to determine beef quality and grade in Korea. Marbling is a very important and valuable trait in the beef cattle industry (Lee et al., 2007). Previous studies have found a relationship between marbling score and percent intramuscular fat (Jeong et al., 2012; Walter et al., 2014). Therefore, the content and distribution of body fats are of special interest for production efficiency and meat quality in farm animal industry (Gondret et al., 2008).
Contents and deposition of intramuscular fat can be influenced by several factors, including sex, age, breed, genotype, nutrition, and environmental factors (Maltin et al., 2003; Hausman et al., 2006). Generally, steers have more intramuscular fat, higher marbling score (Destefanis et al., 2003; Schreurs et al., 2008), and more tender meat (Peachey et al., 2002; Purchas et al., 2002) than bulls. Fat storage in cattle muscle is correlated with intramuscular fat percentage (Guo et al., 2014) because the hormonal status of beef cattle from different sex is related to meat quality characteristics, such as tenderness, fat, and protein distribution (Fritsche and Steinhart, 1998). Particularly, castration dramatically increases intramuscular fat deposition, resulting in improved beef quality in Korean cattle (Park et al., 2002).
The effects of sex steroid hormonal status, including testosterone, androgen, and estrogen, on muscle tissue and myogenic satellite cells (MSCs) have been well studied (Inoue et al., 1994; Arnold et al., 1996; Kahlert et al., 1997; Lee, 2002; Sinha-Hikim et al., 2003; Enns et al., 2008).
MSCs are adult stem cells that activate and differentiate into myotubes. Our previous studies investigated the importance of hormonal components in MSC growth and lipid droplets accumulation (Lee et al., 2011). The effect of natural hormones in adult bovine serum (cow, steer, and bull serum) in MSC proliferation was observed. We found that MSC proliferation was the highest in media supplemented with bull serum followed by cow and steer serum. Lipid droplets accumulation was increased in myotubes when MSCs were treated with 17β-estradiol (E2) followed by E2+testosterone or testosterone treatment alone. This may be due to various hormonal components present in the different sera. Our data have demonstrated that sex hormones are key factors affecting the proliferation of MSCs and lipid accumulation in myotubes (Lee et al., 2011).
However, factors important for the improvement of beef quality grade with sex and hormonal differences are not clearly understood in vivo. Therefore, identification of differentially expressed proteins in adipose depots of different sexes might be helpful in defining the functions of intramuscular fat, therefore providing strategies to control meat lipid content independently of body fat depots. The objective of this study was to determine whether proteins involved in depot specific adipose tissue properties were sex dependent. We analyzed the proteome expression of intramuscular adipose tissue (IMAT) and omental adipose tissue (OMAT) in native Hanwoo Korean beef cattle (cows, steers, and bulls) by liquid chromatography-tandem mass spectrometry (LC-MS/MS)–based proteomic analysis, quantitative polymerase chain reaction (PCR), and western blot analysis.
MATERIALS AND METHODS
Animals and sample collection
All experimental procedures involving animals were approved by the National Institute of Animal Science Institutional Animal Use and Care Committee (NIASIAUCC) and conducted in accordance with the Animal Experimental Guidelines provided by NIASIAUCC in Republic of Korea. We used adipose tissue samples of cows (n = 7), steers (n = 7), and bulls (n = 7). Tissue samples were collected in three animals groups from two different adipose depots (i.e. intramuscular and omental). Slaughter age was approximately 31 months for all cattles. Carcass weight was 406.1±13.4, 452.6±12.3, and 490.9±13.6 kg for cows, steers, and bulls, respectively.
Gel electrophoresis and silver staining
Adipose tissues were collected from cows, steers, and bulls. Total protein isolation was performed using PRO-PREP protein extraction solution (iNtRON Biotechnology, Seoul, Korea) according to the manufacturer’s instructions. Proteins eluted were measured using Pierce BCA Protein Assay Kit (Thermo scientific, Rockford, IL, USA). Equal amounts of protein samples were precipitated with cold acetone. Protein pellets were dissolved in 1× sodium dodecyl sulphate (SDS) sample buffer and separated by 12% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE). Following SDS-PAGE, protein spots were visualized using protocols described in the PlusOne Silver staining kit (GE Healthcare Bio-Sciences, Uppsala, Sweden). Complete protocol was followed for analytical gels. For preparative gels, the protocol was modified. Glutaraldehyde was omitted from the sensitization step. Formaldehyde was omitted from the silver reaction step (Yan et al., 2000). Silver-stained gels were scanned (UMAX PowerLook 2100KL Imaging system, UMAX, Taiwan) and protein profiles were compared.
Liquid chromatography-tandem mass spectrometry (LC-MS/MS)
The resulting tryptic peptides were separated and analyzed using reversed-phase capillary high-performance liquid chromatography directly coupled to a Thermo LTQ Orbitrap mass spectrometer following the procedure described by Zuo et al. (2001) with slight modifications. Briefly, both a 0.075×20 mm trapping column and a 0.075× 120 mm resolving column were packed with C18AQ 218MS low formic acid C18 beads (5 μm in size, 200Å pore size; C18AQ, Michrom BioResources, Auburn, CA, USA) and placed in-line. Peptides were bound to the trapping column for 10 min with 2% (vol/vol) aqueous cetonitrile containing 0.1% (vol/vol) formic acid. The bound peptides were eluted with a gradient of 2% to 90% (vol/vol) acetonitrile containing 0.1% (vol/vol) formic acid at a flow rate of 0.2 μL/min. For tandem mass spectrometry, full mass scan range mode was set at m/z = 50 to 2,000 Da. After determining the charge states of the ion zoom scans, product ion spectra were acquired in MS/MS mode with relative collision energy of 55%. Individual spectrum from MS/MS was processed using Protein discoverer 2.1 software (Thermo scientific, USA). The generated peak list files were used to query either the MSDB or the NCBI database using MASCOT program (http://www.matrixscience.com). We took into account modifications of methionine and cysteine, peptide mass tolerance at 2 Da, MS/MS ion mass tolerance at 0.8 Da, allowance of missed cleavage at 2, and charge states (namely, +1, +2, and +3). Only significant hits defined by MASCOT probability analysis were initially considered.
RNA extraction and real-time PCR analysis
Adipose tissues were collected from cows, steers, and bulls. Total RNA isolation was performed using TRIzol reagent (Invitrogen, Grand Island, NY, USA) according to the manufacturer’s instructions. Briefly, total RNA levels were quantified at absorbance of 260 nm. RNA integrity was evaluated by 1.2% (w/v) agarose gel. Total RNA (2 μg amounts) was reverse-transcribed into cDNA using QuantiTect Reverse Transcription Kit (Qiagen, Chatsworth, CA, USA) according to the manufacturer’s instructions. Real-time PCR was performed with SYBR green Premix Ex Taq II (Takara, Dalian, China) using Applied Biosystems StepOne Plus Real-time PCR System (Applied Biosystems, Carlsbad, CA, USA). The expression of β-actin was used as the endogenous control. Relative quantification analysis was performed using the comparative Ct (2−ΔΔCt) method (Wilting et al., 2010). Primers used in the study are listed in Table 1.
RESULTS AND DISCUSSION
Carcass characteristics
We used cows (636 kg live weight), steers (762 kg live weight), and bulls (832 kg live weight) at normal slaughter age (31 months) in Korea. Generally, Korean beefs are slaughtered routinely at 29 to 32 month of age to increase marbling and quality grade (Choy et al., 2012). Carcass characteristics of a subset data of the cows, steers, and bulls used for proteomic analysis are summarized in Table 2. Bulls had significantly (p<0.05) heavier carcass weight with lower trend backfat thickness. Bulls also had significantly (p<0.05) lower marbling scores, quality grade, and better yield grade. Our result was mostly in consistent with the effect of castration on meat quality in Korean cattle reported in a previous study (Jeong et al., 2013).
Protein profiles in IMAT and OMAT from Hanwoo cows, steers and bulls
To obtain a comprehensive overview of protein components in IMAT and OMAT from individual seven groups (cows, steers, and bulls), protein profiles of whole lysate of IMAT and OMAT were separated by SDS-PAGE and assessed by silver-stained image analysis. The number, marbling score, and quality grade of individuals were showed in Figure 1A. The patterns of total proteins in IMAT and OMAT were similar to each other. However, IMAT components were significantly different from OMAT components (Figure 1B).
Protein identification and gene ontological classification by LC-MS/MS-based proteomic analysis
LC-MS/MS-based proteomic analysis was performed to identify proteins involved in depot specific adipose tissue (i.e. intramuscular and omental) properties associated with sex (cows, steers, and bulls). Of the 55 proteins identified, 44, 40, and 42 proteins were confirmed to be differentially expressed in IMAT of cows, steers, and bulls, respectively. In OMAT of cows, steers, and bulls, 33, 33, and 22 were confirmed to be differentially expressed, respectively (Table 3). All identified proteins were clustered into eight categories based on biological process (BP) using information obtained from the DAVID gene ontology (GO) database (http://david.abcc.ncifcrf.gov) and UniProt (http://www.uniprot.org). Depending on the BP in which the proteins were involved, they were categorized into the following groups (Figure 2A): carbohydrate metabolism (35.7%), glycolysis (19.6%), muscle contraction (10.7%), electron transport (10.7%), protein folding (7.1%), muscle development (5.4%), tricarboxylic acid (TCA) pathway (5.4%), and carbon metabolism (5.4%).
A total of 16 up- or down-regulated proteins between IMAT and OMAT of cows, steers, and bulls were selected. GO analysis was performed using DAVID Bioinformatics Resources 6.7 categories both Reactome-Pathway and Panther-BP. Depending on the Reactome-Pathway in which the protein was involved, the 16 proteins were categorized into the following five groups (Figure 2B): metabolism of carbohydrates (37%), integration of energy metabolism (22.2%), diabetes pathways (22.2%), muscle development (11.1%), and TCA cycle (7.4%). Depending on the Panther-BP in which the protein was involved, they were categorized into the following six groups (Figure 2C): carbohydrate metabolism (33.3%), glycolysis (23.3%), cell structure (13.3%), muscle development (10%), muscle contraction (10%), and cell motility (10%) (Table 4). The expression changes of the up- and down-regulated proteins in IMAT and OMAT of cows, steers, and bulls depending on the Reactome-Pathway were summarized in Table 4. The mRNA expression patterns of the 16 selected proteins were further analyzed by real-time PCR.
Quantitative real-time PCR confirmation for selected genes
To study the patterns of gene expression in IMAT and OMAT associated with sex, we used cows, steers, and bulls. The mRNA expression levels of the selected genes were subjected to quantitative real-time PCR with specific primers (Table 1). Previous studies have reported that fructose-bisphosphate aldolase A (ALDOA) mRNA increases during in vitro myogenesis (Colbert and Ciejek-Baez, 1988) are responsible for significant activation during the differentiation of primary myoblasts, therefore playing important roles in muscle gene transcription (Walsh et al., 1980; Hidaka et al., 1993; Ren et al., 2011). Our data showed that ALDOA had significantly higher expression in IMAT than in OMAT in cows (p = 0.0042) and steers (p<0.0001) (Figure 3A and 3B). However, ALDOA had significantly (p<0.0001) lower expression in IMAT than in OMAT in bulls (Figure 3C). These results demonstrated that ALDOA was differentially expressed depending on sex, suggesting that ALDOA could be one of the factors affecting lipid accumulation in OMAT.
Western blot analysis for selected proteins
We found significant correlations between several factors (including tropomyosin [TPM] 1, TMP2, and TMP3) and gene expression in IMAT and OMAT. TPMs are a family of actin binding proteins in all tissues that are always associated with polymerized actin. TPMs are a diverse group of cytoskeletal proteins found in most eukaryotic cells, with distinct isoforms found in muscle (skeletal, cardiac, and smooth) and various non-muscle cells (Dlugosz et al., 1984; Lin and Lin, 1986). Previous studies have shown that TPM plays a critical role in skeletal muscle development and function (Marston et al., 2013; Zhang et al., 2014). Results of the mRNA levels (upper panels) and protein expression levels (lower panels) of TPM1, TPM2, and TPM3 are shown in Figure 4. Notably, transcriptional and protein levels of TPM1, TPM2, and TPM3 were significantly lower in IMAT of steers compared to cows or bulls. The mRNA and protein levels of TPM1, TPM2, and TPM3 were higher in OMAT of cows than in bulls. In addition, TPM1, TPM2, and TPM3 had higher expression in OMAT than in IMAT in cows and steers, but had lower expression in OMAT than IMAT in bulls. These results demonstrated that TPM1, TPM2, and TPM3 were differentially expressed depending on sex. Adipose depots and TPMs were positively correlated with marbling score and quality grade. Therefore, we suggest that TPM1, TPM2, and TPM3 are key factors closely associated with muscle development and lipid accumulation in Hanwoo cows, steers, and bulls.
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